Cited in Index Medicus/MEDLINE, BIOSIS Previews, SCOPUS
Journal of the Italian Society of Anatomic Pathology
and Diagnostic Cytopathology,
Italian Division of the International Academy of Pathology
Periodico bimestrale – POSTE ITALIANE SPA - Spedizione in Abbonamento Postale - D.L. 353/2003 conv. in L. 27/02/2004 n° 46 art. 1, comma 1, DCB PISA
Aut. Trib. di Genova n. 75 del 22/06/1949
Vol. 102 August 2010
Società Italiana di Anatomia Patologica e Citopatologia Diagnostica,
Divisione Italiana della International Academy of Pathology
IL BOARD DI PPPFAD
Bruno Murer, Mattia Barbareschi, a nome del GIPP
Un servizio di aggiornamento scientifico realizzato con il contributo di
Care Colleghe e Cari Colleghi,
PPPFAD, il programma formazione a distanza web-based
che il Grruppo Italiano di Patologia PleuroPolmonare
(GIPP) ha organizzato lo scorso anno, grazie ad un grant
educazionale non condizionante di Lilly Italia, ha avuto un
grande successo, con quasi 400 colleghi che hanno visitato il nostro sito e affrontato i 24 casi proposti fra il 2009 e
i primi mesi del 2010!
Abbiamo così deciso di proseguire l’attività formativa anche nel corso del 2010, adottando però una nuova formula
che speriamo possa essere gradita a tutti. Si tratta di un percorso che da un lato
propone nuovamente la formula della visione di casi problematici prevalentemente di natura neoplastica con un format più snello e vivace, dall’altro ci offre la
straordinaria possibilità di partecipare a un grande studio di concordanza diagnostica nella applicazione della classificazione degli istotipi dei tumori del polmone,
con la possibilità anche di fare una fotografia reale delle possibilità/difficoltà
diagnostiche sia sulle piccole biopsie che sui pezzi operatori.
Da tale studio di concordanza potremo trarre un lavoro scientifico di sicuro interesse che cercheremo di pubblicare a nome di tutti i partecipanti. Lo studio di concordanza inizierà con una serie di casi su piccole biopsie per poi proseguire nella
seconda metà del progetto con i casi operatori, sui quali cercheremo di applicare
anche i nuovi schemi classificativi che sono attualmente in corso di pubblicazione.
PPPFAD risponde appieno alle caratteristiche educazionali richieste dal nuovo
Sistema nazionale ECM, presso il quale è stato accreditato appunto quale programma di formazione a distanza.
Sul website di www.pppfad.it troverete dunque il doppio percorso formativo col
quale poter conseguire i crediti ECM-FAD.
Con la certezza che possiate gradire e condividere lo sforzo del GIIPP per offrire
ai patologi italiani le migliori occasioni di aggiornamento e di confronto, a Voi tutti
l’augurio di un buon lavoro!
Bruno Murer
Coordinatore GIPP
1. PPPFAD 2010
È il nuovo programma FAD realizzato dal GIPP,
Gruppo Italiano di Studio di Patologia Pleuropolmonare
2. COSA PUBBLICA PPPFAD
Un duplice percorso formativo che prevede:
- 24 casi clinici (2 al mese)
- 100 casi di concordanza diagnostica articolati in 20 moduli da 10 casi ciascuno
- A completamento, la possibilità di partecipare ad una survey finalizzata all’elaborazione
dello studio scientifico promosso dal GIPP
3. COME ACCEDERE A PPPFAD
Accedere a PPPFAD è facile:
- digitare l’indirizzo Internet: www.pppfad.it
- inserire il codice di attivazione fornito
- registrarsi scegliendo le proprie username e
password
4. CREDITI ECM-FAD
- PPPFAD è accreditato quale servizio di Formazione a Distanza presso il Sistema nazionale ECM
- Eroga dunque crediti ECM-FAD: 32 crediti
annui
- Per acquisirli è necessario registrarsi e completare i casi clinici proposti da www.pppfad.it
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Ai sensi dell’art. 13 del DL 196/03 e succ. modifiche e integrazioni La informiamo che i Suoi dati personali verranno trattati dal Titolare con strumenti informatici nel pieno rispetto della normativa applicabile al
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Data Firma
Cited in Index Medicus/MEDLINE, BIOSIS Previews, SCOPUS
Journal of the Italian Society of Anatomic Pathology
and Diagnostic Cytopathology,
Italian Division of the International Academy of Pathology
Editor-in-Chief
Marco Chilosi, Verona
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Vol. 102 August 2010
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COMITATI
PRESIDENTE COMITATO
SCIENTIFICO
Vincenzo Eusebi
COMITATO SCIENTIFICO
Arrigo Bondi
Cesare Bordi
Marco Chilosi
Maria Pia Foschini
Giorgio Gardini
Felice Giangaspero
Walter F. Grigioni
Giovanni Lanza
Antonio Maiorana
Giuseppe Martinelli
Roberto Nannini
Stefano Pileri
Anna Sapino
Gian Luigi Taddei
Giovanni Tallini
PRESIDENTE COMITATO
ORGANIZZATORE
Arrigo Bondi
COMITATO
ORGANIZZATORE
Andrea Ambrosini Spaltro
Gian Piero Casadei
Giovanna Cenacchi
Guido Collina
Antonia D’Errico
Giuseppina Ferro
Michelangelo Fiorentino
Luisa Losi
Luca Morandi
Annalisa Pession
Pier Paolo Piccaluga
Teresa Ragazzini
Donatella Santini
lectures
Pathologica 2010;102:127-235
Wednesday, September 22nd, 2010
Symposium Susan G. Komen: Breast pathology
Moderators: R. Masetti (Roma), V. Eusebi (Bologna)
The role of magnetic resonance in the
surveillance of women at high risk of
hereditary breast cancer
F. Podo, F. Santoro, F. Sardanelli *
Department of Cell Biology and Neurosciences, Istituto Superiore di
Sanità, Rome; * Università di Milano, IRCCS Policlinico San Donato,
Milano, Italy
Background. Breast cancer (BC) affects up to 1:7 to 1:11
women in Western Countries. Although BC is mainly a sporadic disease, about 15% of cases are clustered in families
with highly or moderately elevated BC incidence. Pathogenic
mutations in high-risk genes at autosomic dominant inheritance are held responsible for about 5% of BC cases, in which
the disease may have early onset with a estimated cumulative
lifetime risk as high as 50% to 85%. About 50% of hereditary BC cases can be explained by mutations in BRCA1 and
BRCA2 genes. BRCA1 mutations are frequently associated
with triple negative BC (TNBC), defined by lack of estrogen
and progesterone receptor expression and absence of ErbB2
(or HER2) amplification (reviewed in Bosch A. et al. Cancer
Res and Treatment Reviews 2010; Podo F. et al., Mol Oncol
2010). In women at high risk of breast cancer, screening mammography has shown a lower sensitivity (29-50%) compared
with that of the screening addressed to the general female
population (80%), with higher percentages of interval cancers
(35-50% vs 20-25%) and higher nodal involvement (20-56%
vs 22%). In the last decade a number of prospective, non-randomized studies have been conducted in Europe and North
America to assess the value of dynamic contrast-enhanced
magnetic resonance imaging (MRI) as a screening tool to be
used as an adjunct to ×-ray mammography (XM), or to XM
and ultrasonography (US) for the surveillance of women at
high genetic-familiar risk of BC 1-10. We will summarize the
results of two consecutive multicenter, prospective, non-randomized studies coordinated in Italy by the Istituto Superiore
di Sanità, the ISS-HIBCRIT Study 6 9 11, carried out from June
2000 to March 2008 in 18 Centers, and the four-year ISSINHBCR study, whose screening activities started in 2008 with
the collaboration of a Network of 23 Centers located in 13
regions.
Methods. Both studies prospectively compared clinical breast
examination (CBE), XM, US, and MRI for screening women
at genetic-familial high risk of BC. CBE, XM, US, and MRI
were used for repeated, yearly screening of women either
proven to be BRCA1 or BRCA2 mutation carriers (BRCA+),
or untested first degree relatives of BRCA+, or enrolled only
on the basis of strong family history of breast and/or ovarian
cancer. Histopathology or at least one-year negative follow-up
were used as the reference standard.
Results. The HIBCRIT study enrolled 501 asymptomatic
women (mean age 46.0 ± 11.8 years; median age 45 years);
69% proven carriers of BRCA1 or BRCA2 mutation (or first
degree relatives of proven carriers) with a BRCA1:BRCA2
ratio of 1.3; 43.5% women had previous BC and/or ovarian
cancer. A total of 1592 annual rounds (3.2 rounds/woman)
were performed. A total of 52 breast cancers were detected:
49 screen-detected and 3 interval cancers (all three TNBC); 44
invasive, 8 in situ; only 4 at stage ≥ pT2; 32 G3 grade; 72%
(28 out of 39 patients explored for nodal status) were nodenegative. Of 43 invasive BC for which histopathological findings were reported, 18 (43%) were TNBC (12 associated with
BRCA1 mutation, 3 with BRCA2 mutation and 3 detected
in untested women with a strong family history of BC). The
detection rate per year was 3.0% (95% CI 1.9%-4.0%) for
BRCA1 or BRCA2 mutation carriers and 3.3% overall (95%
CI 2.4%-4.1%). Cancer was detected only with MRI in 15
out of 40 patients examined with all modalities. MRI showed
the highest sensitivity (91%) compared with CBE (18%),
XM (50%), ultrasonography (52%) and to the combination
of mammography plus US (63%) (p < 0.001). Specificity for
CBE, mammography, ultrasonography, mammography plus
ultrasonography and MRI was 99%, 99%, 98%, 98% and
97%; positive predictive value (PPV) was 56%, 71%, 62%,
56% and 56%; negative predictive value 96%, 97%, 98%,
98% and 100%, without significant differences. The area
under the curve at ROC analyses was significantly higher for
MRI (0.97) than for mammography (0.83) and ultrasonography (0.82) (p < 0.001) and was not significantly increased in
the combination of MRI with either mammography or ultrasonography or both modalities.
The ISSIN-HBCR study enrolled 662 women in two years
(1.4 rounds/woman). The population characteristics are not
significantly different from those of the HIBCRIT study in
terms of mean and median age, percentage of BRCA mutation
carriers (70%) and percentage of women with previous breast
cancer. A total of 29 breast cancers have already been detected: 24 screen-detected and 5 interval cancers (four TNBC,
one not yet reported); 28 invasive, 1 in situ. The distribution
of pT stages, G grade and percentage of nodal involvement
are very close to those of the eight-year HIBCRIT study. The
sensitivity of the MRI similarly outperformed that of the other
imaging modalities or their combination; 33% of cases were
detected by MRI only.
Conclusions. In conclusion, the consolidated results of the
HIBCRIT study showed that: a) MRI largely outperformed
XM, US and their combination for screening high-risk women
under and over 50 years of age; b) over 30% of tumors were
detected by MRI only; c) the PPV of MRI reached 56%;
d) over 40% of detected tumors were either in situ or smaller
than 1 cm; e) over 70% of invasive tumors were lymph-node
negative; f) women at high genetic-familial risk with personal
history of BC should be included in a multimodality surveillance including annual MRI; g) the increasing incidence with
age suggests not to reduce intensive surveillance in menopausal women. Preliminary results of the ISSIN-HBCR study
confirm these trends. Further studies are needed: to better define risk-reduction strategies for BRCA1 mutation carriers in
relation to the high risk of TNBC; to identify MRI parameters
related to TNBC diagnosis, prognosis and prediction of therapy response; to evaluate the benefits of surveillance of high
risk women compared with other strategies of risk reduction,
128
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
according to gene mutation and age; to possibly optimize and
simplify the protocols of multimodality surveillance; to evaluate the cost/benefit ratio of extending secondary prevention
programs to women at intermediate risk of breast cancer.
References
1
Kriege M, Brekelmans CT et al. N Engl J Med. 2004;351:427-37.
2
Warner E, Plewes DB, et al. JAMA. 2004;292:1317-25.
3
Leach MO, Boggis CR, et al. Lancet. 2005;365(9473):1769-78.
4
Kuhl CK, Schrading S, et al. J Clin Oncol 2005;20;23:8469-76.
5
Lehman CD, Blume JD, et al. Cancer 2005;103:1898-905.
6
Sardanelli F, Podo F, et al. Radiology 2007;242:698-715.
7
Hagen AI, Kvistad KA, et al. Breast 2007;16:367-74.
8
Riedl CC, Ponhold L, et al. Clin Cancer Res. 2007;13:6144-52.
9
Sardanelli F, Podo F. Eur Radiol 2007;17(4):873-87.
10
Kuhl C, Weigel S et al. J Clin Oncol 2010;28(9):1450-7.
11
Podo F, Sardanelli F et al., submitted.
Preoperative breast mri: which evidence?
F. Sardanelli
Dipartimento di Scienze Medico-Chirurgiche, Università di Milano,
Unità di Radiologia, IRCCS Policlinico San Donato, Milan, Italy
([email protected])
Background. Breast conserving treatment (BCT), comprising breast conserving surgery (BCS) plus radiation therapy,
is equally effective to mastectomy, in terms of survival, for
early-stage cancers as demonstrated in randomized controlled trials (RCTs) and confirmed in a meta-analysis 1. Of
importance, four of the six RCTs of BCS included in this
meta-analysis show a significantly lower risk of locoregional
recurrence in favor of mastectomy (odds ratio 1.561) 1. Thus,
BCS should always aim to completely remove tumoral tissue
and obtain clear margins.
Evidence on MRI’s detection capability. MRI has a superior
sensitivity compared with mammography in assessing index
tumor size and in detecting ipsilateral multifocal or multicenter cancers, as demonstrated also in a multicenter study 2.
However, MRI may fail to detect all cancers when the whole
breast is used as a pathological reference standard 3, especially
when ductal carcinoma in situ (DCIS) is considered 4. The
advantage of MRI has been shown to be non-significant in
fatty breasts, while significant in scattered fibroglandular,
heterogeneously, or extremely dense breasts 3. MRI has also
been shown to detect extensive intraductal component, but
may overestimate or underestimate this finding in 11-28%
and 17-28% of cases, respectively 5-7. In a meta-analysis of
19 studies 8, the impact of pre-operative MRI on ipsilateral
surgical planning was evaluated reporting surgical outcomes
as follows 8:
– 8.1% conversion from wide local excision to mastectomy
due to true positive findings;
– 1.1% conversion from wide local excision to mastectomy
due to false positive findings;
– 3.0% conversion from wide local excision to wider/additional excision due to true positive findings;
– 4.4% conversion from wide local excision to wider/additional excision due to false positive findings.
Furthermore, several studies have shown that MRI can detect
otherwise occult contralateral malignancy in women newly
diagnosed with invasive cancer for about 3% of patients 9.
A meta-analysis of 22 studies 10 showed that MRI yields an
incremental cancer detection rate equal to 4.1% with a positive predictive value of 47.9% due to a false positive detection
rate of 5.2% (true positives/false positives = 0.92). In this
analysis 10 35% of contralateral cancers were DCIS with a
mean diameter of 7 mm, 65% invasive with a mean diameter
of 9.3 mm, the majority of the latter were node negative 10. A
higher probability of an added diagnostic value of MRI for
local staging has been shown for particular patient subgroups.
In a recent systematic review of patients with invasive lobular
cancer, additional ipsilateral lesions were found to be detected with MRI in 32% of cases, contralateral lesions in 7%
while surgical management was changed in 28%.11 In these
patients, MRI showed a 93% pooled sensitivity and a high
correlation with pathologic tumor extent.11 Women with an
inherited high risk for breast cancer have a high probability of
a more accurate local staging with MRI. The rate of multifocal and multicenter cancers in these women was reported as
high as 45-50% 12 13. In one study, the percentage of breasts
with exact detection of the number of malignant lesions was
reported to be 0% for mammography, 33% for sonography,
and 71% for MRI 13. Regarding the assessment of tumor extent, a retrospective analysis by Deurloo et al. 14. reported that
patients younger than 58 years of age with irregular lesion
margins at mammography and discrepancy in tumor extent
(including spiculated lesions and suspicious microcalcifications) by > 10 mm between mammography and sonography,
had a 50% probability of complementary value of MRI over
conventional imaging vs 16% in the remaining patients. Last
but not least, MRI identifies a fraction of candidates for partial breast irradiation (PBI) who are affected with multifocal,
multicentric, or contralateral cancer and may therefore not be
suitable for this approach in treatment, about 5-10% according three recent studies 15-17. This should be considered in the
light that the American Society for Radiation Oncology has
recently established the possibility of using PBI “outside a
clinical trial” at least for patient subgroups 18. Up to recent
times, we have lacked evidence on patient outcomes in favor
of, or against, pre-operative MRI. Conflicting retrospective
studies on outcomes have been reported 19-22, intrinsically
limited by non-randomization. Unfortunately, the results of
the COMICE 23 study, indicating the absence of benefit from
MRI (about 19% of re-excision rate in both the MRI and nonMRI arms), are flawed by relevant limitations mainly due to
very few experience with breast MRI by the large number of
centers involved in that trial 24.
The potential and the drawbacks of MRI. Using tissue
needle sampling of MRI-detected additional findings (through
second-look sonography or MR-guidance), we will potentially
drastically reduce overtreatment due to MRI false positives.
As a consequence, using the estimates of Houssami et al. 8, we
would have only the 11.1% rate of MRI-induced potentially
correct changes of surgical planning for the breast harboring
the index lesion. To place this into context, we should consider the routine rate of positive margins after BCS, ranging from
20% to 40% or more,25 and that of local recurrences after
BCT, usually considered from 5% to 10% at ten years26 and
reported about 9% at 20 years 27. A similar reasoning can be
proposed for the detection of contralateral cancers. Consistent
use of MR-guided biopsy could strongly reduce the surgical
treatment of false positives (about 5%) 10, offering the chance
to treat the synchronous contralateral cancers in about 4% of
the women10 with simultaneous surgery. This rate should be
compared with the 0.5-1% annual risk of contralateral breast
cancer in women with a previous history of breast cancer 28 29.
We could speculate that only ipsilateral recurrences or contralateral cancers which would have appeared in the first years
after BCT might be avoided by pre-operative MRI 26. Thus,
this comparison gives a relatively balanced result for contralateral cancers: with a 0.75% annual rate of contralateral
129
Lectures
cancers and an anticipated MRI diagnosis up to 3-4 years, we
have a 2-3% cumulative rate of contralateral cancers in the
first few years to be compared with a rate of MRI-detected
contralateral cancers of 3-4% 9 10. A larger discrepancy is obtained if we hypothesize a similar cumulative rate (2-3%) for
local recurrences in the first years, to be compared with the
11.1% rate 8 of MRI-induced correct changes of surgical planning for the breast harboring the index lesion. However, the
rate of MRI-detected ipsilateral and contralateral cancers is
probably overestimated due to the fact that pre-operative MRI
has been performed in non-consecutive (selected) series 26, i.e.
through selection of patients with a probable higher likelihood
of ipsilateral and contralateral cancers (for example dense
breasts, or high-risk patients) to MRI. A publication bias is
also hypothesized. Moreover, it is hard to evaluate the combination of the two aspects from a patient-based perspective:
pre-operative MRI could determine an unnecessary wider/additional ipsilateral excision but also anticipate the diagnosis
of contralateral cancer (or vice versa), thus avoiding the second cancer event in future, and receiving treatment for both
breasts upfront; it may be argued that a bilateral advantage or
a bilateral overtreatment could happen as a consequence. This
interpretation considers the fact that systemic therapy may
prevent some of the contralateral cancers 26 detected upfront
by MRI only. At present, potential outcome benefits of preoperative MRI may include a possible reduction in the rate of
the following events: surgical intervention needed to achieve
free margins; ipsilateral recurrences; secondary mastectomies;
and contralateral malignancy. On the other hand, we should
consider that the use of MRI has been reported to be associated with an increased higher rate of mastectomy 22 26 30 31 and
with a treatment delay of 22.4 days 24.
Perspectives on indications for pre-operative MRI. Acceptable indications for pre-operative MRI can be presently
defined for subgroups of patients inwhom a larger potential
benefit in term of local staging might be expected. This approach should be considered also for future RCTs evaluating
pre-operative MRI. In fact, if the advantages of MRI would
be relevant only for particular subgroups, RCTs on the average population of women newly diagnosed with a breast
cancer may dilute the benefit and probably reduce power for
achieving significance in subgroup analysis. Patients with a
potential higher anticipated benefit from preoperative MRI
can be identified as those: 1. with mammographically (heterogeneously or extremely) dense breasts; 2. with a unilateral
multifocal/multicentric cancer or a synchronous bilateral cancer; 3. with a lobular invasive cancer; 4. at high-risk for breast
cancer; 5. with a cancer which shows a discrepancy in size
of > 1 cm between mammography and sonography; 6. under
consideration for PBI.
More limited evidence exists in favor of MRI for evaluating candidates for total skin sparing mastectomy in order to
decide saving or not the nipple32 or for patients with Paget’s
disease 33-35. Further research is needed in particular on these
indications. Irrespective of whether the clinical team routinely uses preoperative MRI or not, the following issues are
paramount: A. women newly diagnosed with breast cancer
should always be informed of the potential risks and benefits
of pre-operative MRI; B. results of pre-operative MRI should
be interpreted taking into account clinical breast examination,
mammography, sonography and verified by percutaneous biopsy; C. MRI-only detected lesions require MR-guidance for
needle biopsy and pre-surgical localization, and these should
be available or potentially accessible if pre-operative MRI is
to be implemented; D. total therapy delay due to pre-operative
MRI (including MRI induced work-up) should not exceed one
month; E. changes in therapy planning resulting from pre-operative MRI should be decided by a multidisciplinary team.
Conclusions. In reality, and considering the detection capability of MRI, we cannot wait for conclusive evidence in favor
of or against preoperative MRI. To deny this examination to
all women newly diagnosed with breast cancer is a questionable decision because the evidence is ‘uncertain’ rather than
against a benefit from preoperative MRI. In this context, to
define general rules to be shared by breast cancer specialists is
the first goal to avoid inappropriate use of this diagnostic step.
To propose pre-operative MRI for subgroups of women as
here defined can be a practical strategy for the present. Finally,
the woman’s preference should be also carefully considered in
order to decide whether to perform or not to perform preoperative MRI, according to evidence-based medicine basic
principles 36. From this standpoint we should also consider that
mastectomy in 2010 is no longer the same surgical approach
performed thirty or forty years ago. Immediate reconstruction,
skin- and nipple-sparing mastectomy changed the scenario at
least in terms of cosmetic results. Part of the reported increase
in mastectomy rate may be due to the availability of these
options. The large meta-analysis of Clarke et al. on the effect
of radiation therapy concludes that “differences in local treatment that substantially affect local recurrence rates would, in
the hypothetical absence of any other causes of death, avoid
about one breast cancer death over the next 15 years for every
four local recurrences avoided, and should reduce 15-year
overall mortality” 37. MRI is not radiation therapy but guiding
a more effective surgery might potentially provide a similar
effect. High-quality clinical research on pre-operative MRI is
needed, especially RCTs.
References
1
Jatoi I, et al. Am J Clin Oncol 2005;28:289-94.
2
Schnall MD, et al. J Surg Oncol 2005;92:32-8.
3
Sardanelli F, et al. AJR Am J Roentgenol 2004;183:1149-57.
4
Sardanelli F, et al. Radiol Med 2008;113:439-51.
5
Schouten van der Velden AP, et al. Am J Surg 2006;192:172-89.
6
Van Goethem M, et al. Eur J Radiol 2007;62:273-82.
7
Kim do Y, et al. Korean J Radiol 2007;8:32-9.
8
Houssami N, et al. J Clin Oncol 2008;26:3248-58.
9
Lehman CD, et al. N Engl J Med 2007;356:1295-303.
10
Brennan ME, et al. J Clin Oncol 2009;27:5640-9.
11
Mann RM, et al. Breast Cancer Res Treat 2008;107:1-14.
12
Kuhl CK, et al. J Clin Oncol 2005;23:8469-76.
13
Sardanelli F, et al. Radiology 2007;242:698-715.
14
Deurloo EE, et al. Eur Radiol 2006;16:692-701.
15
Al-Hallaq HA, et al. Cancer 2008;113:2408-14.
16
Godinez J, et al. AJR Am J Roentgenol 2008;191:272-7.
17
Tendulkar RD, et al. Cancer 2009;15(115):1621-30.
18
Smith BD, et al. Int J Radiat Oncol Biol Phys 2009;74:987-1001.
19
Fischer U, et al. Eur Radiol 2004;14:1725-31.
20
Solin LJ, et al. J Clin Oncol 2008;26:386-91.
21
Pengel KE, et al. Breast Cancer Res Treat 2009;116:161-9.
22
Bleicher RJ, et al. ASCO Breast 2008 [abstract 227].
23
Turnbull L, et al. Lancet 2010;375:563-71.
24
Morris EA. Lancet 2010;375:528-30.
25
Pleijhuis RG, et al. Ann Surg Oncol. 2009;16:2717-30.
26
Houssami N, Hayes DF. CA Cancer J Clin 2009;59:290-302.
27
Veronesi U, et al. N Engl J Med 2002;347:1227-32.
28
Adami HO, et al. Cancer 1985;55:643-7.
29
Rutqvist LE, et al. J Natl Cancer Inst 1991;83:1299-306.
30
Foote RL, et al. Breast 2008;17:555-62.
31
Katipamula R, et al. J Clin Oncol 2008;26(Suppl):a509.
32
Wijayanayagam A, et al. Arch Surg 2008;143:38-45.
33
Frei KA, et al. Invest Radiol 2005;40:363-7.
34
Haddad N, et al. J Radiol 2007;88:579-84.
35
Morrogh M, et al. J Am Coll Surg 2008;206:316-21.
36
Sackett DL, et al. BMJ 1996;312:71-2.
37
Clarke M, et al. Lancet2005;366:2087-106.
130
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
New and old entities in breast pathology
V. Eusebi
Sezione di Anatomia Istologia e Citologia Patologica “M. Malpighi”
Università di Bologna
To use Goethe’s words “we see what we know”. As we know
very little new entities usually emerge as the result of better
technology as well as more accurate methods of analysis. In
addition some lesions tend to become obsolete and periodically are rediscovered and rejuvenated.
In recent years very powerful molecular techniques have appeared which has lead to the statement by some molecular
pathologists that by the year 2020, histopathology is going
to be history and all diagnostic work up is going to be in the
hands of scientists or machines.
This might be the case although, to use the words of Lorenzo
il Magnifico, “del diman non c’è certezza”. For the time being it appears that in spite of great expectations in molecular
techniques, no very consistent achievements have been obtained. One example for all. Perou et al. 1, at the beginning of
this century, using a DNA array technique, reclassified breast
cancer among groups different from those classically used.
After nearly 10 years since the publication of Perou’s article,
it appears that the new classification is not very useful in
routine practice. One for example is the basal like carcinoma.
In spite of myriads of papers published on it, there is still no
a consensus on the definition of this type of tumor. Basal like
molecular profile appears to be in common with a heterogeneous group of tumors which include very aggressive lesions
that are G. 3 and triple negative carcinomas together with
lesions that are quasi benign as well differentiated adenoid
cystic carcinomas.
Therefore we discuss here cases whose definition is mainly
based on morphology.
Breast carcinomas are simulated by a number of inflammatory
conditions.
Nodules either single, multiple or even bilateral are shown
by IgG4-related sclerosing mastitis 2 which is a new entry of
the syndrome of the IgG4-related sclerosing disease. This is a
recently recognized syndrome characterized clinically by tumour-like enlargement of one or more exocrine glands as well
as extra-glandular tissue. There is raised serum IgG4 level and
histologically there is lymphoplasmacytic infiltration together
with sclerosis. There are an increased number of IgG4-secreting plasma cells. The syndrome is believed to be autoimmune
in origin, it was originally observed in autoimmune sclerosing-pancreatitis but a number of different sites have been
reported such as hepatobiliary tree, lachrymal glands, salivary
glands, lymph node, prostate, lung, kidney, retro-peritoneum
and mesentery, mediastinum, meninges and breast 2-5. In this
latter site, of the 5 cases reported 2 were unicentric, 3 multifocal and 1 bilateral. Breast lesions are characterized by dense
masses of lymphocytic infiltrate associated to intense sclerosis
and loss of lobules. Reactive lymphoid follicles can be seen
but granulomas as well as lympho- epithelial lesions are lacking. IgG4+ ought to be no less than 50% of IgG+ elements.
This “inflammatory” lesion has to be distinguished from
low grade B cell lymphoma and Castleman’s disease. In addition an inflammatory quasi neoplastic condition is Rosai
Dorfman’s disease that can affect the breast. Of the 7 cases
reported by Green et al. 6, 3 patients had disease confined to
the breast, one had involvement of the breast and ipsilateral
auxiliary lymph nodes and two had bilateral breast involvement. A xantomatous infiltrate with scattered Touton’s giant
cells and patchy lymphocytic infiltrate are the features of
Erdheim-Chester (E-C) disease that may involve the breast
presenting as bilateral clinically malignant breast masses.
E-C disease is a rare non Langerhans cell histiocytosis of
unknown aetiology. The commonest sites of involvement are
long bones, skin, orbit pituitary and retro peritoneum. A number of granulomatous mastitis can clinically simulate a breast
carcinoma among which idiopathic granulomatous mastititis 7,
sarcoid 8 and cat scratch disease.
Among the lesions that are rejuvenated, the most obsolete
entity that only recently has been brought up again is infiltrating epitheliosis 9.
Infiltrating epitheliosis (IE) was described by Azzopardi in
1979 in Chapter 9 “Overdiagnosis of malignancy” of his book
“Problems in breast pathology” 10 as “a lesion which is not
uncommon but which has not received adequate recognition
in the literature”, a statement very pertinent 30 years later.
Infiltrating epitheliosis (IE) is usually a microscopic lesion,
observed incidentally in cystic disease but which may infrequently present as a palpable lump. The lesion is generally located far from the nipple as epitheliosis (also known as usual
duct hyperplasia-UDH), which is the main component, affects
the acinar, terminal duct lobular unit (TDLU) and small duct
portions of the mammary lobes 10.
The lesion is a complex epithelial-stromal interaction composed of epitheliosis (UDH) which constitutes the bulk of the
lesion, and sclero-elastotic stromal changes.
At low power IE appears as an asymmetrical lesion, with
sclero- elastotic areas located randomly either in the centre
or at the periphery. The borders vary from irregular to circumscribed. In palpable lesions the scleroelastotic areas can
be multiple.
Morphologically the lesion is composed of Epitheliosis (synonym usual duct hyperplasia), and Scleroelastotic areas the
latter characterized by a stromal reaction which is not only
desmoplastic, but may also contain dense sclerotic and hyaline collagenous bands…” not unlike the appearances seen in
a keloid” 10. Finally abundant elastic tissue (elastosis) is seen
intermingled with the desmoplastic reaction or around small
ducts forming nodular foci. Infiltrating epitheliosis has to be
distinguished from Radial Scar (benign scleroelastotic lesion
simulating invasive duct carcinoma) which Hamperl in 1975
described as a microscopic lesion that he named in German
“strahligen narben”. In the summary this was translated as
“radial scar” 11. A few months later, Eusebi et al. 12 independently reported on the mammographic, macroscopic and
microscopic features 4 cases showing a lesion they named in
Italian “lesioni focali scleroelastotiche mammarie simulanti il
carcinoma infiltrante”. In the summary this was translated as
“mammary focal scleroelastotic lesions simulating an infiltrating carcinoma”.
Both papers dealt with the same identical lesion 10, the only
difference being the size. The lesions described by Hamperl 11
were selected on microscopic grounds and therefore were
minute. Those reported by Eusebi et al. 12 were selected at
mammography and all were palpable nodules, the largest
measuring 2.5 cm in greatest axis.
Both reports were in languages (German and Italian) that,
especially 30 years ago, were not readily available to the
scientific community. As a result the histological features of
radial scar (scleroelastotic lesion) were not fully appreciated
and the terms radial scar and infiltrating epitheliosis (and its
synonyms) that describe two different lesions (see later) are
used interchangeably by many authors.
RS has a central zone of sclero-hyaline fibrous tissue mixed
with abundant elastic tissue (elastosis). The sclero-elastotic
131
Lectures
nidus is surrounded by proliferating benign epithelium usually
consisting of sclerosing adenosis and less frequently of epitheliosis, together with dilated small ducts that radiate towards
the periphery. The zoning phenomenon, most evident at low
power, is distinctive and very useful in frozen sections when
distinguishing between invasive duct carcinoma and RS. The
sclero-elastotic center on close analysis is seen to consist of
round to elongated plaques of sclerohyaline tissue surrounded
by elastosis a feature called obliterating mastopathy a phenomenon of involution of ducts as seen in the late stages of
duct ectasia 13.
Finally both RS and IE must be distinguished from tubular
carcinoma (TC) (Tab. I) 14.
In conclusion it seems that IE and RS are two morphologically
and histogenetically distinct lesions. Whilst it may be a controversial view, careful histological evaluation reveals distinct
differences between the two entities allowing for their separation. This is important as although both are clinically benign
they appear to have different relationships with carcinoma. IE
by definition has florid epitheliosis which is a proliferative
process that is so extensive that it has been considered a “low
risk duct intraepithelial neoplasia” 14 and accordingly should
be regarded a probable risk “marker” for carcinoma 15. RS on
the other hand is the result of involutionary processes 16. If a
carcinoma arises in it, it is an infrequent phenomenon comparable to cases of fibroadenomas that harbour CIS but it should
not be included in the list of precancerous lesions.
References
1.
Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of
breast carcinomas distinguish tumor subclasses with clinical implications. Medical Science 2001;98:10869-74.
2
Cheuk W, Chan AC, Lam WL, et al. IgG4-related sclerosing mastitis:
description of a new member of the IgG4-related sclerosing diseases.
Am J Surg. Pathol 2009;33:1058-64.
3
Chan SK, Cheuk W, Chan KT, Chan JK. IgG4-related sclerosing
pachymeningitis: a previously unrecognized form of central nervous
system involvement in IgG4-related sclerosing disease. Am. J. Surg.
Pathol 2009;33:1249-52.
4
Cheuk W, Yuen HKL, Chu SYY, et al. Lymphadenopathy of IgG4related sclerosing disease. Am J Surg Pathol 2008;32:671-81.
5
Cheuk W, Lee KC, Chong LY, et al. IgG4-related Sclerosing disease:
a potential new etiology of cutaneous pseudolymphoma. Am J Surg.
Pathol 2009;33:1713-9.
6
Green I, Dorfman RF, Rosai J. Breast involvement by extranodal
Rosai-Dorfman disease: report of seven cases. Am J Surg Pathol
1997;21(6):664-8.
7
Donn W, Rebbeck P, Wilson C, et al. Idiopatic granulomatous mastitis. Arch Pathol Lab Med 1994;18:822-5.
8
Shapiro JL, Goldblum JR, Petras RE. A clinicopathologic study
of 42 patients with granulomatous gastritis. Am J Surg Pathol
1996;20(4):462-70.
9
Eusebi V, Millis RR. Epitheliosis, infiltrating epitheliosis, and radial
scar. Semin Diagn Pathol 2010;27:5-12.
10
Azzopardi JG, Ahmed A, Millis RR. Problems in breast pathology.
London: W.B. Saunders Company 1979.
11
Hamperl H. Strahlige narben und obliterierende mastopathie. Virchows Arch A Pathol Anat 1975;369:55-68.
12
Eusebi V, Grassigli A, Grosso F. Lesioni focali sclero-elastotiche mammarie simulanti il carcinoma infiltrante. Pathologica 1976;68:507-18.
13
Davies JD. Hyperelastosis, obliteration and fibrous plaques in major
ducts of the human breast. J Pathol 1973;110:13-26.
14
Tavassoli FA, Eusebi V. Tumors of the breast. 4 edn. Washington, DC:
American Registry of Pathology/AFIP 2009.
15
Wellings SR, Alpers CE. Subgross pathologic features and incidence
of radial scars in the breast. Hum Pathol 1984;15:475-9.
16
Jacobs TW, Byrne C, Colditz G, et al. Radial scars in benign breastbiopsy specimens and the risk of breast cancer. N. Engl. J Med
1999;340:430-6.
Tab. I. Infiltrating Epitheliosis (IE), Sclero-elastotic Lesion (RS) and
Tubular Carcinoma (TC).
Epitheliosis
Streaming of
proliferating
epithelial
cells
Zoning
Obliterative
mastopathy
Sclerotic
tissue
Desmoplastic
stroma
Periductal
elastosis
Perivenous
elastosis
Glandular
structures
In situ
carcinoma
Myoepithelial
cells
Basal lamina
IE
Bulk of the
lesion
Present
RS
May be
present at
periphery
Not a feature
TC
Not a feature
Not a feature
Not a feature Present
Not a feature Present
Not a feature
Not a feature
Not a feature Present
May be
present
Present
Present
Not a feature
Not a feature Present
Rare
Rare
May be
present
Frequent
Solid
Entrapped
“fingers”
sometimes
squamoid
features
Not a feature Not a feature
Angulated
“tear drops”
Present
Present
Absent
Present
Present
Absent
Frequent
Molecularly targeted therapies in breast cancer:
a rapidly evolving scenario
F. Montemurro, E. Geuna, A. Milani, M. Aglietta
Divisione Universitaria di Oncologia Medica I; Fondazione del
Piemonte per l’Oncologia/IRCC Candiolo; Strada Provinciale 142,
Km 3.95, 10060 Candiolo
Over the last two decades, a greater understanding of the
heterogeneous biology of breast cancer has resulted in the
development of newer, molecularly targeted therapeutic approaches. Currently, the presence or absence of two main therapeutic targets, hormone receptors and HER2, recapitulates
more complex biological definitions of breast cancer subtypes
based on gene expression profiling 1. Although endocrine
therapy is historically considered the first form of biologically
targeted therapy, the monoclonal antibody trastuzumab was
the first compound that was rationally designed to target a
biologically relevant alteration 2. The evolution of HER2-targeting is a good example on how the concept of “biologically
targeted therapy” has evolved over the years.
HER2 is a tyrosine kinase receptor belonging to the epidermal
growth factor receptor (EGFR) family 3. Other members of
this family include HER2, HER3 and HER4. Overexpression
of HER2, which results from HER2 gene-amplification (from
now on defined HER2-positivity), occurs in some 15-20%
of all breast cancers and characterizes a biologically distinct
subset of this disease 4. HER2-positivity is associated with
adverse histopathological features, propensity to metastasize
to viscera and to the central nervous system, poor prognosis
and resistance to endocrine therapy 4. Compared with chemo-
132
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
therapy alone, the addition of trastuzumab to chemotherapy
boosts response rate, progression-free survival and overall
survival in patients with metastatic disease 5 6. In patients
with operable, HER2-positive breast cancer, the inclusion
of trastuzumab in adjuvant chemotherapy programs reduces
the risk of relapse and prolongs survival 7-9. More recently,
several other HER2-targeting agents have shown clinical efficacy both in trastuzumab-naïve and in trastuzumab-resistant
patients and several others are expected in the near future 10.
This tremendous research effort has become necessary because resistance to HER2-inhibition is a major challenge. In
fact, as a single agent or in combination with chemotherapy,
trastuzumab induces tumor regression in about 20-30% and
60-70% of HER2-positive metastatic breast cancer patients,
respectively 11. Unfortunately, the vast majority of patients,
including those with impressive initial responses, will ultimately show disease progression.
Overcoming primary and acquired resistance to trastuzumab
has been the focus of several preclinical and clinical investigations to increase the efficiency of HER2-targeting.
These studies have clarified several aspects of the high level
of interaction between signal transduction pathways, which
account for the ability of cancer cells to circumvent inhibition.
For example, tyrosine-kinase receptors can be seen as one
layer of a complex, multilayered network 12. Other layers are
represented by extracellular ligands and downstream signalling pathways. By virtue of this architecture, a “core function”
like for example proliferation or survival may be sustained by
different effectors, in a bow-tie structure. This high level of
integration is the result of an evolutionary process that started
with a single ancestral tyrosine-kinase receptor, activated by
one ligand and transmitting signals through a single cascade
of intracellular mediators.
The four EGFR family members have probably originated
from a single receptor through gene duplication. Inactive
monomers form homo- and heterodimeric structures with
other members of the family, resulting in receptor activation and phosphorilation of downstream signalling effectors.
HER2 has an “always-on” structure and lacks the capacity to
interact with growth-factors ligands. HER3 has no tyrosine
kinase activity. Despite this loss of functions, both HER and
HER3 form hetherodimers with other EGFR members that
are capable of generating potent cellular signals 3. Apart from
this “family-specific” cooperation, HER receptors can engage
“external cooperation” with members of other families of
tyrosine-kinase receptors, like for example the Insulin-like
growth 1 receptor or with the estrogen receptor pathway 13 14.
Multiple ligands and intracellular cross-talk between signal
transduction pathways complete this complex evolutionary
network. This architecture has properties that are critical for
both normal and cancer cells 12. Robustness, which is the ability of the system to function despite external (environmental)
and internal (genetic) perturbations, is ensured by modularity
and redundancy. Furthermore, the system is able to learn how
to circumvent common, single-hit perturbations (network
training). It appears more and more evident that simultaneous targeting at several different levels in this multi-layered
biological network is required for maximum clinical efficacy.
Multiple targeting can be accomplished by using single agents
with the ability to inhibit different substrates or by cocktails
of selective or non-selective inhibitors. Furthermore, it can
involve other members of the HER2 family or also connected
“external” pathways. Examples of multiple targeting are
already available in the clinic: pan-HER inhibitors, combinations of HER inhibitors with endocrine agents, antiangiogenic
compounds and heat shock protein inhibitors 15. HER2- negative tumors can be targeted successfully with antiangiogenetic
agents 15. Even “triple negative tumors” (hormone-receptors
and HER2 negative) are no-longer a “targetless” subgroup
since the therapeutic success achieved by PARP-inhibitors 16.
Due to several unanswered questions on the optimal use of
these agents, this rapidly evolving scenario requires rigorously conducted clinical studies to select patients who are
most likely to benefit from treatments.
References
1
Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human
breast tumours. Nature 2000;406:747-52.
2
Baselga J, Tripathy D, Mendelsohn J, et al. Phase II study of weekly
intravenous recombinant humanized anti-p185HER2 monoclonal
antibody in patients with HER2/neu-overexpressing metastatic breast
cancer. J Clin Oncol 1996;14:737-44.
3
Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network.
Nat Rev Mol Cell Biol 2001;2:127-37.
4
Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu
oncogene. Science 1987;235:177-82.
5
Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus
a monoclonal antibody against HER2 for metastatic breast cancer that
overexpresses HER2. N Engl J Med 2001;344:783-92.
6
Marty M, Cognetti F, Maraninchi D, et al. Randomized phase II trial
of the efficacy and safety of trastuzumab combined with docetaxel
in patients with human epidermal growth factor receptor 2-positive
metastatic breast cancer administered as first-line treatment: the
M77001 study group. J Clin Oncol 2005;23:4265-74.
7
Smith I, Procter M, Gelber RD, et al. 2-year follow-up of trastuzumab
after adjuvant chemotherapy in HER2-positive breast cancer: a randomised controlled trial. Lancet 2007;369:29-36.
8
Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant
chemotherapy for operable HER2-positive breast cancer. N Engl J
Med 2005;353:1673-84.
9
Joensuu H, Bono P, Kataja V, et al. Fluorouracil, epirubicin, and cyclophosphamide with either docetaxel or vinorelbine, with or without
trastuzumab, as adjuvant treatments of breast cancer: final results of
the FinHer Trial. J Clin Oncol 2009;27:5685-92.
10
Metzger-Filho O, Vora T, Awada A. Management of metastatic
HER2-positive breast cancer progression after adjuvant trastuzumab
therapy ΓÇô current evidence and future trends. Expert Opin Invest
Drugs 2010;19:S31-9.
11
Montemurro F, Valabrega G, Aglietta M. Trastuzumab-based combination therapy for breast cancer. Expert Opin Pharmacother
2004;5:81-96.
12
Citri A, Yarden Y. EGF-ERBB signalling: towards the systems level.
Nat Rev Mol Cell Biol 2006;7:505-16.
13
Nahta R, Yuan LX, Zhang B, et al. Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization
contributes to trastuzumab resistance of breast cancer cells. Cancer
Res 2005;65:11118-28.
14
Bender LM, Nahta R. Her2 cross talk and therapeutic resistance in
breast cancer. Front Biosci 2008;13:3906-12.
15
Rosen LS, Ashurst HL, Chap L. Targeting signal transduction pathways in metastatic breast cancer: a comprehensive review. Oncologist
2010;15:216-35.
16
Fong PC, Boss DS, Yap TA, et al. Inhibition of poly(ADP-ribose)
polymerase in tumors from BRCA mutation carriers. N Engl J Med
2009;361:123-34.
133
Lectures
Standardization of techniques in anatomic pathology
Moderators: G. Bussolati (Torino), G. Stanta (Trieste)
Nucleic acids extraction from ffpe tissues
S. Bonin, G. Stanta
ACADEM Department-Cattinara Hospital-University of Trieste, Italy
Background. Archival formalin-fixed and paraffin embedded tissues are the most abundant supply of clinical material.
Formalin fixation and paraffin embedding represents the standard methods used in any hospital to process clinical tissues,
allowing permanent preservation of tissues, with easy storage
and optimal histologic quality. However, nucleic acids extractions and analysis from formalin-fixed and paraffin embedded
tissues still remains a hang-up issue, as formalin induces crosslinkage of nucleic acids to proteins and covalently modifies
the bases of nucleic acids, impairing extraction of macromolecules 1. Nevertheless, it is possible to extract and analyse
nucleic acids from formalin fixed paraffin embedded tissues
(FFPE). Mostly, PCR based assays are performed in FFPE
samples. At the present time most of the methods applied in
molecular pathology are laboratory-based assays and commercial kits not directly intended for diagnostic purposes. Therefore there is a need to establish guidelines with standardised
procedures for molecular methods, starting from an analysis
of the currently used methods of nucleic acids extraction. A
collaborative study including 13 European laboratories of the
IMPACTS group (www.impactsnetwork.eu) has been carried
out to evaluate the performance of nucleic acids extractions
using the same formalin-fixed paraffin-embedded specimens
to assess if the different methodologies used for nucleic acids
extraction in different laboratories might affect the results 2.
By the use different protocols, but the same tests for quality
controls, the authors demonstrated that most of the homemade
protocols and commercial kits allow the extraction of nucleic
acids, but for data comparison quality tests are needed 2.
Materials and Methods. The extractions procedures for
DNA and RNA differ for some main aspects related to the
different characteristics of the two macromolecules. The
extraction protocols we analysed for DNA could be mainly
divided in three groups:
1.DNA extraction with alcohol precipitation of the DNA.
2.DNA extraction without further precipitation or purification
(crude extract).
3.DNA extraction using commercial kits following silica
based adsorption columns for DNA purification.
For RNA the methods could be roughly divided into:
a.RNA extraction with phenol extraction and isopropanol
precipitation (homemade protocols and commercial solutions).
b.RNA extraction using silica based columns for purification.
Nucleic acids quantity was detected by means of spectrophotometer measurements, while nucleic acids’ quality was
assessed by means of PCR and RT-PCR analysis with increasing lengths amplicons.
Results. As a general consideration for both DNA and RNA,
the purity and quantity assessment by spectral photometry did
not correlate with the maximum amplifiable length.
DNA extraction protocols that used purification of the extracted DNA, gave comparable results in terms of yield and
purity of the DNA. DNA quality, assessed by PCR-amplifi-
ability was not drastically affected by the use of different
DNA-extraction protocols, without a single DNA-extraction
protocol prevailing on others 2.
For RNA, the isolation methods affected the yield of the
extracted RNA, even when extraction conditions are similar.
Regarding the amplifiability, all extraction methods resulted
in RNA of useful quality for expression analyses in archival
and diagnostic tissues, since shorter amplicons are sufficient
for quantitative PCR 2. However, column based methods provided best RNA quality assessed by RT-PCR.
In any case to overcome the inter-laboratory variability, further standardization of the techniques, especially quality control procedure, is recommended for research and diagnostic
applications in molecular pathology.
References
1
Dotti I., Bonin S., Basili G., et al. Effects of formalin, methacarn,
and fineFIX fixatives on RNA preservation. Diagn Mol Pathol
2010;19:112-22.
2
Bonin S., Hlubeck F., Benhattar J., et al. Multicentre Validation Study
of Nucleic Acids Extraction from FFPE Tissues. Virchow Archiv (in
press).
Role of microRNAs in defining tissue of origin of
metastatic cancer
A. Vecchione
Roma
MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, small non-coding RNA molecules processed from
precursors and in their mature forms, serve as important
gene regulators that have the capacity to down-regulate gene
expression through translation inhibition and promotion of
miRNA degradation mediated by specific target site binding
to the 3’-untranslated region of target genes. According to
the most recent version of miRBase (v.15.0), 4000 different
mature miRNA sequences have been identified in humans
to date. A unique attribute of miRNAs that renders them potentially useful for the molecular diagnosis of tumors is their
tissue and cell lineage specificity. Many of the miRNAs are
highly specific in their expression in specific tissues and cell
types, and this specificity is often retained in the corresponding tumor tissues. Identification of cell origin by profiling of
miRNAs is more efficient compared with global analysis of
mRNAs, because the former is not confounded by such a large
pool of irrelevant genes because of the relatively small number
of miRNA species. Therefore, miRNAs could facilitate the
accurate diagnosis of tumors that are difficult to classify with
respect to the tissue origin by conventional means, for example, metastatic cancer of unknown primary origin, a highly
aggressive malignancy that poses diagnostic and management
difficulties. Deregulation of miRNAs occurs frequently during
tumorigenesis, making them attractive candidates for molecular detection of malignancy. A subset of miRNAs can often
be found up- or down-regulated in tumors compared with the
normal tissues in a specific tumor type or more globally in a
number of different tumor types. Not only is the identification
of these miRNAs critical in the understanding of the pathogenetic role of miRNAs in cancer development, it can also
provide a diagnostic methodology for distinguishing tumors
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
from normal tissues of different cell origins. In the current
setting of clinical diagnostic practice, where morphological
and antigenic evaluation appears to be adequate for accurate
diagnostic separation between tumor and normal tissues in
the vast majority of biopsies, the utility of miRNA analysis in
this arena may not be too apparent. However, the differential
expression of miRNAs between tumors and normal tissues
may be exploited in the diagnosis of samples where cells are
scant or poorly preserved, which may render diagnosis of a
malignancy by traditional methods difficult.
Despite the many exciting developments that demonstrate the
potential capacity of miRNAs for tumor classification and
prognosis, their practical use as biomarkers in a routine clinical setting is still in its infancy. To ascertain and accelerate its
advancement beyond the developmental phase, efforts should
be made to perform retrospective and prospective studies in
global and gene-specific analysis of miRNAs on multiple
independent cohorts of patient samples using standardized
methodologies.
Fish
C. Marchiò, P. Gugliotta, C. Botta, L. Verdun di Cantogno,
A. Sapino
Department of Biomedical Sciences and Human Oncology, Turin,
Italy
In Situ hybridization (ISH), is a molecular technique that has
been available since 1969 and over time it has become part
of the diagnostic armamentarium of pathologists in different
fields (Lambros et al, 2007). Unlike other molecular biology
techniques ISH is unique as it is based on a visual assessment
of probe copy numbers directly at the microscope and can
be performed on interphase nuclei, i.e. on tissue sections of
archival samples (Lambros et al, 2007). Two modalities of
in situ hybridisation have already been introduced in pathology laboratories: fluorescent in situ hybridisation (FISH)
and chromogenic in situ hybridisation (CISH). FISH is most
widely used and has already made prime time in terms of
diagnosis, prognosis and prediction in several diseases (sarcomas, lymphomas and leukaemias, breast cancer, oligodendrogliomas, gastric cancer, melanomas).
The success of this techniques stems from the fact that they
allow for semi-quantitative assessment of gains, losses and
amplifications directly on tissue sections, combining molecular genetics with traditional pathology (Marchiò and Reis
Filho, 2008). FISH can be performed on both histological
and cytological specimens, according to distinct protocols. In
particular, for histological samples the pre-analytic phase of
tissue collection, preservation and preparation is of paramount
importance in order to obtain a good performance of the assay.
Ideally tissue specimens should be fixed immediately after
gross sampling (5mm thick tissue sample slices); neutralbuffered formalin is recommended, alcohol-based fixatives
can be used as well, however they often give a fluorescent
background which can be troublesome for interpretation of the
results; the time of fixation should range from 6 to 24 hours.
A second and crucial step in represented by section cutting: in
order to be able to appreciate the signals and not to underestimate chromosome abnormalities, sections are recommended
to be 2-5 µ thick. It has to be mentioned that FISH analysis is
also feasible on Tissue Micro Array (TMA) sections, following a specific protocol (Sapino et al, 2006): once again, the
pre-analytical phase, with proper sampling of tumour area and
high quality tissue cutting, plays a central role.
The analytic phase includes different variables, such as assay
validation, equipment calibration, use of standardized laboratory procedures, training and competency assessment of staff,
type of pre-treatments, test reagents, use of standardized
control materials and automated laboratory methods (Wolff
et al, 2007). These variables are closely related with laboratory competency and efficiency, thus suggesting such type
of analyses should be delegated to specific laboratories with
long-standing experience and high work-load of the assay.
Automated machines for FISH analysis have been recently
introduced and are of assistance to the pathologist in scanning
and scoring areas of interest.
Post-analytic phase issues are represented by interpretation
criteria, use of image analysis, reporting elements, laboratory accreditation, pathologist competency assessment and
quality assurance procedures. Undoubtedly, quality controls
(QCs) represent a very important mechanism, even though
a consensus has not been reached on the way QCs should be
organized. In Italy, we have recently set up a “ring study”
for QC of FISH analysis 11 institutions are have adhered to:
single Institutions make cases with different amplification
status circulate among the others to perform FISH in their
own laboratories. Afterwards data are centrally collected and
analysed by a referral centre, with particular attention to consensus on reported results.
Taken together, all variables discussed in different phases of
FISH analysis play a role in the definition of a good FISH experiment, therefore standardization, competency of operators
(technicians, biologists, pathologists) and quality check are
eagerly warranted, as also pointed out in different guidelines
by experts of single diseases (see for example (Wolff et al,
2007)).
References
Lambros MB, Natrajan R, Reis-Filho JS. Chromogenic and fluorescent in
situ hybridization in breast cancer. Hum Pathol 2007;38:1105-22.
Marchiò C, Reis Filho JS. Molecular diagnosis in breast cancer. Diagnostic Molecular Pathology 2008;14.
Sapino A, Marchiò C, Senetta R, et al. Routine assessment of prognostic
factors in breast cancer using a multicore tissue microarray procedure. Virchows Arch 2006;449:288-96.
Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast
cancer. J Clin Oncol 2007;25:118-45.
135
Lectures
Thyroid pathologies:
non-conventional thyroid lesions and emerging diagnostic problems
Moderators: M. Papotti (Torino), G. Gardini (Reggio Emilia)
Clinical-pathological and molecular features of
papillary thyroid carcinomas smaller than 2 cm
2
F. Basolo, L. Torregrossa, R. Giannini, M. Miccoli *,
C. Lupi, E. Sensi, P. Berti, R. Elisei **, P. Vitti **, A. Baggiani *,
P. Miccoli
3
Department of Surgery, University of Pisa, Italy; * Department of
Experimental Pathology B.M.I.E., Biostatistics Research Unit, University of Pisa, Italy; ** Department of Endocrinology, University of
Pisa, Italy
Background. The actual incidence of thyroid cancer is predominantly characterized by the increased detection of small
PTCs: about 87% of cases consist of cancers measuring 2 cm
or smaller 1. According to TNM staging system, evaluation of
the degree of neoplastic infiltration beyond the thyroid capsule
remains a unique parameter that can be evaluated by histopathologic examination to label a Papillary Thyroid Carcinoma
(PTC) measuring ≤ 2 cm in size as a pT1 or pT3 tumor 2. PTCs
smaller than 2 cm in size that are limited to the thyroid gland
and without lymph node metastasis are considered low-risk tumors and can be successfully treated with total thyroidectomy
alone, whereas PTCs of the same size but with extrathyroidal
extension justify a more aggressive treatment 3.
In recent years, BRAF V600E has emerged as a promising
prognostic factor in the risk stratification of PTC 4. Many studies have demonstrated significant correlations between BRAF
mutation and high-risk clinical-pathological features of PTCs
in overall analyses of tumors of all sizes 4.
In the current study, we correlated the BRAF V600E mutation with both clinical-pathological features and the degree of
neoplastic infiltration to redefine the reliability of the actual
system of risk stratification.
Methods. The presence of BRAF mutations was examined
in a large group of PTCs smaller than 2 cm (overall 1,060
patients: 254 males and 806 females, mean age 44.6 ± 13.3
years) divided into four degrees of neoplastic infiltration:
a) “totally encapsulated”; b) “not encapsulated without thyroid capsule invasion”; c) “thyroid capsule invasion”; and
d) “extrathyroidal extension.”
Results. The overall frequency of the BRAF V600E mutation was 44.6%. In both univariate and multivariate analyses,
BRAF mutations showed a strong association with PTC variants (classical and tall cell), tumor size (1,1-2 cm), multifocality, absence of tumor capsule, extrathyroidal extension, lymph
node metastasis, and higher AJCC stage. In PTCs staged as
pT1 with thyroid capsule invasion, the frequency of BRAF
mutations was significantly higher than in pT1 tumors that did
not invade the thyroid capsule (67.3% vs. 31.8%, respectively,
p < 0.0001). No statistically significant difference in BRAF alterations was found between pT1 tumors with thyroid capsule
invasion and pT3 tumors (67.3% and 67.5%, respectively).
In conclusion, we suggest that evaluation of BRAF status
even in pT1 tumors would be useful to improve risk stratification and patient management, although follow-up data
are necessary.
References
1
Davies L, Welch HG. Increasing incidence of thyroid cancer in the
United States, 1973-2002. JAMA 2006;295:2164-7.
4
Edge SB, Byrd DR, Carducci MA, et al. American Joint Committee on
Cancer (AJCC). Cancer staging manual. Seventh edition. New York:
Springer: 2009.
Pacini F, Schlumberger M, Dralle H, et al. European consensus for the
management of patients with differentiated thyroid carcinoma of the
follicular epithelium. Eur J Endocrinol 2006;154:787-803.
Xing M. BRAF mutation in papillary thyroid cancer: pathogenic role,
molecular bases, and clinical implications. Endocr Rev 2007;28:74262.
Mitochondrial DNA changes and oncogenic
mutation of nuclear genes in thyroid oncocytic
nodules: BRAFv600e and RET/PTC are associated
with papillary carcinomas showing oncocytic
features, but RAS mutations are uncommon in
oncocytic follicular adenomas and carcinomas
D. De Biase, E. Bonora *, L. Morandi, G. Gasparre *, G. Romeo *, G. Tallini
Sezione di Anatomia, Istologia e Citologia Patologica “M. Malpighi”,
Univerisità di Bologna, Ospedale Bellaria, Via Altura 3, 40139 Bologna, Italy; * U.O. Genetica Medica, Dipartimento di Scienze Ginecologiche, Ostetriche, Pediatriche Policlinico “S. Orsola-Malpighi”
Background. Oncocytic neoplasms are tumors composed of
cells characterized by an aberrant amount of mitochondria that
is responsible for their ‘swollen’ (i.e. ‘oncocytic’, from the
greek onkoustai, to swell) appearance 1. These neoplasms may
occur at various sites but are particularly common in the thyroid gland. Thyroid oncocytic tumors (with the exception of
the rare oncocytic variant of medullary carcinoma) originate
from follicular cells 1. They can be benign (oncocytic adenomas) or malignant (oncocytic carcinomas) and have been the
subject of both fascination and controversy for pathologists.
One area of disagreement has concerned the definition of
thyroid oncocytic tumors as a separate tumor category 1. It is
now accepted that oncocytic tumors in the thyroid should be
viewed as special subtypes or variants, since their features
are distinct enough to set them apart from corresponding neoplasm lacking accumulation of mitochondria 2. Accordingly,
oncocytic thyroid carcinomas are now classified as variants
of follicular carcinomas (commonly) or of papillary carcinomas (less commonly) 2. The obvious cellular derangement of
oncocytic cells, with complete dysregulation of the mitochondrial mass and metabolism, have spurred some investigators
to study the molecular mechanisms underlying the genesis
of this peculiar phenotype 3. Disruptive mitochondrial DNA
(mtDNA) mutations in complex I subunits of the respiratory
chain have recently been shown to be very common in thyroid
oncocytic lesions, after the entire mtDNA of many cases has
been sequenced, while in vitro experiments have demonstrated that complex I mtDNA mutations actually cause the
decreased production of ATP associated with the oncocytic
phenotype 3-5.
Somatic alterations of various nuclear genes are known
to occur in thyroid tumors, including point mutations and
rearrangements 6. The most frequent oncogenic alterations
involve the RET, RAS and BRAF genes 6. These genes code
for proteins involved in the linear signalling that goes from
the tyrosine kinase receptors at the plasma membrane and
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
RAS to cytoplasmic Ser/Thr kinases like BRAF, MEK, ERK
- the MAPK pathway - whose activity is pivotal in controlling
cell proliferation 6. While these oncogenic events in thyroid
tumors that are not oncocytic have been well studied, their
prevalence in oncocytic thyroid lesions is unclear. Unclear is
also their relationship with mtDNA mutations.
Methods. H-, K-and N-Ras mutations as well as BRAF exon
15 mutation and RET/PTC rearrangements were analyzed in
45 thyroid oncocytic tumors (15 hyperplastic adenomatous
nodules, HANonc; 8 follicular adenomas, FAonc; 14 follicular carcinomas, FConc; 8 papillary carcinomas with oncocytic
features, PConc) that had been previously characterized for
their mtDNA abnormalities 4. Nucleic acids were extracted
from paraffin-embedded neoplastic tissue after examination
of the corresponding Hematoxylin and Eosins stained sections using a commercial kit (RecoverAll Total Nucleic Acid
Isolation, Applied Biosystems/Ambion – Austin, TX). Direct
sequencing was used to analyze H-, K- and N-Ras and BRAF
exon 15, qRT-PCR to identify RET/PTC1 and RET/PTC3 rearrangement. The entire mtDNA was sequenced and mtDNA
mutations were classified as disruptive, possibly/probably
damaging and absent.
Results. We found three cases with RAS mutations, two
cases with a BRAFV600E activating mutation, one case with a
RET/PTC1 rearrangement. All the above nuclear DNA alterations did not overlap in any given tumor. Of the RAS mutated
cases, one had a NRASQ61R mutation and was diagnosed as a
minimally invasive FConc; the second case had a KRASQ61R,
and was diagnosed as FAonc; a third case had HRASQ61R mutation, was diagnosed as a follicular variant PConc; mtDNA
mutations were identified in the last two cases, and in both
the mtDNA mutations were classified as possibly/probably
damaging. Both cases with the BRAFV600E activating mutation
were diagnosed as PConc, tall cell variant and did not have
mtDNA mutations. The single RET/PTC1 mutated case was
a Warthin-like PConc, with no mtDNA alterations. mtDNA
mutations classified as disruptive were identified in 5/14
(35.7%) FConc, 2/8 (25.0%) FAonc, 4/15 (26.7%) HANonc,
1/8 (12.5%) PConc. mtDNA mutations classified as possibly/
probably damaging were identified in 3/14 (21.4%) FConc,
4/8 (50.0%) FAonc, 3/15 (20.0%) HANonc, 3/8 (37.5%)
PConc.
Conclusion. RAS, BRAFV600E mutations and RET/PTC rearrangement have been identified in malignant oncocytic tumors
and in one follicular adenoma. RAS mutations are uncommon in oncocytic follicular neoplasms (both carcinomas and
adenoma), suggesting that other tumorigenic events may play
a role in their development. BRAFV600E mutations are associated with tall cell variant papillary carcinomas with oncocytic
features. Pathogenic mtDNA alterations may overlap with the
oncogenic mutations commonly found in non-oncocytic thyroid tumors. Disruptive mtDNA mutations are more common
in oncocytic follicular carcinomas than in papillary oncocytic
carcinomas. They are also more common in oncocytic follicular neoplasms – both carcinomas and adenomas – and
hyperplastic adenomatous nodules with oncocytic features,
when compared with papillary oncocytic carcinomas.
References
1
Tallini G. Oncocytic tumors. Virchows Archives A (Anat Pathol)
1998;433:5-12.
2
World Health Organization Classification of Tumors-Pathology and
Genetics, Tumors of Endocrine Organs. Lyon (France): IARC Press,
2004.
3
Gasparre G, Bonora E, Tallini G, et al. Molecular features of thyroid
oncocytic tumors. Mol Cell Endocrinol. 2010;321:67-76.
4
5
6
Gasparre G, Porcelli AM, Bonora E, et al. Disruptive mitochondrial
DNA mutations in complex I subunits are markers of oncocytic phenotype in thyroid tumors. Proceedings of the National Academy of
Sciences USA 2007;104:9001-6.
Bonora E, Porcelli AM, Gasparre G, et al. Defective Oxidative Phosphorylation in Thyroid Oncocytic Carcinoma Is Associated with
Pathogenic Mitochondrial DNA Mutations Affecting Complexes I and
III. Cancer Research 2006;66:6087-96.
Knauf JA, Fagin JA. Role of MAPK pathway oncoproteins pathogenesis and as drug targets. Current Opinion in Cell Biology 2009;21:296303.
Trabecular neoplasms of the thyroid
M. Volante, I. Rapa, M. Papotti.
Department of Clinical and Biological Sciences at San Luigi Hospital, University of Turin, Orbassano, Turin, Italy
Follicular and papillary growth patterns represent the most
common architectural features within thyroid nodules, in both
benign and malignant settings. Alternative to these, nodules
having a non follicular-non papillary structure may be encountered, being solid/trabecular arrangement the most common feature. In general, irrespective of the biological nature of
the lesion under analysis, trabecular growth is represented by
sheets of cells regularly arranged in one or few rows or more
irregularly anastomosing, separated by usually scarce connective tissue and a thin vascular network. The solid growth is an
extreme of the trabecular architecture, being represented by a
more nodular arrangement with a wider thickness of cellular
islands and a more irregular and dispersed vascular network.
However, the border between compact trabecular growth and
solid pattern is poorly defined and since this latter is usually
mixed with and represents an architectural arrangement parallel to the trabecular one, they will be considered together.
When dealing with a trabecular lesion in the thyroid, a wide
range of differential diagnoses exists, representing one of the
major diagnostic problems in the routine thyroid practice 1,
and include the following, among others:
a) Lesions derived from the follicular epithelium with
papillary carcinoma-type nuclear features. When follicular
cell derivation is morphologically or immunophenotypically
evident, the nuclear features – as conventionally considered
for follicular/papillary lesions – should be carefully examined
to check the presence of the diagnostic features of papillary carcinoma. If clear-cut papillary-type nuclei are recognized, the following two entities have to be considered. The
solid variant of papillary carcinoma is a rare and still poorly
characterized variant of papillary thyroid carcinoma, most
commonly found in children and young adults especially in
radiation-exposed individuals; the presence of irregular clear
nuclei with grooving and pseudo-inclusions is the cytological
hallmark whereas the solid growth pattern is accompanied
by vascular invasion and extra-thyroidal extension in about
one-third of cases. The clinical behaviour of the solid variant
of papillary carcinoma is characterized by a slightly higher
frequency of distant metastases and less favourable prognosis
than classical papillary carcinoma 2. Hyalinizing trabecular
tumor (HTT) is a trabecular neoplasm of follicular derivation with peculiar nuclear, architectural and histochemical
features, with a benign behaviour in the vast majority of
cases reported so far 3. HTT is a well circumscribed lesion,
lacking morphological signs of capsular or vascular invasion.
Two principal features are diagnostic of HTT: a uniform
and diffuse solid and trabecular architecture, with markedly
hyalinized deposits containing basal membrane-type material,
typically located within the trabeculae rather than in the inter-
137
Lectures
posed stroma together with nuclear features, including clear
nuclei with grooves and pseudoinclusions, resembling those
of papillary carcinoma.
b) Lesions derived from the follicular epithelium with
dark round/convoluted nuclei. Trabecular (embryonal)
adenoma is a variant of follicular adenoma, both of conventional and oncocytic types, with predominant/pure trabecular
growth pattern associated to high cellularity and oedematous
modifications of the stroma. The name embryonal adenoma is
related to the morphologic resemblance of this tumor to the
early stages of the developing thyroid. Follicular carcinoma
with solid/trabecular growth pattern: focal or predominant
trabecular growth may be observed in follicular carcinoma,
similarly to follicular adenoma but with a higher frequency,
especially in oncocytic forms. The differential diagnosis with
adenomas relies on the identification on capsular and/or vascular invasion, whereas its distinction from poorly differentiated carcinoma is outlined below. Poorly differentiated carcinoma is characterized by a predominant trabecular, insular
and/or solid growth together with the presence of unequivocal
high grade histology, high mitotic count and necrosis 4.
c) Primary thyroid tumors not derived from the follicular
epithelium. Medullary carcinoma. As stated in the WHO
classification, the diagnosis of medullary carcinoma should
be considered in any thyroid nodule showing unusual features. Its histological appearance is widely variable, being
tumor cells most commonly arranged in nests or trabeculae
with a variable amount of fibrovascular stroma. The presence
of amyloid deposition is characteristic but not constant. Cytological features suggestive of medullary carcinoma are the
presence of round to oval nuclei, without prominent nucleoli
and with coarse chromatin. Primary thyroid paraganglioma
is a very rare tumor showing a striking female predominance,
it is usually confined to the thyroid gland and composed of
neoplastic cells arranged in the typical lobular growth with
fine connective tissue interposed. Architecture and cytology
are similar to those of medullary carcinoma which shares a
common neuroendocrine origin and represents the major differential diagnosis, but the absence of calcitonin immunoreactivity rules out the diagnosis of the latter.
d) Extra-thyroidal lesions. Parathyroid lesions: intra-thyroidal parathyroid tissue is not uncommon and should be
searched for during surgical interventions for primary or secondary hyperparathyroidism. When clinical hyperparathyroidism is not evident, hyperplastic or adenomatous parathyroid
tissue showing typical trabecular arrangement might be confused with follicular cell-derived nodules or even medullary
carcinoma. In the presence of a follicular patterned associated
component, the recognition of bi-refringent crystals is useful
in distinguishing thyroid from parathyroid gland tissues. More
complicated is the differential diagnosis between malignant
thyroid nodules and parathyroid carcinoma involving the
thyroid gland, which present vascular and capsular invasion,
trabecular growth with sometimes oncocytic changes, and in
a fraction of cases necrosis and mitotic activity. Metastases:
despite its high vascularisation, the thyroid is not a frequent
site of tumor spread, according to clinical evidence. Metastases have as most common primary sites the kidney, lung,
breast and gastrointestinal tract. In most instances, thyroid
metastases present as solitary masses thus entering in the
differential diagnosis with primary thyroid nodules. With special reference to kidney primaries, the clear cell/oncocytoid
features and frequent trabecular growth might be source of
misdiagnosis with other benign and malignant trabecular lesions primary of the thyroid.
References
1
Volante M, Papotti M. A practical diagnostic approach to solid/trabecular nodules in the thyroid. Endocr Pathol 2008;19:75-81.
2
Nikiforov YE, Erickson LA, Nikiforova MN, et al. Solid variant of
papillary thyroid carcinoma: incidence, clinical-pathologic characteristics, molecular analysis, and biologic behavior. Am J Surg Pathol
2001;25:1478-84.
3
Carney JA, Hirokawa M, Lloyd RV, et al. Hyalinizing trabecular
tumors of the thyroid gland are almost all benign. Am J Surg Pathol
2008;32:1877-89.
4
Volante M, Collini P, Nikiforov YE, et al. Poorly differentiated thyroid carcinoma: the Turin proposal for the use of uniform diagnostic
criteria and an algorithmic diagnostic approach. Am J Surg Path
2007;31:1256-64.
Clinicopathological and prognostic features
of well-differentiated capsulated carcinomas
S. Piana, G. Gardini
Department of Pathology, Arcispedale “Santa Maria Nuova”, Reggio
Emilia, Italy
Background. There is a conspicuous number of well-differentiated thyroid neoplasms of follicular cells characterized
by encapsulation and a follicular pattern of growth (“follicular-patterned tumors”) which are currently designated as
malignant if they show evidence of capsular/vascular invasion
and/or exhibit the nuclear features of the papillary family of
neoplasms 1 2.
The vast majority of these tumors have an excellent prognosis,
but an aggressive therapy is often unnecessarily carried out.
This group comprises firstly the minimally invasive follicular
carcinoma (MIFCa) and the encapsulated follicular variant of
papillary carcinoma (FV-PTC). The category of MIFCa continues to be controversial and a consensus regarding the minimal criteria for its diagnosis is still missing 3. However, some
studies have indicated that capsular invasion in the absence of
vascular invasion does not appear to significantly affect the
outcome of these tumors 4.
The FV-PTC is currently defined as a thyroid malignancy
with a predominant or exclusive follicular growth pattern displaying the characteristic nuclear features of papillary thyroid
carcinoma 5. Multiple studies have demonstrated great interobserver variability in the diagnosis of these tumors, even
among experts in thyroid pathology, thus emphasizing the difficulties in properly defining the criteria for the diagnosis of
this particular type of papillary thyroid carcinoma 6. The most
difficult circumstance for diagnosis arises when these tumors
are well-circumscribed an encapsulated.
To these categories, the Chernobyl Pathologists Group 7 has
proposed the addition of the well-differentiated carcinomas,
not otherwise specified (WDC) for tumors with features
intermediate between FCa and FV-PTC, and well-differentiated/follicular tumor of uncertain malignant behaviour
(WDT-UMP and FT-UMP) for tumors with “incomplete”
nuclear changes and/or “incomplete” capsular invasion, respectively. This alternate terminology reflects our current
incomplete knowledge and it offers the advantage of avoiding
unnecessary aggressive treatment for a tumor that shows an
overwhelmingly innocuous behaviour following conservative
surgery.
Methods. If the above mentioned types of well-differentiated
encapsulated carcinomas have an excellent prognosis, they
should not be represented in a series of fatal thyroid carcinomas comprising all histologic types. Therefore, the files of
the Department of Pathology of the Arcispedale Santa Maria
Nuova in Reggio Emilia, Italy, were searched for all cases di-
138
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
agnosed as thyroid carcinoma of any type from 1979 to March
2004. A total of 1039 cases was found. Representative slides
from each case were selected and re-classified according to
the criteria recommended by standard texts 1 2 with the addition of the categories suggested by the Chernobyl pathology
group 7. Follow-up and clinical information were obtained
from the Reggio Emilia Cancer Registry and from the files of
the Pathology and Endocrinology Department. Follow-up was
available in 1009 cases and ranged from 4.5 to 29 years (median, 9.8 years; mean, 11.9 years) or until death. Among the
1009 cases, 159 patients had died; thyroid carcinoma was the
cause of death for 67 of the 159 patients, and these 67 cases
are the focus of the study.
Results. Among the 67 patients deceased as a consequence of
thyroid carcinoma, there were none of the tumors belonging to
any of the categories above mentioned, that is MIFCa, FV-PTC,
WDT-UMP, and FT-UMP. In fact, the vast majority of these
tumors shows a shows an exceedingly innocuous behaviour
following conservative surgery. The overdiagnosis of this condition may lead to excessive treatment, including total thyroidectomy followed by radioactive iodide therapy. This acquires a
particular importance with the encapsulated variant of FV-PTC,
which is associated with an excellent prognosis and for which
distant blood-borne metastasis has been rarely documented 8.
The results of this study and a critical review of the pertinent
literature indicate that tumors with these features are associated with an extremely favourable outcome and that they
do not play a significant role in the fatality rate of thyroid
carcinoma 9 10.
References
1
DeLellis RA, Lloyd RV, Heitz PU, et al. Tumours of Endocrine Organs, World Health Organization Classification of Tumours; Pathology and Genetics. Lyon: IARC Press 2004.
2
Rosai J, Carcangiu ML, DeLellis RA. Tumors of the thyroid gland,
Atlas of Tumor Pathology, Third Series, Fascicle 5, Washington, D.C:
Armed Forces Institute of Pathology, AFIP 1992.
3
Franc B, De La Salmoniere P, Lange F, et al. Interobserver and intraobserver reproducibility in the histopathology of follicular thyroid
carcinoma. Hum Pathol 2003;34:1092-100.
4
Van Heerden JA, Ray ID, Goellner JR, et al. Follicular thyroid carcinoma with capsular invasion alone: a non-threatening malignancy.
Surgery 1992;112:1130-8.
5
Carcangiu ML, Zampi G, Pupi A, et al. Papillary carcinoma of the thyroid. A clinicopathologic study of 241 cases treated at the University
of Florence, Italy. Cancer 1985;55:805-28.
6
Lloyd RV, Erickson LA, Casey MB, et al. Observer variation in the
diagnosis of follicular variant of papillary thyroid carcinoma. Am J
Surg Pathol 2004;28:1336-40.
7
Williams ED (on behalf of the Chernobyl Pathologists Group). Two
proposals regarding the terminology of thyroid tumors. Intern J Surg
Pathol 2000;8:181-4.
8
Chan JKC. Strict criteria should be applied in the diagnosis of encapsulated follicular variant of papillary thyroid carcinoma. Am J Clin
Pathol 2002;117:16-18.
9
Piana S, Frasoldati A, Di Felice E et al. Encapsulated well-differentiated follicular-patterned thyroid carcinomas do not play a significant
role in the fatality rates from thyroid carcinoma. Am J Surg Pathol
(E-pub ahead of print).
10
Rosai J. The encapsulated follicular variant of papillary thyroid carcinoma; back to the drawing board. Endocr Pathol 2010;21:7-11.
Vascular lesions of the thyroid
M. Papotti, M. Volante.
Department of Clinical and Biological Sciences, University of Turin
at San Luigi Hospital, Orbassano (Torino), Italy
Vascular lesions of the thyroid gland include benign endothelial proliferations of reactive (Masson’s “hemangioma”),
benign neoplasms (cavernous hemangioma) and the rare
malignant angiosarcomas. These latter occur in pure form
or combined with anaplastic carcinoma (angio-sarcomatoid
carcinoma).
Reactive endothelial proliferations. In long standing nodular goiter, regressive changes are common, including oedema, fibrosis and calcification. Haemorrhage is an additional
event, which can be associated to complete nodule infarction,
followed by reparative processes such as granulation tissue
and reactive endothelial hyperplasia, closely resembling
intravascular papillary endothelial proliferations (Masson’s
phenomenon). In these cases, intraluminal papillary projections in vascular spaces are lined by plump endothelial cells
with occasional atypias possibly leading to a suspicion of
malignancy. This condition may be an uncommon consequence of fine needle aspiration biopsy or the result of spontaneous intranodular haemorrhage/infarction. Completely
infarcted goiter nodules are a challenge for clinicians, radiologists and pathologists: at ultrasound investigation, such
nodules having prominent vascular endothelial hyperplasia
are typically hyporeflecting and unhomogeneous and/or
calcified, all features simulating malignancy. At light microscopy, the diagnosis of goiter may be missed (especially
in fine needle aspiration cytological material) in the absence
of residual micro- or macro-follicles due to haemorrhage and
endothelial hyperplasia.
WHAFFT. Another condition associated to vascular proliferation in the thyroid gland was described under the acronym
WHAFFT, which stands for “Worrisome Histologic Alterations Following Fine needle aspiration of the Thyroid”. The
alterations caused by the fine needle aspiration passages
included haemorrhage,, fibrosis, calcification and worrisome
lesions, like nuclear atypia, squamous metaplasia, capsular
pseudoinvasion, and plump endothelial hyperplasia in vascular spaces, mimicking vascular tumors.
Thyroid hemangioma. It is very rare and generally results
from subsequent organization of intranodular hemorrhagic
events in goiter, thus suggesting their reactive rather than
neoplastic nature.
Angiosarcoma and Sarcomatoid carcinoma. Thyroid angiosarcoma (or malignant hemangioendothelioma) was originally described in mountain areas and associated to endemic
goiter. Grossly, a large extensively hemorrhagic mass is
recognized in the presence of multinodular goiter in the
surrounding parenchyma. Microscopically, elongated cells
either lining vascular spaces and protruding into them, or
arranged in small solid sheets are identified. Eosinophilic cytoplasm, polygonal shape, large and hyperchromatic nucleus,
prominent nucleoli and numerous mitoses are typically present, with occasional intracytoplasmic lumina. Tumor cells are
reactive for FVIII-related antigen, CD31, CD34 and vimentin, as well as for cytokeratin (focally). The histogenesis of
thyroid angiosarcomas is controversial being the hypothesis
that all such tumors are indeed (angio)sarcomatoid anaplastic
carcinomas contrasted by the alternative evidence on the
existence of rare true angiosarcoma cases of the thyroid.
Whether reactive endothelial hyperplasia in long standing
139
Lectures
goiter nodules represents an intermediate step in the development of malignant vascular growths remains to be defined.
From a microscopic point of view, the distinction between a
benign (pseudoangiomatous) lesion and a malignant vascular
tumor is extremely difficult on both surgical and fine needle
aspiration materials.
Slide seminar: Breast
Moderators: A. Sapino (Torino), M.P. Foschini (Bologna)
Breast hamartoma with apocrine glandular
structure without myoepithelium
I. Castellano, L. Macrì, G. Canavese, A. Sapino
Torino
We describe a case of a 46-years old woman with painless
mass of the left breast slowly enlarging in the last year. There
was no family history of breast cancer and the patient was not
under any pharmacological or hormonal treatments. Clinical
examination reveals in the upper external quadrant a well-circumscribed, mobile, round nodule similar to a fibroadenoma.
Ultrasound examination demonstrated a heterogeneous lesion
with lobulated contour and a thin capsule, measuring 5 cm in
diameter. The patient underwent fine needle aspiration with a
diagnosis of hypercellular lesion, categorized as C3. Core biopsy of the mass, performed in another institution, revealed a
fibroadenomatous epithelial lesion with usual ductal epithelial
hyperplasia classified as B3. Quadrantectomy was performed.
Grossly the mass was bilobated firm and rather circumscribed,
grey-white on cut surface. Histologically, the nodule showed
a stromal proliferation of interlobular elongated fibroblasts
with lobular structures surrounded by sclero-hyaline stroma or
stroma with a pseudo-angiomatous appearance. Lobular structures showed a histological pattern similar to the so called
“gynecomastia-like hyperplasia” with columnar cells. In
distinct foci ductal structures disposed in an organoid pattern
or small cysts were formed by cytologically normal apocrine
cells. No myoepithelial cells were seen at the periphery of
these apocrine glands. Staining for p63 confirmed the absence
of myoepithelial cells. The final diagnosis was of Hamartoma
of the breast. The peculiar appearance of the apocrine gland
without myoepithelila cells was described as a possible event
mimicking pseudoinvasion.
Hamartoma is generically defines as “a malformation that
resembles a neoplasm, grossly and even microscopically, but
results from faulty development in an organ; it is composed of
an abnormal mixture of tissue elements, or an abnormal proportion of a single element normally present at that site…”.
Breast Hamartoma is uncommon, with incidence of 0.7% of
benign breast tumors. It presents as a painless slow growing
breast mass, not attached to the underlying structures, in patients predominantly in 5th or 6th decade of life. The mammographic appearance corresponds to a “breast in breast” mass,
generally without microcacifications. Although the lesion is
almost always benign, rare case reports describe cancer inside
hamartoma, so complete excision is the only way to rule out
malignancy. However, recurrences have been described in
almost 10% of patients, mainly due to multifocal disease. The
most interesting feature of the present case was the presence
of several distinct foci of apocrine glands devoid of myoepithelial cells often arranged in an organoid (lobular) pattern.
Cserni G. (Histopathology 52:239-262) described a similar
pattern in apocrine glands of a low-grade phyllodes tumour.
With the exception of microglandular adenosis, lack of myoepithelium is generally considered a hallmark of malignancy
and invasion in breast pathology. However, as discussed by
Cserni “the absence of myoepithelial cells around apocrine
glandular structures of the breast does not necessarily imply
malignancy, and may also be seen in some benign lesions”.
To rule out malignancy the following criteria were taken into
account: the presence of an organoid, lobular growth pattern
which is generally associated with benign changes, lack of
cellular atypia, monomorphic nuclei and absence of mitotic
activity in apocrine cells, which are instead typical features
of apocrine carcinomas. The patient is free of disease at one
year follow-up.
Head and neck pathologies
Moderators: M.P. Foschini (Bologna), E. Maiorano (Bari)
Undifferentiated and poorly differentiated
sinonasal malignancies
A. Franchi
Division of Anatomic Pathology, Department of Critical Care Medicine and Surgery, University of Florence Medical School, Florence,
Italy
Malignant tumours of the nasal cavities and paranasal sinuses
represent about 3.6% of all malignancies arising in the head
and neck area. In this complex anatomic region a significant
number of neoplasms may present with “undifferentiated”
light microscopic morphology. In general, they represent a
group of clinically aggressive neoplasms, although the knowledge has progressively evolved towards the need for careful
differential diagnosis, because some of these entities present
distinct clinico-pathologic features and biologic behaviour,
warranting individualized treatment strategies. In addition,
a number of studies have recently defined the use of novel
diagnostic markers for sinonasal carcinomas, and there is
increasing evidence of the importance of the role of immunophenotyping and genotyping for differentiating among these
neoplasms. This presentation will focus on recent acquisitions
140
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
concerning the diagnosis of sinonasal undifferentiated and
poorly differentiated carcinomas, neuroendocrine carcinoma
and olfactory neuroblastoma.
Sinonasal poorly differentiated and undifferentiated carcinomas
The group of high grade poorly differentiated and undifferentiated sinonasal carcinomas include nasopharyngeal-type
undifferentiated carcinoma (lymphoepithelioma), sinonasal
undifferentiated carcinoma (SNUC), NUT midline carcinoma,
and poorly differentiated keratinizing and non-keratinizing
variants of squamous cell carcinoma.
Nasopharyngeal-type undifferentiated carcinoma is typically
associated with EBV infection, and this is a useful feature
to separate this entity from other sinonasal undifferentiated
carcinomas, which are typically EBV negative 1.
SNUC is a rare highly aggressive tumour of uncertain histogenesis. The term “undifferentiated” has been applied inconsistently in the past, but should now be applied more selectively with better methods of cell study. By definition SNUC
does not show any overt squamous or glandular differentiation, whereas neuroendocrine features have been frequently
noted, both histologically and immunohistochemically 2.
SNUC can be distinguished from poorly differentiated
squamous cell carcinoma variants for the different pattern
of cytokeratins subtypes expression, since SNUC is positive
for simple epithelia cytokeratins and lacks the expression
of cytokeratins 5/6 and 13, which instead are expressed by
squamous cell carcinoma variants 3. In addition, SNUC has a
limited expression of p63, which is present in squamous cell
carcinoma variants 4.
NUT midline carcinoma (NMC) is a rare, clinically aggressive carcinoma, which is defined by a translocation involving the NUT (nuclear protein in testis) gene on chromosome
15q14 and, in most cases, the BRD4 gene on chromosome
19p13.1. Initial cases were reported in young patients affected by intrathoracic carcinomas, but it is now well established that these tumours may occur in adults and involve
other anatomic sites, including the sinonasal tract 5. So far
less than ten cases have been described in the nasal cavity
and paranasal sinuses. These tumours affected young adults
of both sexes and showed an aggressive clinical behaviour.
However, there is certainly an underestimation of their occurrence due to the lack of specific diagnostic features.
Histologically, these carcinomas are composed of undifferentiated basaloid cells with focal, often abrupt, squamous
differentiation. Therefore, the diagnosis of NMC requires
the demonstration of the NUT translocation, which can be
achieved by karyotyping, reverse transcription polymerase
chain reaction (RT-PCR), and FISH. Recently, a monoclonal
antibody to NUT has been developed, which showed a sensitivity of 87%, a specificity of 100%, a negative predictive
value of 99%, and a positive predictive value of 100% when
tested in a large panel of carcinoma tissues 6. Moreover, the
expression of normal NUT protein is limited to the germ cells
of the testis and ovary, thus increasing the reliability of the
use of immunohistochemistry in the diagnosis of NMC. The
use of this antibody may help to separate NMC from other
poorly differentiated sinonasal carcinomas, thus contributing to their clinico-pathologic characterisation. In addition,
it appears that the distinction of NMC from other sinonasal
carcinomas is of clinical relevance, in view of the favourable
response to certain treatment regimes, including chemotherapy according to Ewing’s sarcoma protocols 7 or docetaxel
and radiotherapy 8.
Small cell carcinoma, neuroendocrine type (SCCNET)
Currently, WHO classification of head & neck tumours,
places SCCNET in the category of neuroendocrine tumours
together with carcinoid tumour, which can be further sub-classified into typical and atypical 9. SCCNET of the nasal cavities
and paranasal sinuses is a very uncommon neoplasm of which
only small series and isolate case reports have been reported
in the English literature 10. A critical review of these reports
reveals that in some cases the clinico-pathological features
of the lesions described were more consistent with other diagnoses, including olfactory neuroblastoma and SNUC. This
underlines the current lack of criteria, including a definition
of a panel of immunohistochemical markers, to make the diagnosis of SCCNET. Small cell neuroendocrine carcinoma of
the sinonasal tract is histologically indistinguishable from its
pulmonary counterpart. Immunohistochemically, it is positive
for cytokeratins and neuroendocrine markers such as NSE
(neuron specific enolase), synaptophysin, and chromogranin,
although with variable intensity 10. As small cell neuroendocrine carcinomas of other sites, sinonasal tumours express
CD57 11. These features allow the distinction from SNUC,
malignant melanoma, olfactory neuroblastoma, lymphoma,
Ewing’s sarcoma/PNET and rhabdomyosarcoma.
Olfactory Neuroblastoma (ON)
ON is a rare neoplasm occurring in a broad age range, which
most commonly originates in the region of the cribriform plate
from the olfactory mucosa 12. More frequently, the tumour
grows in nests separated by fibrovascular septa, or sometimes
it may show a diffuse growth pattern. The neoplastic cells
typically have small and round nuclei with stippled chromatin, absent or small nucleoli, and scanty cytoplasm. They are
embedded in a fibrillary background formed by cell processes.
Homer-Wright type of rosettes, or more rarely Flexner rosettes
can be found. Immunohistochemically, ON shows diffuse
positivity for NSE and synaptophysin, while chromogranin,
GFAP and leu-7 are less often positive. S-100 protein stains
sustentacular cells around neoplastic nests, but in less differentiated tumours there may be few scattered S-100 protein
positive cells. Neurofilament protein and other markers of neural differentiation are more often expressed in tumours with
diffuse, sheet-like pattern. Cytokeratins are generally negative,
although in ON with nesting pattern a few tumours cells may
exhibit staining for low molecular weight cytokeratins. A subgroup of ON with gland-like formations and more widespread
cytokeratin positivity has been designated “olfactory neuroepithelioma” 13. EMA is consistently negative, as they are CD99,
CD45, HMB-45 and muscle markers. Ultrastructural analysis
shows evidence of neuroblastic differentiation, including the
presence of dendritic processes containing dense core granules
and neurotubules, and occasional synaptic junctions. ON lacks
the t(11; 22) translocation of Ewing’s sarcoma/PNET.
References
1
Cerilli LA, Holst VA, Brandwein MS, et al. Sinonasal undifferentiated
carcinoma: immunohistochemical profile and lack of EBV association.
Am J Surg Pathol 2001;25:156-63.
2
Mills SE. Neuroectodermal neoplasms of the head and neck with emphasis on neuroendocrine carcinomas. Mod Pathol 2002;15:264-78.
3
Franchi A, Moroni M, Massi D, et al. Sinonasal undifferentiated carcinoma, nasopharyngeal-type undifferentiated carcinoma, and keratinizing and nonkeratinizing squamous cell carcinoma express different
cytokeratin patterns. Am J Surg Pathol 2002;26:1597-604.
4
Bourne TD, Bellizzi AM, Stelow EB, et al. p63 expression in olfactory
neuroblastoma and other small cell tumors of the sinonasal tract. Am
J Clin Pathol 2008;130:213-8.
141
Lectures
5
6
7
8
9
10
11
12
13
French CA, Kutok JL, Faquin WC, et al. Midline carcinoma of
children and young adults with NUT rearrangement. J Clin Oncol
2004;22:4135-9.
Haack H, Johnson LA, Fry CJ, et al. Diagnosis of NUT midline carcinoma using a NUT-specific monoclonal antibody. Am J Surg Pathol
2009;33:984-91
Mertens F, Wiebe T, Adlercreutz C, Mandahl N, French CA. Successful treatment of a child with t(15;19)-positive tumor. Pediatr Blood
Cancer. 2007;49:1015-7.
Engleson J, Soller M, Panagopoulos I, et al. Midline carcinoma with
t(15;19) and BRD4-NUT fusion oncogene in a 30-year-old female with
response to docetaxel and radiotherapy. BMC Cancer 2006;6:69.
Perez-Ordonez B. Neuroendocrine tumours. In: Barnes L, Eveson JW,
Reichart P, Sidransky D (eds.). WHO Classification of Tumours. Pathology and Genetics of Head and Neck Tumours. Lyon: IARC Press
2005, pp. 26-7.
Perez-Ordonez B, Caruana SM, Huvos AG, et al. Small cell neuroendocrine carcinoma of the nasal cavity and paranasal sinuses. Hum
Pathol 1998;29:826-32.
Morice WG, Ferreiro JA. Distinction of basaloid squamous cell carcinoma from adenoid cystic and small cell undifferentiated carcinoma
by immunohistochemistry. Hum Pathol 1998;29:609-12.
Dulguerov P, Allal AS, Calcaterra TC. Esthesioneuroblastoma: a meta
analysis and review. Lancet Oncol 2001;2:683-90.
Sugita Y, Kusano K, Tokunaga O, et al. Olfactory neuroepithelioma:
an immunohistochemical and ultrastructural study. Neuropathol
2006;26:400-8.
Minor salivary gland tumors
G. De Rosa
Department of Biomorphological and Functional Sciences, Pathology Section, University of Naples Federico II, School of Medicine,
Naples, Italy
Background. Minor salivary gland tumors (MSGT) are uncommon, representing approximately 10-15% of all salivary
neoplasms and 3-5% of all head and neck tumors. Despite this
relatively low frequency, these neoplasms show a high variety
in clinical behavior and morphology, constituting a heteroge-
neous group with a broad range of histological types. All the
histotypes of WHO classification of salivary gland tumors
may arise in minor salivary gland, but some tumors, as canalicular adenoma, polymorphous low grade adenocarcinoma,
and sialadenoma papilliferum are exclusive or predominant
in these sites. Unlike parotid and submandibular tumors, most
MSGT are malignant, with different rates among the various
anatomical locations.
Results. In our Department we identified 91 cases of
MSGT from 1993 to 2009; in agreement with the data
of the literature, the most frequent benign type was the
pleomophic adenoma, while among the malignant tumors,
polymorphous low grade adenocarcinoma was the most
common, followed by adenoid-cystic and mucoepidermoid
carcinoma.
The diagnosis of MSGT may be difficult, because many histotypes show overlapping morphological features; cribriform
areas, clear cells, bilayered and papillary pattern are present in
many different benign and low grade neoplasms complicating
the differential diagnosis. In this setting, the search of stromal
and perineural invasion is one of the most important feature
of malignancy; however, near always the specimen are small
incisional biopsy and is not possible to assessing the tumor
borders and the tumor interface with adjacent tissues, and then
differentiate between benign tumor and malignancies. In these
cases, a definitive diagnosis should be deferred to complete
excision or a larger-size biopsy.
At the present, the role of immunohistochemistry in diagnosis of MSGT is limitated; in fact the immunohistochemical
staining may demonstrate the coexistence of glandular and
myoepithelial components, and the presence of dual luminalabluminal cell differentiation, but most benign and low grade
tumors exhibit these same features. On the contrary, Ki67
proliferative index may be useful in distinguishing a benign
lesion from a malignant tumors, being 10% the reliable cut-off
value for malignancy.
Gynaecological pathologies in Lynch syndrome
Moderators: M.L. Carcangiu (Milano), C. Riva (Varese)
Clinicopathologic features of gynecologic
tumors in Lynch syndrome
M.L. Carcangiu
Dipartimento di Patologia e Laboratorio, Fondazione IRCSS Istituto
Nazionale Tumori, Milano, Italia
Background. Women with hereditary non-polyposis colorectal
cancer (HNPCC)/Lynch Syndrome have a high risk for gynaecological cancer and in particular for endometrial cancer (EC).
Methods. The pertinent literature on the field from 2000 to
present was retrieved trough a med-line search and critically
reviewed.
Results. Lynch syndrome (LS), also known as hereditary polyposis colorectal cancer syndrome (HNPCC), is an autosomal
dominant inherited cancer susceptibility syndrome characterized by a high risk of colorectal, endometrial, and other
tumors, including ovarian, gastric, urinary, and biliary tract
cancer. The genetic basis of this syndrome is a mutation in one
of the known DNA mismatch-repair genes: MLH1, MSH2,
and MSH6. Women with LS have a 20%-60% lifetime risk of
endometrial cancer and approximately 50% of them present
first with endometrial cancer.
Clinical-pathologic data on LS-related EC are scanty. Most
authors have stated that they occur in a younger age group
and that most of them show low grade endometrioid histology. A different picture emerges from the series of Broaddus
et al., in which the non-endometrioid types made up 14% of
the LS-related EC, as opposed to 2.4% in the control cases; no
details were given on the histologic subtypes comprising the
non-endometrioid group. In our series of endometrial carcinomas from 23 patients (mean age 46.2 years) with MSH2 (16),
MLH1 (6) and MLH1/MSH2 (1) constitutional mutations,
there were 13 (56.5%) endometrioid endometrial carcinomas (EECA) and 10 (43.4%) non-endometrioid endometrial
carcinomas (N-EECA) with a predominance of the clear cell
type frequently combined with an endometrioid carcinoma
as opposed to the control group made by 66 sporadic tumors
in same age patients where 44 (95.6%) were of endometrioid
type and 2 (4.3%) non-endometrioid (OR 0.59, 0.013-0.27).
Furthermore the endometrioid cancers in women with a germ-
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
line LS mutation had a higher FIGO grade and more frequent
vascular invasion than their sporadic counterparts. In our series we also identified 2 cases of Malignant Mixed Mullerian
Tumor in 2 MSH2-mutated sisters, a finding that has also been
recently reported in a MLH1-mutated patient
Regarding the FIGO grade, 46.1% of the LS women with
EECA in our study had a grade III tumor, a frequency appreciably higher than that seen in the control group.
As far as the FIGO Stage is concerned, Boks et al. and Broaddus et al. had commented on the relatively high number of
Stage III tumors they found in their series of LS-related EC,
especially in view of the relatively young age of the patients;
regrettably, no information was provided on the stage distribution according to histologic type. In our group of patients,
the tumor stage distribution at presentation of both EECA and
N-EECA paralleled those observed in their sporadic counterparts, with the majority of EECA presenting at stage I and
most N-EECA (with or without an endometrioid component)
presenting at higher stages.
The overall survival of our patients at the end of the followup was lower than that reported in previous studies and was
strongly influenced by histologic type and tumor stage, as
shown by the fact that 4 of the 5 patients who died of EC had
a N-EECA. By contrast, the EECA were characterized by
a uniformly favourable outcome, with only a tumor-related
death in a 67-year-old woman who had a grade III, stage IIB
tumor.
LS associated ovarian carcinomas account for 10-15% of
hereditary ovarian carcinomas. Histologically, they tend to be
more frequently of endometrioid and clear cell types. Findings
from a study by Crijnen et al. on 26 patients with LS associated ovarian cancer showed that the survival rate for ovarian
cancer was not significantly different between these patients
and controls with sporadic ovarian cancer.
References
Boks DE, Trujillo AP, Voogd AC, et al. Survival analysis of endometrial
carcinoma associated with hereditary nonpolyposis colorectal cancer.
Int J Cancer 2002;102:198-200.
Broaddus RR, Lynch HT, Chen LM, et al. Pathologic features of endometrial carcinoma associated with HNPCC: a comparison with sporadic
endometrial carcinoma. Cancer 2006;106:87-94.
Carcangiu ML, Radice P, Casalini P, et al. Lynch syndrome--related
endometrial carcinomas show a high frequency of non-endometrioid
types and of high FIGO grade endometrioid types. Int J Surg Pathol
2010;18:21-6.
Crijnen ThEM, Janssen-Heijnen MLG, Gelderblom H, et al. Survival of
patients with ovarian cancer due to a mismatch repair defect. Fam
Cancer 2005;4:301-305.
Lynch HT, Casey MJ, Snyder CL, et al. Hereditary ovarian carcinoma:
heterogeneity, molecular genetics, pathology, and management. Mol
Oncol 2009;3:97-137.
Molecular and immunohistochemical features
of endometrial carcinoma in HNPCC/LS
C. Riva
Dept of Human Morphology, University of Insubria, Varese
Endometrial carcinoma (EC) is the most common extracolonic neoplasia in HNPCC/Lynch Syndrome (LS) patients.
Men with LS have a 74% lifetime risk of colorectal cancer
(CRC), in women the risk is over 30%, but more than 40% to
50% will develop EC.
LS is characterized by germline mutations of DNA MMR
genes. The MMR system repairs DNA replications errors
that are not immediately corrected by DNA polymerase and,
therefore, it plays a crucial role in DNA replication accuracy.
Human (h) MMR genes and corresponding proteins include
hMSH2, hMSH6, hMLH1, and hPMS2.
Functional inactivation of MMR genes by mutations or epigenetic changes leads to the accumulation of insertions/deletions. These are easily identified in short DNA tandem repeat
sequences (microsatellites) and this phenotype is known
as microsatellites instability (MSI). MSI can be present in
tumors from LS patients but it is also observed in a fraction
(15-25%) of various sporadic neoplasms, including EC.
Among germline mutations are recognized 1) truncating mutations leading either to a loss or to a easily degradable protein
and allowing to a negative immunohistochemical (IHC) staining of MMR protein and 2) missense point mutations leading
to an amino acid substitution, affecting chemical stability or
functionality; in point mutations often MMR protein is still
immunoreactive and atypical clinical scenarios are observed.
Analysis for germline mutations in MMR genes is confirmatory for LS diagnosis. Mutational analysis is very expensive
and time consuming, therefore pre-selection of high risk
patients for mutation search is very important. This purpose
can be achieved by employing MSI tumor DNA analysis (by
PCR), IHC for 4 MMR proteins (hMLH1, hMSH2, hMSH6
and PMS2) and methylation assays.
An epigenetic (sporadic) cause of MSI is more common than
LS. Most of sporadic MSI CRCs and ECs arise via hMLH1
promoter methylation, therefore methylation analysis seems
useful to distinguish between sporadic and heritable cases.
MSI analysis is performed using the NCI panel of 5 PCR
microsatellite markers (BAT-25, BAT-26, D2S123, D5S346
and D17S250). Tumors are classified as MSI-H (two or more
markers show MSI), MSI-L (one marker shows MSI) and
MSS (none marker shows MSI). Not all LS associated ECs are
MSI-H, while most MSI-H ECs are sporadic tumors, therefore
MSI analysis may fail to detect a number of ECs arising in a
hereditary setting.
DNA-MMR protein IHC is an effective method to detect MSI
and it is useful as a screening of LS. Infact MLH1, MSH2,
MSH6 and PMS2 are lacking in tumor cell nuclei by IHC in
up 1/3 of ECs; this derives in most cases from MLH1 promoter
ipermethylation, while mutations accounts for the rest. IHC
interpretation can be problematic: in general only a complete
loss of expression in the setting af a positive internal control
(endometrial stroma, normal glands, lymphocytes, etc) seems
to be reliable. In rare cases with inconclusive IHC results and a
clinical LS suspicion, an alternative test should be performed.
IHC with MLH1, MSH2 and MSH6 antibodies shows an high
sensitivity (91%) and a specificity of only 83%, due to lack of
correlation between loss of MSH6 and MSI-H. In fact LS-associated EC is fivefold more frequently associated with MSH6
mutations compared to CRC and these mutations usually do
not leave to MSI-H. The positive predictive value of IHC for
detect a germline mutation, especially with absence of MSH2
and MSH6 is very high.Therefore it has been suggested that
IHC loss of these proteins may be a sufficient evidence of LS.
On the other hand, IHC can be easy performed in the majority
of pathology laboratories, is a convenient test and, in addition,
it can address specific genes sequencing.
Since LS detection methods cannot be applied routinely to every EC, various screening algorithms have been proposed. In
particular some data suggest the application of IHC for DNA
MMR proteins in ECs patients with strong personal or family
history, age of onset less than 50 years, lower uterine segment
localisation, a synchronous ovarian clear cell carcinoma and,
finally, morphological features characterized by peritumoral
or tumor infiltrating lymphocytes and tumor heterogeneity
including dedifferentiated areas.
143
Lectures
The EC patients with abnormal IHC results are referred for a
genetic evaluation including MSI analysis, if indicated a methylation test and mutational analysis of the screened genes.
References
Garg K, Leitao M, Kauff N, et al. Selection of endometrial carcinomas
for DNA mismatch repair protein immunohistochemistry using patient
age ansd tumor morphology enhances detection of mismatch repair
abnormalities. Am J Surg Pathol 2009;33:925-33.
Garg K, Soslow RA. Lynch Syndrome (hereditary non-polyposis colorectal cancer) and endometrial carcinoma. J Clin Pathol 2009;62 67984.
Karamurzin Y, Rutgers KL. DNA Mismatch Repair Deficiency in endometrial carcinoma. Int J Gynecol Pathol 2009;28,3;239-52.
Resnick K, Straughn JM, Backes F, et al. Lynch Syndrome screening
strategies among newly diagnosed endometrial cancer patients. Obstet
Gynecol 2009;114:530-6.
Lynch syndrome and new model of
individualized gynaecological cancer prevention
M.G. Tibiletti
U.O. Anatomia Patologica Ospedale di Circolo, Università
dell’Insubria Varese
Lynch syndrome (LS), or hereditary non-polyposis colorectal
cancer (HNPCC), is an autosomal dominant syndrome (MIM)
that predisposes its carriers to multiple malignancies including
colorectal cancer (CRC), endometrial cancer (EC), ovarian
cancer (OC), and cancer of the renal pelvis and ureter, stomach, pancreas, small bowel and brain.
Traditionally Lynch syndrome has been perceived as a CRC
dominated syndrome. However, in women with Lynch syndrome, the incidence of EC equals or exceeds that of CRC,
and in more than 50% of cases, these women present a gynaecological cancer as their first malignancy.
LS is caused by a germline mutation in one of the DNA
mismatch repair genes: MLH1, MSH2, MSH6, and PMS2.
Deficient mismatch repair protein activity leads to DNA microsatellite instability (MSI) and absent immunoistochemical
protein expression in tumour tissue. This pattern of abnormal
staining provides guidance as to which of the MMR genes is
likely to harbour a germline mutation.
The initial (1991) and revised (1998) Amsterdam criteria
were developed to identify families at high risk for LS.
These criteria required colorectal or other LS-associated
cancers in three first- degree relatives, occurring in at least
two successive generations, and in one individual under the
age of 50 years. These criteria were recognized to have poor
sensitivity in identifying individuals carrying an LS gene
mutation. Therefore, the Bethesda guidelines were introduced
to broaden testing recommendations and to identify a greater
proportion of affected individuals. The Bethesda guidelines
recommended molecular testing for LS in different groups
of patients including two gynaecologic cancer populations:
patients with endometrial cancer diagnosed before 45 years of
age and those with two LS-related cancers.
In 2006 an European group of experts in LS established guidelines for the clinical management of LS (Mallorca guidelines
J Med Genet 2007) in order to improve the identification and
the care of these families.
Identification of LS in affected individuals has important
implications for screening in individuals as well as family
members, as close screening and surveillance has been shown
to reduce the mortality of colorectal cancer by over 60%.
The frequency of germline DNA mismatch repair gene mutations among unselected patients with EC has been found to
be 1.8% to 2.1% which is similar to the frequency of LS in
colorectal carcinoma. In patients younger than 50 years, the
incidence is increased up to 9%. The identification of these
patients is important for several reasons. Affected patients are
at risk for multiple synchronous and metachronous tumors.
These individuals would therefore benefit from surveillance
measures to detect other LS associated tumours; their family
members may benefit from genetic testing to determine carrier
status and to have adequate surveillance measures.
References
Meyer LA, et al. Endometrial cancer and Lynch Syndrome: Clinical and
pathological considerations. Cancer Control 2009;16:14-22.
Vasen H, et al. Guidelines for the clinical management of Lynch syndrome (HNPCC). J Med Genet 2007;44;353-62.
Walsh CS, et al. Lynch syndrome among gynecologic oncology patients meeting Bethesda guidelines for screening. Gynecon Oncol
2010;116:516-21.
Pathologica symposium: new frontiers in immunohistochemistry
Immunoprofile of renal tumors
D. Segala, M. Brunelli, G. Martignoni
Department of diagnostic pathology, University of Verona, Verona,
Italy
The WHO 2004 classification of renal cell neoplasms includes
numerous entities characterized by different prognosis and the
correct subtyping is an important procedures to predict the
behavior of these tumors.
Among major renal histotypes, oncocytoma has the best prognosis followed by chromophobe, papillary, clear cell and collecting duct renal cell carcinomas (RCCs) 1-4. The overlapping
morphological features and the increasing description of novel
potential entities determine the difficulties of some histologic
distinctions. That is why traditional histology needs today the
support of more specific markers that should be consistently
detected also on small biopsies. In fact, new minimal invasive
forms of therapies, such as criotherapy and diathermocoagulation will require a pre-operative diagnosis 5 6.
Moreover, reliable predictive factors are essential for the
stratification of patients into clinically meaningful categories. Staging has recently improved with the development of
integrated systems 7 8, however the information obtained by
molecular tumor markers are expected to revolutionize the
staging of RCC.
Immunohistochemistry is the most easily available and not
expensive ancillary technique used by pathologists, therefore
it is the most important field to be improved.
Diagnostic immunohistochemical markers
Clear cell renal cell carcinoma. Clear cell RCC is immmunohistochemically characterized by a high positive rate for
CD10 (82%) 9-11. In our experience also CD13 is a good immunohistochemical marker of clear cell renal cell carcinoma
being positive in 81% 12. Most clear cell RCCs typically show
144
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
a restricted expression pattern of cytokeratins (CK) with limited cases expressing CK7, CK8, CK19, high weight CKs and
a large portion of cases positive for CK18 13 14. Parvalbumin
was found to be constantly absent 10. Alpha-methylacyl-CoA
racemase (AMACR) positivity has been detected in 25%
whereas S100A1 immmunostaining has been observed in 75%
of the cases 15 16. Around more than a half of the clear cell
RCCs reveals immunoreactivities for vimentin, RCC Marker
and Epithelial Membrane Antigen (EMA) 9 14 17.
Papillary renal cell carcinoma. Papillary RCCs typically
express CK7 (87%), 8, 18 and 19, Vimentin (90%) and they
constantly show AMACR immunostain 14 15 18. CK7 expression is more frequently observed in type 1 (87-100%) than
type 2 (20-50%) 18 such as EMA (type 1 ranging from 72 to
100% and type 2 from 13 to 17%) 17 19 whereas E-cadherin
is reported predominantly in type 2 17. An high incidence
of positivity for CD10 (59-90%), BerEP4, EMA and RCC
Marker is reported 10 20. S100A1 is reported in 92% of papillary RCCs 16 which occasionally express high molecular
weight CKs (26%) 11.
Chromophobe renal cell carcinoma. Chromophobe RCCs
is strongly positive for parvalbumin in all primary and
metastatic tumors 21-23. Chromophobe RCCs are also positive for CK7 in 73-100% of the samples 13 14. CD10 expression has been found in 26% of chromophobe RCCs, five of
the seven (71%) showing aggressive features 10. CKs 8, 18,
EMA and E-cadherin are frequently positive whereas chromophobe RCCs do not usually express vimentin 13 14 17 24.
Immunohistochemical membrane expression of c-KIT
is frequently found in chromophobe RCC however c-kit
mutation has not been found 25. AMACR is usually not
expressed and only 4% of chromophobe RCCs are positive
for S100A1 15 16.
Collecting duct carcinoma. The immunohistochemical profile of these carcinomas shows high molecular weight CKs,
EMA, vimentin, lectin Ulex europaeus agglutinin and peanut
lectin agglutinin (Arachis hypogaea) immunostain 26.
Oncocytoma
Most of oncocytomas are immunoreactive for CKs (86%),
EMA (86%), E-cadherin (71%), parvalbumin (70%) and cKIT (100%) 13 14 17 21 27 28. Vimentin and RCC marker are usually not expressed 14. Althought contrasting results have been
reported for CK7 in renal oncocytoma, it actually seems that
only a focal immunoreactivity of a few cells can be found 29 30.
S100A1 is expressed in 92% of this neoplasm 16.
Renal mucinous tubular and spindle cell carcinoma. This
histotype immunostains for CKs (CK5/6, 7, 8, 13, 14, 17, 18,
19, 20), high molecular weight CKs 1, 5, 10, 14, E-cadherin
and vimentin, but CD10 and RCC marker are usually not
expressed 31-33. Immunoreactivity for AMACR (93%), CK7
(81%) and EMA (95%) have also been reported 33.
TFE-family translocation renal cell carcinomas. TFE-family translocation renal cell carcinomas bear specific translocations that results in overexpression of TFE3 or TFEB,
genes that are strictly related to microphtalmia transctiption
factor (MiTF). Different translocations involving chromosome Xp11.2 bring TFE3 fusion gene product overexpression,
whereas TFEB overexpression is the result of the specific
translocation t(6;11)(p21;q12).
Immunohistochemistry for TFE3 and TFEB is the most reliable test able to distinguish TFE-family translocation renal
cell carcinomas from formalin-fixed and paraffin-embedded
archive tissue, but sometimes troubles using these antibodies
have been reported. These tumors were also consistently im-
munoreactive for the RCC antigen and CD10 and negative or
focally positive for citokeratins 34-37.
Our group have recently described the immunohistochemical
expression of Cathepsin-K, a protein described in osteoclasts
to be modulated by the expression of MiTF, in 17 cytogenetically demonstrated TFE3 and TFEB renal cell carcinomas and
in a large group of renal tumors 38. Cathepsin-K was positive
in all TFEB renal cell carcinoma and in 60% of TFE3 renal
cell carcinomas, whereas all other renal tumors were negative.
Therefore cathepsin-K could be a useful marker alternative to
TFE3 and TFEB.
End-stage renal disease associated tumors. Tumors arising in kidneys with end-stage renal disease include those
resembling sporadic renal tumors such and tumors distinct
from them that Tickoo at al named “acquired cystic diseaseassociated renal cell carcinoma” and “clear cell papillary renal
cell carcinoma of the end-stage renal kidneys” 39. This last
neoplasms seem to display distinctive histologic features not
easily referable to the histotypes described in the WHO 2004
classification system.
Clear-cell papillary renal cell carcinoma of the end-stage
kidney, unlike papillary RCC, were costantly negative for
AMACR, but unlike clear-cell RCC all tumors tested showed
strong immunoexpression for CK7 39. Gobbo et al. found similar tumors in normal kidneys 40. They also observed the lack of
immunoexpression of CD10.
Tumors with a strict related immunohistochemical pattern and
similar morphological features have also been recently described and called RCC with prominent angioleiomyomatous
proliferation 41. This tumors are characterized by a various
grade of stromal proliferation beside the epithelial structures.
To date the correlation between these two entities is not already demonstrated.
Prognostic molecular markers
Nomograms assigning numerical scores to various clinical
and pathological prognostic indicators, excluding molecular
markers, has been proposed, however a wide variety of molecular markers have been examined and some seem promising to legitimize further research to prove their value as
prognostic tools.
Among tumour suppressor genes p53 overexpression has been
described as a significant molecular predictor of tumor recurrence, especially in clear cell RCC and the loss of p27/kip1
expression is described as a possible prognostic and diagnostic marker of tumor development and/or progression 42-44.
Ki-67 proliferation index has been shown to be a prognostic
factor in both univariate and multivariate analysis, although
conflicting evidence has challenged these findings 45 46.
COX-2 expression in patients with renal cell carcinoma is
associated with several clinicopathological factors, and appeared to play an important role in tumor cell proliferation,
but is not a significant prognostic factor 47 48.
The adipose differentiation-related protein (ADFP) is a lipid
storage droplet-associated protein and its transcription is considered to be regulated by the von Hippel-Lindau/hypoxia-inducible factor pathway. ADFP expression status may provide
useful prognostic information as a biomolecular marker in
patients with clear cell RCC 49.
Decreased carbonic anhydrase IX (CAIX) levels are independently associated with poor survival in advanced RCC. CAIX
reflects significant changes in tumor biology, which should
be used to predict clinical outcome and identify high-risk patients in need for adjuvant immunotherapy and CAIX-targeted
therapies 50.
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Lectures
Epithelial growth factor receptor (EGF-R) positivity is more
common in clear cell (81%) than in papillary tumours (40%).
Membranous location of EGF-R immunostaining is associated with good prognosis in renal cell carcinoma 51 52.
Vascular endothelial growth factor (VEGF) protein expression
is a significant independent predictor of outcome and suggests
that VEGF is involved in angiogenesis in clear RCCs 53.
Patients with EpCam expressing clear cell RCC showed a
trend toward a better prognosis in a Cox regression analysis
including stage, grade, and necrosis 54.
Conclusions
Among the large number of molecular markers proposed
in recent years, CD10, parvalbumin, AMACR, CK7 and
S100A1 seem the more promising immunostains for an accurate diagnostic panel. Immunohistochemical prognostic
markers useful in the daily routine work are lacking to date
and TNM staging, grading sec. Fuhrman and necrosis appear as prognostic information to include in the pathological
report.
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Molecular diagnosis of solid tumours.
A practical approach for organ pathologies
Moderators: G. Tallini (Bologna), G. Stanta (Trieste)
Molecular diagnosis in solid tumor: the breast
A. Sapino, C. Marchiò
Dipartimento di Scienze Biomediche e Oncologia Umana. Torino.
Italy
Molecular techniques are, nowadays, in common use in pathology laboratories, especially in the field of cancer diagnosis. In breast pathology, molecular testing continues to expand
as requests by the oncologists of more precise prediction on
response to treatment and risk of recurrence increase.
In situ hybridization techniques, such ad FISH/CISH, SISH
to test HER2 gene status, are the basic and most widely used
molecular tests applied to breast cancer diagnosis. However,
following the results of the first studies of microarrays used
as prognostic/ predictive tools, countless prognostic and/or
predictive signatures have been developed. Two of these
signatures, the MammaPrint® (Agendia BV, Amsterdam,
Netherlands) and the Oncotype DX® (Genomic Health Inc.,
Redwood City, CA, USA) have achieved the FDA approval.
In Italy, both of them are commercially available only for
patients’ use. In particular, the first assay is based upon a
multi-gene prognostic predictive score comprising 70 genes
and works on mRNA extracted form fresh cancer tissues.
This signature segregates patients in two categories: one of
good prognosis (“low-risk” group), and one of poor prognosis
(“high-risk” group). The Oncotype DX® is an RT-PCR based
test that is based on the mRNA expression levels of only 21
genes (16 cancer related genes and 5 reference genes) and is
presented as single Recurrence Score, which is a continuous
variable ranging between 0 and 100 divided into three risk
groups: low (< 18), intermediate (18-31) and high (RS ≥31),
for clinical decision-making. The main goal of both signatures
is to safely spare patients at “low molecular risk” with border
line biological risk from chemotherapy. However extensive
validation of MammaPrint® and of Oncotype DX® represents
the main challenge in integrating them in the standard of
breast patients care. Combining molecular assay results with
the pathological and clinical features will pave the way to a
new era in breast oncology.
Molecular diagnosis of lung cancer
A. Marchetti
Sezione di Diagnostica Molecolare, Dipartimento di Oncologia e
Medicina Sperimentale, Università “G. D’Annunzio”, Chieti, Italia
Lung cancer is the most frequent cause of cancer-related
morbidity and mortality in industrialised countries, and about
80% of primary lung cancers are non-small cell lung carcinomas (NSCLCs). The two most common subtypes of NSCLC,
squamous cell carcinoma (SCC) and adenocarcinoma (AC)
derive from different compartments in the lung. The main
molecular pathways involved in the pathogenesis of NSCLC
include: a) growth promoting pathways (EGFR, KRAS PI3K,
ALK), b) growth inhibitory pathways (p53, Rb, P14ARF,
STK11), c) apoptotic pathways (Bcl-2, Bax, Fas/FasL), d)
pathways involved in DNA repair and immortalisation processes. A number of epigenetic changes, including DNA
methylation, histone/chromatin protein modification, and
micro-RNA expression can also contribute to tumour develop-
Lectures
ment. Cumulative information suggests that the SCC and AC
subtypes progress through different carcinogenic pathways,
but the genetic aberrations promoting such differences are
poorly understood.
The recent advent of targeted therapies in the management of
NSCLC patients have greatly enhanced the interest for predictive molecular markers that could allow to select patients
maximising efficacy and avoiding toxic effects of treatments.
The identification of predictive biomarkers that can guide
treatment decisions is an important step for individualized
therapy and in ultimately improving patient outcomes.
Monoclonal antibodies and small-molecule tyrosine kinase
inhibitors (TKIs) targeting the Epidermal Growth Factor
Receptor (EGFR) and the Vascular Endothelial Growth Factor (VEGF) have recently emerged as effective agents for
the treatment of patients with advanced NSCLC. In addition, several novel agents have been developed which may
overcome acquired resistance to these treatments or target
other deregulated cell pathways. Potential biomarkers for
the selection of patients with NSCLC most likely to benefit
from tyrosine kinase inhibitors include mutations, gene copy
number increase and single-nucleotide polymorphisms of
the EGFR gene, EGFR protein expression and oncogenic
mutation on the KRAS gene. Additional biomarkers that may
predict response to other recently developed targeted therapies
are under investigation.
A number of different techniques including fluorescence in
situ hybridization (FISH), PCR amplification followed by
sequencing or other mutation detection assays, and reversetranscription PCR, have been used to rapidly and efficiently
characterize these biomarkers. The current weight of evidence
for using these methods to analyse biomarkers for personalized therapy for a rapid characterization of NSCLCs to be
treated with targeted agents will be presented.
Integration of molecular diagnostics into thyroid
cytological practice
G. Troncone
Dipartimento di Scienze Biomorfologiche e Funzionali, Università di
Napoli “Federico II”
Background. Although thyroid Fine-needle aspiration (FNA)
is much more accurate than the clinical, biochemical or radiological assessments, the method is highly dependent on
the operator experience 1. Conventional wisdom dictates that
FNA is more efficient when an experienced cytopathologist
ensures the proper smearing technique and the rapid interpretation of air-dried Diff-Quick-stained smears 2. Molecular
testing of thyroid nodules for a panel of mutations refines the
cytological diagnosis of a thyroid cell malignancy 3. In particular the V600E BRAF mutation, highly specific for papillary carcinoma, is also emerging as an independent marker of
clinical aggressiveness 4 5. Preoperative knowledge of BRAF
mutation may be helpful to tailor the surgical treatment for
any individual patient 5. However, the full application of this
test from dedicated research labs to cytopathology outpatient
settings has not completely been accomplished 6. A number
of pratical issues have not been investigated, as most of the
studies were retrospective. To widespread the use of this test,
sample collection procedures have to be standardized step by
step. In particular, the way in which the aspirated samples is
aliquoted into routine smears and the buffer for DNA extraction is crucial; in this step the “informativeness” of the material both for cytopathological and molecular diagnosis needs
147
to be carefully preserved. Here we present a study recently
undertaken to assess whether our method of FNA preparation
is suitable to implement BRAF testing without interfering
with routine cytology.
Methods. One-hundred and twenty-eight cases were picked
up consecutively without any selection among the FNAs
routinely performed in the outpatient clinic, at the University
“Federico II” of Naples. Totally, three needle passes were
taken in each case. As usual Diff-Quick smears were prepared
from the first two passages by the nodule. When the adequacy
criteria were fulfilled, the whole tissue material from the
third pass was collected into a tube containing 500 µl of the
nucleic acids preservative solution (RNA later. Ambion). In
the case that the first two needle passes failed to provide a
fully satisfactory sample, the third needle pass was used for
direct smears and the remainder material was collected for
molecular testing. Cases were classified according to scheme
suggested by the NCI Thyroid FNA State of the Science Conference1. Regardless of the collection method, all samples
were similarly processed and exon 15 BRAF mutational
analysis was performed as previously described 6.
Results. Basing on a satisfactory on-site evaluation, a BRAF
dedicated third pass was performed in 44 (34%) cases; concordance between preliminary impressions and the final diagnosis was found in 42/44 (96%) cases. Conversely, in 84 (66%)
cases additional smears were prepared from the third pass.
This latter group included two cases (2,3%) in which the final
diagnosis could not be rendered due to scant cellularity. Final
cytological diagnosis of most nodules 110/128 (86%) cases
was benign; this category included nodular goiter (n = 61),
colloid nodules (n = 39), goiter with Hurtle changes (n = 4),
goiter with associated chronic lymphocytic thyroiditis (n = 4)
and hyperplastic/adenomatoid nodule in goiter (n = 2). In six
cases (4,6%) mixed features of both hyperplastic/adenomatoid
nodules and follicular neoplasm were observed; in these case
a diagnosis of follicular lesion of undetermined significance
(FLUS) was issued. In two cases (1,5%) follicular neoplasms
features were observed. In three cases (2,3%), there was a suspicion of papillary thyroid cancer. Five cases (3,9%) showing
clear-cut PTC nuclear changes were diagnosed as malignant.
DNA was isolated from 128 consecutive samples collected
during thyroid FNA. In 44 cases the whole tissue material
obtained from a dedicated pass was extracted, whereas in the
remaining 84 cases only the remainder material was employed
for DNA extraction. The quantity of isolated nucleic acids
ranged from 500 pg/µl to 309 ng/µl in the first group and from
500 pg/µl to 16,5 ng/µl to in the second group. Higher average
of extracted DNA concentration was observed in the dedicated pass group (25,9 ng/µl vs 7,9 ng/µl). Benign FNA had
a BRAF dedicated pass in 32%, whereas more often (68%)
the third pass was dedicated to the preparation of additional
smears. Conversely, the dedicated dedicated pass was often
performed in the FLUS (50%), follicular lesions (100%),
suspect (33%) and malignant (60%) groups. The vast majority
of samples (95.3%) showed successful exon 15 BRAF amplification. Only 6 samples (4.6%), nearly all from the needle
rinsing group (5/6), had insufficient and/or poor quality DNA
and were excluded from the analysis. Two of these cases
were inadequate for cytological diagnosis too. BRAFV600E
mutation was found in three cases. One case had a cytological
diagnosis of suspect for PTC, whereas other two cases had a
diagnosis of PTC. All other diagnostic group (benign, FLUS,
follicular neoplasms) showed wild type exon 15 BRAF in all
examined cases. We conclude that the FNA collection protocol here shown proved to be highly efficient. Cytological
148
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
and molecular tests gave adequate results respectively in the
98,4% and in 95,3%. In particular our method of aliquoting
the aspirated samples into routine smears and the buffer for
DNA extraction did not affects the “informativeness” of the
cytopathological diagnosis. Recently, Xing suggested that,
for prognostic purpose, perhaps all patients with cytologically
diagnosed PTC should be preoperatively tested for BRAF
mutation. In this respect this test provides information that
are additional and non redundant to those provided by a well
taken and correctly interpreted thyroid FNA. Our data showed
that in a routine clinical setting FNA specimens can properly
be handled to provide both morphological and molecular information. Although a large number of studies have reported
BRAF analysis of thyroid nodules aspirates only recently a
prospective study, on a large of number and with a complete
molecular analysis, was published. However on site-evaluation was performed only in a minority of cases and the issues
of sample collection was not taken into account 7. Our study
focusing on the single steps required to aliquot the aspirated
material into routine smears and DNA extraction buffer may
help to implement BRAF testing in the prognostic evaluation
of PTC diagnosed by FNA. Our proposed method ensures
that this test does not interfere with conventional cytology
diagnostic accuracy.
References
1
Baloch ZW, et al. Cytojournal 2008;5:6.
2
Alexander EK, et al. J Clin Endocrinol Metab 2002;87:4924-7.
3
Cohen Y, Xet al. J Natl Cancer Inst 2003;95:625-7.
4
Xing M, et al. J Clin Endocrinol Metab 2005;90:6373-9.
5
Xing M. Endocr Rev 2007;28:742-62.
6
Troncone G, et al. Cytojournal 2008;5:2.
7
Nikiforov YE, et al. J Clin Endocrinol Metab 2009;94:2092-8
Molecular diagnostic of solid tumors: a practical
approach for systematic pathology. Urinary
system
D. Segala, M. Brunelli, G. Martignoni
Department of diagnostic pathology, University of Verona, Verona,
Italy
Clinically robust molecular tests are necessary in current
clinical management of urological malignancies in order to
improve the faculties of choosing the right therapy and screening patients for target therapies.
Several promising biomarkers for diagnosis, prognosis and
target therapy are now under evaluation.
Urothelial carcinoma of the bladder. The five-years survival rate for localized bladder cancers and distant metastasis are
94% and 6%, respectively. This fact highlights the importance
of detection and appropriate therapeutic intervention at early
stages of disease.
Two forms of noninvasive bladder cancer are well known
to have a distinct histology and clinical behaviour: papillary
urothelial carcinoma rarely invades and metastasized, whereas
flat carcinoma in situ (CIS) is known to have a high rate of
invasion and metastasis.
Molecular evidences of the presence of distinct pathogenesis
for this two phenotype of urothelial carcinoma are now increasing. Both tyrosine kinase receptor FGFR-3 and H-RAS
oncogene are primarily involved in the pathogenetic pathway
of low-grade papillary urothelial carcinoma 1. Flat carcinoma
in situ and invasive urothelial carcinoma predominantly involve tumour suppressor genes p53, p16 and Rb 2-4. Tumor
angiogenetic factors are also involved the tumor-promoting
extracellular environment 5 6.
Chromosomal aberrations are also described in the pathogenesis of bladder cancer. Chromosome 9 alterations are established to be the earliest events in both pathways of urothelial
carcinoma 7 8. Moreover, gains of chromosome 3p, 7p, 17q,
and 9p21 deletions (p16 locus) are of special interest given
their potential diagnostic value.
The diagnostic process is usually supported by cistoscopy,
while urine cytology is the most widely used non-invasive
test to detect urothelial tumors. However, the letter is limited
by its low sensitivity. Recently the FDA approved a new
technique which seems to show better specificity ranges, the
multitarget multicolor fluorescence in situ hybridization assay (UroVysionTM) 9-12, that is based on frequent numerical
chromosomal alterations detection and it consists of fluorescently labelled DNA probes to the pericentromeric regions
of chromosome 3 (red), 7 (green), 17 (aqua) and to the locus
9p21 (gold). With the exception of one study 13, UroVysionTM
appears to enhance the sensitivity of routine cytology analysis
and it can be used in combination with routine cytology in
case with atypical cytology.
Diagnostic applications of UroVysionTM on histology has also
been suggested, such as the distinction of inverted urothelial
papilloma and bladder carcinoma with endophytic growth pattern. In fact, a study described chromosomal abnormalities in
72% of bladder carcinomas, in contrast to the absence of gains
and deletions in inverted papilloma 14.
Since an increasing number of data suggests the presence of
the same typical urothelial carcinoma chromosomal aberration
also in rare histologic subtypes 15 16 (e.g. clear cell adenocarcinoma and small cell carcinoma of the bladder), the possible
use of UroVysionTM could be a valid tool in the diagnosis of
these rare entities.
Adenocarcinoma of the prostate. The challenge in the years
to come will be to introduce new gene-based diagnostic and
prognostic tests in algorithms integrating the other known
clinical and pathological factors to better manage diagnostic
and therapeutic strategies.
In seek of this purpose, in the last decade an extensive list of
molecular biomarkers has been evaluated. One of the most
notable discoveries is presence of recurrent chromosomal rearrangements, which lead to a fusion of the androgen-responsive
promoter elements of the TMPRSS2 gene (21q22) to one of
the three of the ETS transcription factors family members ERG
(21q22), ETV (7p21) and ETV4 (17q21) 17. The prognostic role
of these rearrangements remains controversial, but this discovery has a great implication in terms of providing new markers
for molecular diagnostic and target therapy 18 19.
A large number of prognostic molecular markers still waits
for additional studies before eventually undergoing clinical
trials. p27 and p53 tumour suppressor genes expression has
been demonstrated to have a correlation with progression after
prostatectomy 20-23. Furthermore, several recent studies have
established the importance of PTEN/PI3K/mTOR (mammalian
target of rapamycin) in cell growth, proliferation and oncogenesis of prostate cancer 24-29. Finally, some papers suggest the potential usefulness of proliferation index (ki-67) 30, microvessel
density 31 and nuclear morphometry 32, while some others lack
to confirm their prognostic validity 23 33 34.
Gene expression profiling studies using cDNA microarrays
identified three genetic-differentiated subclasses of prostate
tumours 35. High grade, advanced stage and recurrent tumours
where much more represented among two of the three subtypes, one of which also included most lymph node metastases. Another study, using array-based comparative genomic
Lectures
hybridization (array CGH), identified a series of recurrent
DNA aberrations 36. Deletions at 5q21 and 6q15 were associated with favourable outcome group; 8p21 (NKX3-1) and
21q22 (TMPRSS2-ERG fusion) deletion group, 8q24 (MYC)
and 16p13 gains and loss at 10q23 (PTEN) and 16q23 groups
correlating with metastatic disease.
Germ cell tumors of the testis. The etiopathogenesis of germ
cell tumors of the testis depend on both environmental and
genetic factors acting on the primordial germ cells/gonocyte
that led to the precursor lesion called intratubular germ cell
neoplasia. This precursor can progress to invasive components divide into seminomas and nonseminomas, with different histology and therapeutic response.
Several immunohistochemical markers are described as useful in differential diagnosis of TGCTs. Briefly, seminoma is
characterized by the expression of OCT3/4 37 38, PALP 39 40,
c-KIT 41 and the variable expression of citokeratins 42, embryonal carcinoma stains for CD30 41, OCT3/4 37 38, PLAP, SOX2 43
and citokeratins, yolk sac tumor shows positivity for AFP 44
and PALP but it is negative for OCT3/4 38. Choriocarcinoma
is lighted by the immunoexpression of β-hCG 40.
Studies of familial cases and genome-wide analysis do not
reveal a constant genetic origin, suggesting that multiple
foci must contribute to the development and progression of
TGCTs.
In line with the origin of TGCTs, individuals with disorders
of sex development show an increased risk for this kind of
cancer, in which they Y chromosome genetic material is crucial 45 46. In fact, the recently described microdeletion of the
long arm of the Y chromosome, involving the AZoospermia
Factor (AZF)c region 47, seems to be a significant risk factor
for impaired spermatogenesis 48.
Among somatic chromosomal changes in TGCTs the only
recurrent structural imbalance appears to be the gain of
chromosome 12p 49 50, mostly as isochromosomes, that is described as related to tumor progression. Possible diagnostic
applications of Interphase Fluorescence In Situ Hybridization
(FISH) analysis of chromosome 12p abnormalities have been
proposed, such as the distinction of Epidermoid Cysts of the
testis, a benign lesion that lack the gain of 12p, to pure mature
Teratomas 51.
Rarely primitive and metastatic germ cell malignancies present with histologic features of somatic-type origin. A FISH
study demonstrated the presence of 12p abnormalities in 6 out
of 10 metastatic somatic-type malignancies in patients with
history of testicular or mediastinal germ cell tumors, suggesting the utility of this genetic marker in differential diagnosis
of metastatic tumors 52.
Renal Cell Carcinoma (RCC). Histological subtyping of
renal cell tumors is now considered an important prognostic factor in order to plan appropriate follow-up strategies.
Moreover, numerous targeted agents have been developed
for treatment of patients with renal cell carcinoma. For these
reasons cytogenetical analyses are becoming an essential improvement in the routine diagnostic practice.
Clear cell RCC is characterized by the mutation of the Von
Hippel-Lindau syndrome (VHL) gene mapping in the chromosomal region 3p25 and the deletion of chromosome 3p,
easy detectable by FISH analysis 53. Papillary RCC shows trisomy of chromosomes 7, 17 and loss of the Y chromosome 54.
The distinction of clear cell papillary RCC 55 56, a recently
described rare entity, from Clear cell RCC and papillary RCC
can be difficult; FISH can resolve this differential diagnosis,
in fact this lesion don’t show gains of chromosomes 7 and
17 and 3p deletion 56. Metanephric adenoma and mucinous
149
tubular spindle cell carcinoma, two others rare entities present in WHO 2004 classification, could be misdiagnosed as
papillary RCC. FISH analysis for chromosome 7 and 17 is
helpful since there are no numerical alterations of these chromosomes in these tumors 57-60. Among oncocytic neoplasms,
chromophobe RCC is characterized by a combination of loss
of chromosomes Y, 1, 2, 6, 10, 17 and 21, both classic and eosinophilic variants and renal oncocytoma, a benign lesion that
sometimes enters in differential diagnosis with eosinophilic
variant of chromophobe RCC, showed normal karyotype in
the majority of cases 61.
On the other hand, the information obtained by molecular
tumor markers are expected to revolutionize the staging of
RCC. A large set of immunohistochemical markers such as
Ki-67 62 63, p53 64-66, CAIX 67, ADPF 68, EGFR 69 and VEGR 70
have been investigated. A negative prognostic role different
mTOR pathway members was recently underlined 71 72. Moreover, some prognostic chromosomic aberrations are recently
observed in clear cell RCCs: gains or losses of 5q21 73 and
loss of chromosome 14q 74 have been shown a correlation with
progression (5p21), higher stage, higher histologic grade and
poorer outcome (14q). Loss of chromosome 9p, observed in
18% of clear cell RCC, is described as an independent negative prognostic factor 75.
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Carcinoma: A Distinct Histopathologic and Molecular Genetic Entity.
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Cossu-Rocca P, Eble JN, Delahunt B, et al. Renal mucinous tubular
and spindle carcinoma lacks the gains of chromosomes 7 and 17 and
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Sauter G, Epstein JI, Sesterhenn IA (eds). World Health Organization
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Brunelli M, Eble JN, Zhang S, et al. Metanephric adenoma lacks
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Brunelli M, Eble JN, Zhang S, et al Eosinophilic and classic chromophobe renal cell carcinomas have similar frequent losses of multiple
chromosomes from among chromosomes 1, 2, 6, 10, and 17, and this
pattern of genetic abnormality is not present in renal oncocytoma.
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expression to clinical outcome in renal clear cell carcinoma. Urology
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Dudderidge TJ, Stoeber K, Loddo M, et al. Mcm2, Geminin, and KI67
define proliferative state and are prognostic markers in renal cell
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marker in histologic subtypes of renal cell carcinoma: a systematic
analysis of primary and metastatic tumor tissue. Urology 2004;63:6515.
Shvarts O, Seligson D, Lam J, et al. p53 is an independent predictor of
tumor recurrence and progression after nephrectomy in patients with
localized renal cell carcinoma. J Urol 2005;173:725-8.
Rioux-Leclercq N, Turlin B, Bansard J, et al. Value of immunohistochemical Ki-67 and p53 determinations as predictive factors of
outcome in renal cell carcinoma. Urology 2000;55:501-5.
Bui MH, Seligson D, Han KR, et al. Carbonic anhydrase IX is an
independent predictor of survival in advanced renal clear cell carcinoma: implications for prognosis and therapy. Clin Cancer Res
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Moch H, Sauter G, Buchholz N, et al. Epidermal growth factor receptor expression is associated with rapid tumor cell proliferation in
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Jacobsen J, Grankvist K, Rasmuson T, et al. Expression of vascular
endothelial growth factor protein in human renal cell carcinoma. BJU
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Pantuck AJ, Seligson DB, Klatte T, et al. Prognostic relevance of the
mTOR pathway in renal cell carcinoma: implications for molecular
patient selection for targeted therapy. Cancer 2007;109:2257-67.
Pantuck AJ, Thomas G, Belldegrun AS, et al. Mammalian target of rapamycin inhibitors in renal cell carcinoma: current status and future
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Nagao K, Yoshihiro S, Matsuyama H, et al. Clinical significance of allelic loss of chromosome region 5q22.3 approximately q23.2 in nonpapillary renal cell carcinoma. Cancer Genet Cytogenet 2002;136:23-30.
Herbers J, Schullerus D, Muller H, et al. Significance of chromosome
arm 14q loss in nonpapillary renal cell carcinomas. Genes Chromosomes Cancer 1997;19:29-35.
Brunelli M, Eccher A, Gobbo S, et al. Loss of chromosome 9p is an
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carcinoma. Mod Pathol 2008;21:1-6.
SIAPEC-IAP meets SICI
Moderators: P. Maioli (Ravenna), A. Bondi (Bologna)
The new test of the screening for the prevention
of cervical carcinoma: experience in Abruzzo
region
C. Angeloni
Coordinator of Screening Project on Cervical Carcinoma in Abruzzo
Background. The scientific evidence that the infection of
Human Papilloma Virus (HPV) is the main cause of the cervical carcinoma has opened a new scenery in terms of primary
prevention with vaccination and secondary prevention with
the introduction of new screening technologies. The test of
HPV DNA as the test of the main screening demonstrated
indeed a sensibility that is absolutely higher than the Pap test
both for women aged between 25 and 34 and for women of superior years without showing any significant over-diagnosis.
These recent data and the consideration of the raised predictive negative value of HPV DNA test can make one consider
the possibility to use this test as main test for the screening,
reserving to the Pap the triage in secondary level for positive
HPV DNA test cases of high risk. Another element in favour
of the introduction of HPV DNA test is the higher reproducibility comparing to Pap test.
Therefore the National Centre for Disease Prevention and
Control (CCM) by the Health Ministry is considering to
modify further the Guidelines. It is fundamental that the pilot
projects co-ordinate activities among themselves and share
data, results and protocols to produce a series of conclusive
data for the applicability of this strategy.
The GISCi (Italian Study Group on Cervical Carcinoma)
shares this position that foresees the introduction of HPV test
in the main screening with controlled applications to test it in
practice and it has arranged a proper document by consensus.
Methods. The main objective of the study is to evaluate the
applicability of the screening programme based on HPV test
in a regional territory, already under coverage with a traditional methodology and particularly characterized by centralized
management of the programme and deep experience of new
technologies and computer based systems. The study represents an important diagnostic instrument of a new protocol of
screening in a scenery with different complexity. The results
obtained from this study might represent a preliminary and
necessary element for the introduction of HPV test as routine
test within screening programmes.
Women between 25 and 64 years old resident in Abruzzo
region and suitable to the screening, will be asked to have
another HPV test done, after three years from the previous
screening.
In our Regional Project we have decided to adopt the strategy
HC2 as main screening followed by the triage with cytology
for all ages organized by the screening (25-64 years old) for a
major adaptability and simplicity of using the programme and
also for having a lowest cost, as a double strategy of screening is not forseen, so the number of professional staff can be
limited and the centralization of the cytological triage can be
facilitated. We are expecting to modify the actual strategy of
the screening presupposing a long period of the rescreening
(5 years?) with a consequent reduction of costs and a more
favourable model of costs/benefits.
The area of l’Aquila and its surroundings, devastated by last
year earthquake, will be reached in different ways, such as
mobile medical vehicles.
The study will involve around 60.000 women in 2010.
The samples will be taken in decentralized sampling centres
in Abruzzo, using the vial for the ThinPrep (Hologic). Women
will be shown the procedures of HPV test, along with its clinical and preventive importance, however, they will be asked
to fill in an agreement form. In the agreement form signed by
the women there will be expected the creation of a biological
bank to preserve the residual rates of the material taken from
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
HPV test that, as it introduced by the protocol, could be used
for successive cytological tests of triage and for further elaborations of studies with a particular attention for the markers
of the specificity of the infection caused by HPV and of the
progression of the neoplastic pathology.
The samples will be sent to the medical centres of Atri and
Sulmona, chosen to perform the test for the research of high
risk DNA HPV (Hybrid Capture 2, cut-off 1pg/ml), using
an automated system which will make it possible to process
a high number of samples a day, with very fast turnaround,
costs saving and quality improvement. Women with negative
results will receive a letter with the test results along with an
invitation to a new screening test after 3 years. At the moment, the 3 years time lag is prudentially recommended, but
it is more likely that it will be extended to a 5-6 years, when
all the evidences on the duration of protective effects will
be proved and when the information will be updated by the
Ministry of Health.
The typing of positive results of high risk is centralized in
laboratories of molecular biology in Atri and Sulmona; in fact
we have forseen a triage with a specified type typing to value
better HPV positive women considering that also women
HPV 16 and 18 positive have a high risk to develop a Cin3+.
HPV HG test with positive results of high risk will be reported
to the Centre of Lanciano, entitled to cytological readings
of second level which will take care of slides preparation,
colouring and reading slides. Women with positive cytology
will be asked to take a colposcopy examination. Women with
negative cytology will receive an invitation letter for a further
examination in one year.
To guarantee the necessary sharing of results in order to create
conclusive data on the application of this strategy, we have
adopted a protocol analogous of other studies ongoing.
The software used for the screening is the only one in Abruzzo: it’s based on Web servers and LAN networks connected
with the colon rectum screening. The software managed by
Winsap on Web, adopted by Abruzzo region for the screening that uses the base of traced records of the regional vital
statistics and is continuously updated by our operators, has
been adjusted by the creation of a HPV module. A particular
element of quality has been presented by the traced record
produced individually, transmissible through the New Sanitary Information Service (NSIS) to the national Data Ware
House that allows to equalize, to centralize and to simplify
statistical analysis.
A new screening methodology, which involves a first level
test different from the traditional one, and which detects a
viral infection sexually transmitted, needs of course a different approach in terms of communication strategies. To
avoid useless over-treatments, it’s necessary to introduce a
new way of communication, which has to be scientific, but
at the same time easy to understand without creating anxiety
in women.
For these reasons the project introduces, for the new screening type, the use of information material, scientifically correct
and easy to understand by population, people involved in the
medical centers and doctors of general medicine. Each invitation letter for the test comes with a brochure, written with
simple and clear terms.
The given information illustrates the concept of the cervical
oncogenic risk underlining how the virus test results negative
for 90% of women over 30 and therefore allows to include
the tested subject among those of extremely reduced at risk
to develop a significant cervical pathology whereas the persistent positivity in the virus test represents a simple indicator
of a probable development of cervical pathology in years
analogously of any other test of screening usually made in the
medical prevention (weight, nutrition, etc.).
For all the levels of our regional project of screening there will
be settled a Quality Assurance programme. The creation of
a biological bank will allow to study molecular mechanisms
especially with regard to the determinants of progression and
regression of the infection itself and of the intraepithelial
cervical lesions.
We have already known that only persistent infections of HPV
are associated with a high risk of precancerous lesions. At this
moment persistent infections can only be valued by repeating
the test after 12 months whereas a clinical validation is necessary for the study and the characterization of markers of the
integration HPV-DNA and DNA cells that could signal the
latency state, the persistence of the infection and the progression to cancer.
At the moment there are not biomarkers of specificity in the
algorithms such as p16 and p16 Plus dual kit or mRNA, which
are extremely encouraging, but still under specific experimental studies: it will be the person in charge of reading the
cytological triage to decide whether to use it or not.
In case of a CIN diagnosis, it is up to the pathologist, to guarantee a more accurate diagnosis, to search for a confirmation
with the p16 histological test.
Results. The cost of the strategy of the screening with HPV
test as first level will be established regarding to the costs
met in the last decade with the use of a traditional strategy
of screening (Pap test I level) and with the adoption of new
technologies and computerized systems of cytological reading
(see attached: study ARINT of Abruzzo region and the project
of the research applied for programmes of screening by law
138 approved and financed by the CCM of the Health Ministry for the year 2009) considering also the possible saving
derive from the eventuality of the expected extension of the
interval of the screening.
The heterogeneity of accounting systems and even more the
lacking criterions of analytic accounting stand in the way of
an activity based costing system that would be essential for
estimating the financial requirements.
On the other hand, the necessary overcoming of the criterion
obsoleted by the historic cost requires analysis and applications of alternative systems. A recent decree Calderoli (known
as decree on ‘Federalism’ converted in law recently) has
moved in this direction establishing that costs having reference according to the letter m) of the second paragraph of the
article 117 of the Constitution (that concerns Essential Levels
of Assistance including screenings) ‘should determine with
respect the standard costs associated on essential levels of
services established by the state law, to be distributed in terms
of efficiency and appropriateness in all the national territory’
(articles 6, paragraph 1, letter b).
It is about a sector of studies not having been yet explored and
not lacking of difficulties also because the decree does not
make clear what ‘standard costs’ means and therefore how it
should be calculated.
Moreover, for some economists it seems to be an unrealistic
idea that the efficient specific cost could be calculated for
every singular service of the National Health Service (SSN)
and then have by a simple summation the costs of services
of the Essential Level of Assistance (LEA) in the decision of
the total requirements. They think: ‘it appears substantially
out of reach for services of prevention and for those of territorial medicine’ to prevent a tariff system analogous of that
of hospitals (for regional decree DRG), also because of the
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Lectures
heterogeneity of accounting systems and the lacking valuation
systems for cost centres.
Even believing that the determination of an ‘efficient cost’
is hardly an achievable desire, sometimes it is possible to
prevent a charging of non-hospital services and programmes
of screenings, just like our regional one that arranges an
analytical accounting system for cost centres, represent a possible application of innovative systems in the accounting and
organizational system.
Therefore coherently of what has been arranged recently, using the study of the valuation of costs by law 138, we are going to perform also the innovative valuation of standard costs
in our regional project of screening.
Our preliminary data, based on about 12.000 HPV DNA test,
show 9% positive rate with a positive PAPtest triage of 30%.
High-throughput type-specific detection of HPV
using massarray technology
V. Mantovani, E. Marasco, P. Garagnani, M. Cricca *,
M. Zerbini *, P. Chieco
Centro Ricerca Biomedica Applicata (CRBA); * U.O. Microbiologia e
Virologia, Policlinico “S. Orsola-Malpighi”, Bologna
Background. The persistent infection of oncogenic high
risk Human Papillomavirus (HPV) is one of the major risk
factors for cervical cancer and the presence of HPV DNA is
detected in nearly 100% of invasive cervical cancers. Several
studies showed that the HPV DNA can be included in the
screening for the prevention of cervical cancer in addition
to, or in substitution of the current cytological analysis (PAP
test), increasing the sensitivity and discovering earlier the
carcinogenesis process. However, this diagnostic approach is
not yet extensively applied because of the high costs of the
molecular tests.
Methods. Aim of our project is the development and the
evaluation of a reliable, low-cost method based on mass spectrometry (MALDI-TOF) for type-specific detection of HPV
DNA. The protocol has been optimized on the MassARRAY
platform (Sequenom) located in the CRBA laboratories.
The mass-spectrometry is a precise technology, often taken
as gold standard for biochemical analyses. The highthroughput MassARRAY platform can simultaneously test
384 samples and it is well suitable for large screening. The
methodology doesn’t need expensive immunometric reagents, it can simultaneously perform several tests and it is
easily automatable.
Results. The test here proposed identifies the twelve high risk
HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52,56, 58 and 59),
in addition to the six probably high risk (26, 53, 66, 68, 73
and 82). Sensitivity and specificity are very high for the major
types. In addition, our method identified some HPV infections
missed by standard hybridization test.
A low cost detection of the most relevant HPV types may be
an important advance in assessing the impact of HPV vaccines, allowing the monitoring of the future changes in typespecific prevalence in infection and cancer. Our approach is
very innovative in comparison with the existing methods,
combining high efficiency, high sensitivity and low costs,
suitable for routinely diagnostic activity, as well as for current
screening programs.
Streamlining the urinary oncology cytological
service in the metropolitan area
R. Rapezzi, A. Bondi
Oncological Science Department, U.O. Anatomy, pathological histology and cytodiagnostic departmentOspedale Maggiore, Bologna Local Health Unit (AUSL)
Background. Bladder carcinoma in men ranks fourth in
terms of frequency after prostate, lung and colorectal cancer,
accounting for 5.5% of all cancer cases; among women it
ranks eighth in terms of frequency, accounting for 2.3% of
all female neoplasias. The higher incidence among men than
women (4:1 ratio) is presumably associated with greater exposure to risk factors in the workplace such as chemical agents
(2-naftilamine, benzidine, 4-aminobiphenile), as well as with
a more widespread cigarette smoking habit.
Cigarette smoking increases by two to five times the risk associated with bladder carcinoma; those who quit reduce such
risk by 30-60%.
Other risk factors associated with this type of carcinoma are
recurring infections; the genetic-hereditary risk causes, on
the contrary, play only a marginal role. From a histology
viewpoint, in Europe and North America, bladder carcinomas
are transitional in 90-95% of cases, in 3% of cases squamous,
while 2% are adenocarcinomas and other histotypes are less
than 1%. Out of the total transitional carcinoma cases, 70%
are superficial and about 30% invasive. Transitional carcinomas present a high risk of relapse; five-year survival rates
are closely correlated to staging; they range from 85-65%
for stage 0-I to 14% in the case of metastatic carcinomas. In
superficial bladder carcinomas, the histological grading provides important clues as to how the disease is progressing: low
grades show a 4-5% progression, while in high-grade cases
the figure reaches 39%. Good survival rates and the subsequent follow-up mean that the citological urine examination
– in spite of its limitations – is important both for the initial
diagnosis of a urinary system pathology, and for the follow-up
of oncological patients.
Bladder carcinomas may be asymptomatic, but in 80% of cases they are associated with haematuria:persistant macrohaematuria or microhaematuria, therefore, are the most frequent
indicators of the need for a cytological test. In Europe the
highest incidence of bladder carcinoma (Clinical guidelines
2005 Boccardo and Silvestrini CNR-MIUR) was reported in
Italy, with 14,000 new cases among men and 3,000 among
women, followed by Spain and Switzerland; the highest mortality rates, on the other hand, are reported in Denmark,Spain,
Poland and Malta. According to the Mortality Register of the
Emilia Romagna Region (1998-2004) this type of tumour
causes about five hundreds deaths a year, of which just over
one hundred are women and almost four hundred men. The
relative risk, in the Municipality of Bologna only, is > 1.3
and the distribution pattern is extremely uniform, especially
among males. This tumour seldom appears before the age of
forty; this is why the reorganisation proposed for the province
of Bologna mainly involves citizens over sixty, who account
for more than 30% of the resident population.
Methods. Health care for citizens in the Bologna provincial
area is guaranteed by the Bologna and Imola local health
units. The provincial user base on 31/12/2008 consisted of
1,105,764 people; nine hundred thousand of them refer to the
Bologna Local Health Unit which includes fifty municipalities (Imola covers ten) and is one of the largest health trusts
in Italy.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
In total there are twelve hospitals, one health hub, nine accredited clinics and seven districts; this means that any action or
change regarding the system requires substantial organisation
work. The privatization of health trusts and the complexity
of the established facilities mean that it is becoming increasing urgent to plan, streamline and control the health services
rendered. Within this framework and in order to meet these
requirements, a reorganisation process has been started as regards urinary cytology in the whole province of Bologna, involving all stakeholders in the process, first and foremost the
Pathological Anatomy and Unified Booking (CUP) services.
The project included all four Pathological Anatomy departments dealing with this type of test: in Bologna these are the
Ospedale Maggiore, the Ospedale Bellaria and the S. Orsola
Hospital; in Imola it is the Pathological Anatomy department
of the Imola Hospital. The project started with an assessment
of the various process phases.
The analysis revealed differences both regarding the sampling
indications and the way the test was booked through the CUP;
on the other hand, consistent features emerged both as regards
the test’s setting-up, a single filtering layer with polycarbonate membranes, and the waiting times which could be as long
as 60 days. The old method required delivery of a fresh urine
sample for three days; this means that the patients had to go
through five “steps” before getting the result (one booking
from the CUP, three deliveries to the sample delivery Point,
one report collection from the office in charge), and that the
Pathological Anatomy departments needed to receive and
process the three samples separately and then draft the reports
on different days. The new method was introduced in April
2009; it involves the use of an alcohol-based fixative liquid to
correctly preserve the cellular elements in the sample which
can be stored in a cool place for up to a week. The appropriate amount of fixative is stored in the three jars contained in
the sponge housings which are part of the Kit provided by the
manufacturer. The Kits are delivered both to CUP offices and
to the chemists’ authorised to book these tests (about three
hundred booking points). The Kit is delivered to the patient
when the test is booked, together with a fact sheet explaining
both the precautions and sampling method to be followed,
plus the simplified questionnaire on the patient’s medical history which he/she has to fill in. This information is necessary
in order to reconstruct a clinical record of patients who, in the
case of the Bologna area, can choose between three different
Pathological Anatomy departments which are not part of a
network.
Results. The patient returns the Kit, all of it at the same time,
after having collected the urine at home for three days, following the method illustrated; an efficient transport system connects the delivery point to the Pathological Anatomy service
in charge of the area, making sure that the material is promptly
delivered. Here the material is all received together, which
means that the Service secretariat has less work to do in the
process; the setting-up involves completely filtering the three
samples, all together, for each patient, then collecting the cells
onto a single slide. As a consequence the process is less timeconsuming for the professionals involved, and it also allows
for the production of a single report. The patients collect their
reports on the set date from the booking structure; as a whole
their number of visits to public facilities when a urine cytology examination is required have been reduced to three (one
booking from the CUP, one journey to the delivery point, and
one to collect the report). The delivery point staff has reduced
the amount of time necessary for each user because there is
only one delivery to be received, as opposed to three for the
same number of days. The new process is proving advantageous for everybody, with the exception of the laboratory staff
in charge of setting up the material because the filtration time
has increased. In any case, the implementation of the system,
after the necessary trial period, has led to easier accessibility to
the test, and at the same time to a reduction of the waiting time
required. The waiting times for a urine cytology exam now
range between 3 and 15 days. Moreover, an agreement with
the CUP2000 company, allows the Pathological Anatomy services to directly manage the booking schedules. A statistical
report, provided on a regular basis by the CUP booking office
management, makes it possible for the Pathological Anatomy
departments to monitor the relevant trends, thus extending or
reducing the booking schedules in order to meet the demand.
The new method has been extended to hospital wards and
homogeneously covers the whole provincial area.
Emerging prognostic and predictive factors
in breast cancer: where are we?
M. Mottolese, A. Di Benedetto, E. Melucci, S. Buglioni,
L. Perracchio.
Pathology Department, Regina Elena National Cancer Institute,
Rome, Italy
Background. Breast cancer (BC) is the most commonly
occurring malignancy in women and is responsible for approximately 500 000 deaths per year worldwide. Due to the
remarkable heterogeneity of BC, mostly driven by genetic
variability, a number of clinical and bio-pathological factors
are routinely used to determine prognostic predictions of
clinical relevance. Furthermore, decisions about the adjuvant
chemotherapy (CT), mainly in early stage of the disease, are
affected by a complex interplay of factors and guidelines
stratify BC patients into prognostic subsets suggesting treatment protocols on the basis of the reported estimates of efficacy 1 2. Classical prognostic parameters include patient age,
axillary lymph node status, tumor size, histological features
(especially histological grade and lymphovascular invasion),
estrogen receptor (ER)-progesterone receptor (PgR), and
HER2 status. In addition, a recent report from the St Gallen
International Expert Consensus recommends the use of proliferation markers (eg, Ki-67 and mitosis) and multigene assays
when choosing appropriate systemic CT 3. Although these factors may be of great clinical value, their role in determining
prognosis and evaluating risk in an individual patient with BC
is more limited, since patients with similar combinations of
features may have very different clinical outcomes. In recent
years gene expression profiling have been increasingly used
aimed to improve BC classification and to assess prognosis
and response to therapy 4. Although the precise role of these
novel molecular techniques in the routine management of BC
patients is yet under investigation, certainly they may provide
prognostic and predictive information often more useful than
those provided by the traditional clinical and pathological
factors 5. In addition, thanks to this extended biological knowledge, we have the opportunity of identifying genes involved
in responsiveness to therapy acquiring relevant information
on drug resistance mechanisms. This may lead to the characterization of new therapeutic targets and the subsequent availability of more treatment options for patients with resistant
disease 6.
HER2 Expression and Response toTrastuzumab and
Chemotherapy. It is well established that expression of
HER-2 is predictive of response to trastuzumab 7. Retrospec-
Lectures
tive analysis of several adjuvant randomized studies indicated
that HER-2 overexpression is associated with greater benefit
from adjuvant anthracycline (AC)-containing regimens than
from cyclophosphamide, methotrexate, and 5-fluorouracil
(CMF)-type regimens 8. Furthermore, the addition of paclitaxel to AC-based CT with AC-based chemotherapy alone
also showed that the benefit from inclusion of paclitaxel was
largely restricted to HER-2–overexpressing tumors 9. Based
on the available evidence, the American Society for Clinical
Oncology Expert Panel on Tumor Markers in BC concluded
that high levels of HER-2 expression may identify patients
that will particularly benefit from AC-based adjuvant therapy,
although HER2 negativity should not be used alone to exclude
patients from this treatment 10.
Topoisomerase (TOP2A) Expression and Response to
Anthracyclines. TOP2A, an essential component of the cell
division machinery, is the molecular target of AC and high
levels of the enzyme cause the formation of large amounts of
AC:TOP2A complexes within the nucleus. In a retrospective
analysis of two randomized studies, TOP2A amplification
was a significant predictive factor for greater benefit from cyclophosphamide, epirubicin, and 5-fluorouracil therapy than
from CMF 11. These results were confirmed in a population of
patients with metastatic BC who participated in a randomized
clinical trial of AC-based CT with or without trastuzumab 12.
The FDA recently approved a TOP2A fluorescence in situ
hybridization (FISH) assay (TOP2A FISH pharmDx™; Dako,
Glostrup, Denmark) to measure amplification of this gene in
BC specimens.
Molecular Classification. Microarray studies, using an intrinsic gene set, have now shown that differences in gene
expression can account for much of the diversity in BC 4 13 14.
The largest difference in overall gene expression profile is
observed between tumors that were ER positive or negative. ER negative tumors are further sub-divided into HER2
positive and negative 4. Therefore, hierarchical clustering of
microarray data classified BC into four main groups: Luminal
(LA, LB), HER2 and Basal-like/Triple negative subtypes.
Luminal-type cancers are mostly ER positive, and patients
with LA BC have the most favorable long-term survival (with
endocrine therapy) compared with the other types, whereas
HER-2–positive tumors are more sensitive to CT associated
to anti HER2 trastuzumab therapy 15.
Gene expression profiling and prognosis. Gene-expression
profiling has shown promise to distinguish between patients at
low and high risk for developing distant metastases and identify those who are likely to benefit from adjuvant therapy 16.
The Rotterdam gene set, one of the prognostic genetic tests
now commercially available, is a single 76-gene prognostic
signature, able to predict distant metastatic recurrence with a
sensitivity of 93% and a specificity of 48% 17. The gene signature known as wound response indicator (WRI) identifies
BC patients with a significant shorter overall and disease free
survival than patients whose tumors did not express this gene
signature 18. The oncotype DX™ is a 21-gene indicator which,
through an algorithm based on the expression levels of these
genes, allows a Recurrence Score™ (RS) to be computed for
each specimen correlated with the rate of distant recurrence at
10 years. The assay uses fixed tumor specimens, rather than
frozen tissue 19. The MammaPrint-70-gene profile was developed from patients with lymph-node negative £55 years of
155
age BC. The assay uses frozen tumor specimens and separates
patients developing distant metastases from those disease free
within 5 years. The internal and external validation of this
gene set led to the clearance of this test by the U.S. Food and
Drug Administration (FDA), allowing the test to be marketed
as a prognostic marker to be used with other clinicopathologic
factors 20.
Conclusions. Current BC treatment guidelines are based on
clinical trial evidence obtained in defined patient populations,
and treatment algorithms are developed by relating clinical
trial findings to specific patient subgroups. Nevertheless, better prognostic and, in particular, predictive factors are needed
to assist in treatment decision-making on an individual patient
basis. Considering prognostic markers, gene profiling seems
promising, although further validation, particularly with
respect to potential ‘predictive interactions’, is mandatory.
For predictive testing, emerging evidence suggests TOP2A
amplification or deletions may be appropriate factors for
selecting patients for AC dosing. Gene expression profiling
may become important as a tool to define predictive factors;
however, more accurate statistical approaches able to analyze
gene expression profiles, based on functional hypotheses
about gene networks, will be essential. Finally, the recent
discovery of a class of small noncoding endogenous RNA
molecules, namely microRNA, which are frequently dysregulated in cancer has uncovered an entirely new repertoire of
molecular factors upstream of gene expression. The potential
role of miRNAs in BC management, particularly in improving
current prognostic tools and achieving the goal of individualized cancer treatment is under investigation 21.
References
1
Early Breast Cancer Trialists’ Collaborative Group. Lancet
2005;365:1687-717.
2
Lonning PE, Knappskog S, Staalesen V, et al. Ann Oncol 2007;18:1293306.
3
Goldhirsch A, Ingle JN, Gelber RD, et al. Ann Oncol 2009;20:131929.
4
Sorlie T, Perou CM, Tibshirani R, et al. PNAS 2001;98:10869-74.
5
Parker JS, Mullins M, Cheang MC, et al. J Clin Oncol 2009;27:11607.
6
Geyer FC, Reis-Filho JS. Int J Surg Pathol 2009;17:285-302.
7
Ménard S, Balsari A, Tagliabue E, et al. Ann Oncol 2008;19:170612.
8
Pritchard KI, Shepherd LE, O’Malley FP et al. N Engl J Med
2006;354:2103-11.
9
Hayes DF, Thor AD, Dressler LG, et al. N Engl J Med 2007;357:1496506.
10
Harris L, Fritsche H, Mennel R, et al. J Clin Oncol 2007;25:5287312.
11
Knoop AS, Knudsen H, Balslev E, et al. J Clin Oncol 2005;23:748390.
12
Press MF, Sauter G, Buyse M, et al. J Clin Oncol 2007;25(suppl
18S):524.
13
Lonning PE, Sorlie T, Borresen-Dale AL. Nat Clin Pract Oncol
2005;2:26-33.
14
Sotiriou C, Neo SY, McShane LM, et al. PNAS 2003;100:10393-8.
15
Schnitt SJ. Mod Pathol 2010;23:S60-4.
16
van de Vijver M. The Oncologist 2005;10(Suppl 2):30-4.
17
Wang Y, Klijn JG, Zhang Y, et al. Lancet 2005;365:671-9.
18
Chang HY, Sneddon JB, Alizadeh AA, et al. PLoS Biol 2004;2:E7.
19
Paik S, Tang G, Shak S, et al. J Clin Oncol 2006;24:3726-34.
20
van’t Veer LJ, Dai H, van de Vijver MJ, et al. Nature 2002;415:5306.
21
Heneghan HM, Miller N, Lowery AJ, et al. J Oncol 2009;doi:10.1155
/2010/950201
156
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
SIAPEC-IAP meets the Adriatic Society of Pathology. 25th year
Moderators: M. Del Vecchio (Ascoli Piceno), V. Pisac Presutic (Spalato)
Back to histological subtyping of nsclc in the
era of personalized treatments
M. Papotti, L. Righi, M Volante
Department of Clinical and Biological Sciences, University of Turin
at San Luigi Hospital, Orbassano (Torino), Italy
Background. Lung cancer classification was devised to
work especially on surgical specimens and recognizes four
major histological subtypes, namely squamous cell carcinoma
(SQC), adenocarcinoma (ADC), large cell (LCC) and small
cell carcinomas (SCLC). Such classification is more difficult
to apply on cytological samples or small biopsies, especially
in the presence of poorly differentiated tumors or of limiting
factors, including tumor heterogeneity, extent of necrosis,
limited number of viable cells, marked artifacts. Since in past
years all non-small cell lung cancers (NSCLC), were generally treated with similar chemotherapy regimens, irrespective
of the histotype, as opposed to SCLC, accurate lung cancer
subtyping became less relevant for clinical purposes, a fact
that lead pathologists to concentrate their efforts on the correct recognition of SCLC, only. As a matter of fact, on cytological or small biopsy samples, most pathologists are able
to correctly differentiate SCLC from NSCLC, and within the
NSCLC group to identify well- or moderately-differentiated
SQC or ADC. However, a high percentage of cases are still
simply diagnosed as NSCLC, as they are poorly differentiated
tumors, lacking clear-cut morphologic signs of differentiation.
In recent years, the advent of targeted therapies and novel
chemotherapeutic agents showing differential efficacy or toxicity on specific NSCLC subtypes required a sudden call back
to histological subtyping, which is becoming the milestone
for personalized therapy (especially for unoperable patients,
whose treatment will eventually be based on the biopsy diagnosis, only).
A useful, rapid and cheap tool to identify squamous or glandular differentiation and characterize poorly differentiated
NSCLC could be immunohistochemistry. Although according
to the WHO classification “…classification is largely based
on standard hematoxylin & eosin sections…”, the immunophenotypic profile may provide information in terms of probability level that a given neoplasm has squamous or glandular
differentiation. This latter information may be of predictive
value, assisting the clinician in selecting the most appropriate
treatment for advanced NSCLC affected patients.
Lung cancer histological subtypes that are morphologically recognizable on small biopsy fragments or cytological samples are
basically three, i.e. ADC, SQC and SCLC. Other tumor types
of lung carcinomas such as large-cell carcinoma (LCC) and
its variants (eg large-cell neuroendocrine carcinoma, etc), or
sarcomatoid carcinomas can be definitely diagnosed on surgical
specimens, only. However, ADC variants cannot always be easily identified, with special reference to non invasive subtypes
(former bronchiolo-alveolar carcinoma, now Adenocarcinoma
in situ/minimally invasive adenocarcinoma), in the absence of
the whole tumor specimen available for thorough examination.
Molecular tests may be applied also in biopsy material and may
help to refine the diagnosis, since some correlations were observed between molecular profile and histological subtype (eg
EGFR mutations in BAC or mixed or papillary ADC, or K-RAS
mutations in mucinous ADC).
As stated, immunohistochemistry is also very helpful to identify the three most frequent lung tumor phenotypes: glandular,
squamous and neuroendocrine. The group of large cell carcinomas frequently has an heterogeneous immunohistochemical
profile, probably reflecting divergent differentiation mainly
along squamous and glandular lineages. Adenocarcinomas are
generally positive for TTF-1, surfactant apo-protein A (SPA),
napsin-A, and cytokeratin 7 (CK7) and negative for CK5/6,
CK20 and p63 (an exception is mucinous adenocarcinoma,
which expresses CK20 rather than TTF-1 or SPA). Squamous
cell carcinoma consistently and strongly react with p63, CK5
and desmocollin-3, and virtually never for TTF-1. Neuroendocrine large and small cell carcinomas are known to express
chromogranin A, synaptophysin and CD56, among others.
In the daily practice a panel of immunohistochemical markers is generally employed, based on availability of reagents
and the pathologist’s personal experience. The most widely
applied panel for NSCLC sub-classification includes TTF1, p63, CK7 and CK5. The former two are nuclear markers
and seem more reliable in poorly cellular samples. In such
cases, cocktails of antibodies can also be used, to reduce the
number of necessary glass slides (for example p63+CK5 vs
TTF1+CK7). With regard to the interpretation of results,
TTF-1 is virtually never expressed in SQC, but stains only
70-80% of ADC (depending on tumor grade and the presence
of mucinous features). By contrast, p63 expression in SQC is
robust and not influenced by tumor grade, although p63 immunoreactivity has been observed in a small subset of ADC
(p40 seems a more squamous carcinoma specific marker, in
this respect). Finally, in the presence of ambiguous phenotypes or discrepant marker reactivity, additional antibodies
may be used. Promising results were obtained with napsinA, MUC5AC or desmocollin-3 in discriminating pulmonary
ADC from SQC.
Once a morphological and/or immunohistochemistry-assisted accurate lung cancer subtyping has been obtained, the
final step of pathological characterisation of lung tumors
is their molecular profile. This can be optimally defined in
surgical specimens, but can be assessed in small cytological
or biopsy samples, too. EGFR or K-ras mutational status is
the most common requirement for personalizing treatments,
but new targets are emerging for specific drugs including
c-met mutations, ALK fusion products and the levels of
specific enzymes, such as ERCC1, thymidilate synthase or
topoisomerase II.
Conclusions. 1) An accurate histological subtyping of so
called “NSCLC” may further improve the efficacy or reduce
the toxicity associated to novel therapeutic options; 2) although lung cancer diagnosis is generally based on haematoxylin/eosin-stain, immunohistochemistry can be helpful, if not
mandatory, in defining the histotype (or the most likely differentiation lineage) of poorly differentiated tumors; 3) TTF-1 &
CK7 and p63 & CK5 seem to date the most valuable markers
for ADC and SQC, respectively; 4) novel diagnostic markers
may allow to abandon the “NSCLC” category, thus reducing
as much as possible the number of unclassified cases.
Lectures
Mutation analyses of KRAS in colorectal cancer
and egfr in non-small cell lung cancer: a task
for pathologists?
M. Dietel
Institute of Pathology, Charité Universitätsmedizin Berlin, Germany
Background. Colorectal cancer (CRC) and non-small cell
lung carcinoma (NSCLC) are the two most common malignancy in the western world with estimated 350,000 new cases
reported for the United States in 2008 1. Since both tumors
are being predominantly diagnosed at advanced stage, the option of a curative therapy then does no longer exist in many
instances and chemotherapy is often the treatment of choice.
However, conventional anti-cancer drugs have been shown
to be of limited value accompanied by strong side effects.
This situation was the driving force to search for new more
specific drugs.
The membrane bound epidermal growth factor receptor
(EGFR1) molecule was found to be of major importance in
growth stimulation und thus was identified as a possible target
which inhibition may lead to reduced tumor growth. Meanwhile there exist two types of EGFR-inhibitors which are
approved for clinical application, i.e. the therapeutical antiEGFR antibodies cetuximab (Erbitux®)) and Panitumumab
(Vectibix®) as well as the tyrosine kinase inhibitors (TKI)
erlotinib (Tarceva®)) and gefitinib (Iressa®). In several clinical
studies it became obvious that not all tumors respond equally
but that certain genetic characteristics are the prerequisite to
clinical benefit. This was the background to link the application of the drugs to pre-therapeutic eligibility tests and that
in Europe KRAS mutation testing as well as EGFR mutation
testing became a prerequisite for anti-epidermal growth factor
receptor therapy of metastatic colorectal cancer since the end
of 2007 2 3 and metastatic non-small cell lung cancer (NSCLC)
in 2009, respectively.
Since the analyses have to be performed using carefully selected tumor tissue the tests should be done only in Institutes
of Pathology where pathologists are able to check istology,
select the tumor tissue adequate for molecular testing and sign
out a combined morphological/molecular report. In addition
each institute should participate in interdisciplinary ring trials
(round robin tests) which should be lead by an independent
organisation. For that purpose the German Society of Pathology (DGP) and the German Association of Pathologist have
(BDP) created the QuiP (Quality in Pathology) – initiative
which organizes interlaboratory tests and confers the respected certification. Only if a reliable morphological diagnosis
is combined with a solid genetic analysis a robust basis for
targeted therapy is given.
Methods. A multitude of procedures and methods are available for detecting KRAS/EGFR mutations in tumor samples
(Weichert 4-11). In the Berlin Institute the following selection
of techniques has been found useful, reliable and applicable
for routine molecular pathology.
Tissue selection. As shown in several studies 12 13 a precise selection of the tissue to be analysed is mandatory. This should
be done by an experienced pathologist indicating the area on
H&E-stained slides estimating the percentage of tumor in relation to the whole tissue section. Subsequently a manual microdissection has to be done by the technician. Tissue samples
can be stored for years prior to molecular analyses.
DNA preparation. For DNA preparation the three unstained
slides were used. The putative tumor areas corresponding to
157
the marks on the H&E slide were microdissected. DNA preparation was done as described previously 14. In brief, microdisseceted tissue was transferred to 180 µl ATL-buffer (Qiagen,
Hilden, Germany) and kept for 10 min at 95°C. After cooling
down to room temperature, 20 µl of proteinase K solution
were added. After gentle mixing, the sample was incubated at
55°C until complete lysis (after about 2 h). The further steps
of isolation of DNA follow the protocol “Tissue ProtocolQIAamp DNA Mini Kit” (Qiagen). The nucleic acids were
eluted at a volume of 60-100 µl and DNA content was estimated with a Nanodrop 1000 (PeqLab, Erlangen Germany).
For sequence analyses the techniques of Sanger sequencing,
pyro-sequencing, chip analyses and melting curve analyses
were applied (technical details see Weichert et al. 15).
Results. Institute of Pathology, Berlin. For the analysis of
the somatic KRAS genotype of 263 patients we used Sangersequencing, pyrosequencing, melting curve analysis and chip
hybridization in parallel. We used Sanger sequencing as the
reference method. For all cases DNA of sufficient quality was
prepared. Overall mean (± SD) DNA yield was 196.8 ng/µl
(± 142.5 ng/µl). Array analysis, melting curve analysis and
pyrosequencing, using DNA from the same preparation, was
performed in 233, 188 and 136 cases, respectively.
Using Sanger sequencing 108 out of 260 cases (41.5%) were
found to have a mutation in either codon 12 (31.9%) or codon
13 (9.6%) of the KRAS gene. The most frequent mutations
were p.G12D (12.7%), p.G12V (10.8%) and p.G13D (9.2%).
Similar distributions were seen with the other three methods.
The array analysis identified 104 out of 223 cases (44.6%) to
harbor somatic mutations, melting curve analysis found 77
out of 188 cases (41%) to be mutated, and by pyrosequencing
51 out of 136 cases (37.5%) were reported to carry mutations.
A crossover comparison of the four methods yielded k values
exceeding 0.9 (for more details see 15).
Analogue test comparisons were done for EGFR mutation
analyses using tissue from NSCLC after surgical tumor resection revealing similar results.
QuiP-Initiative. Detailed descriptions of the results regarding
round robin tests for KRAS- and EGFR-testing are published
elsewhere. In summary, around Germany currently there exist
over 60 Institutes of Pathology certified for KRAS testing and
53 certified for EGFR testing (for details visit the home page
of the DGP 17).
Discussion. Somatic gain-of-function mutations in the KRAS
gene of CRCs predict the lack of response to anti-EGFR therapy with cetuximab and panitumumab, and KRAS mutational
screening prior to treatment with either drug has become mandatory in Europe since the end of 2007. A similar situation
become relevant in 2009 when clinical approval of gefitinib
was limited to “tumours that have tested positive for EGFR
with an activating mutation” (EMEA 2009;2,3). This resulted
in a fast growing completely new branch of routine predictive
diagnostic molecular pathology.
The pre-therapeutical analyses which guide patients’ therapy
are chance and challenge for pathologists as a new personal
responsibility is given. To fulfil this following points have to
be considered:
1.The test has to be done only in certified Institutes of Pathology, e.g. with a QuiP-certicate, for details see ref. 17.
2.The responsible pathologists should be experienced in morphology and molecular testing.
3.The responsible pathologists should:
• re-confirm the histological diagnosis on an H&E slide
and
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
• he should identify and mark the tumor area for enrichment of tumor cells.
4.This is followed by manual microdissection to assure that
at least 40% of the material for the molecular analysis is
indeed tumor tissue.
5.The selected tumor tissue then should be analyzed following the procedure and recommendations described above
and by others.
6.Finally the responsible pathologists should prepare a combined report giving details on the histology and the molecular result.
If these criteria are met molecular pathology is facing an excellent future coming closer to clinical decisions and thus to
the patients.
References
1
Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J
Clin. 2008;58:71-96.
2
http://www.emea.europa.eu/humandocs/Humans/EPAR/erbitux/erbitux.htm
3
http://www.emea.europa.eu/humandocs/Humans/EPAR/vectibix/
vectibix.htm
4
Simi L, Pratesi N, Vignoli M, et al. High-resolution melting analysis
for rapid detection of KRAS, BRAF, and PIK3CA gene mutations in
colorectal cancer. Am J Clin Pathol 2008;130(2):247-53.
5
Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing
method for KRAS mutation detection by Pyrosequencing. J Mol Diagn.
2005;7:413-21.
6
Clayton SJ, Scott FM, Walker J, et al. K-ras point mutation detection
in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification. Clin Chem
2000;46:1929-38.
Lilleberg SL, Durocher J, Sanders C, et al. High sensitivity scanning
of colorectal tumors and matched plasma DNA for mutations in APC,
TP53, K-RAS, and BRAF genes with a novel DHPLC fluorescence
detection platform. Ann NY Acad Sci 2004;1022:250-6.
8
Rothschild CB, Brewer CS, Loggie B, et al. Detection of colorectal
cancer K-ras mutations using a simplified oligonucleotide ligation
assay. J Immunol Methods 1997;206:11-9.
9
Emanuel JR, Damico C, Ahn S, et al. Highly sensitive nonradioactive
single-strand conformational polymorphism: detection of Ki-ras mutations. Diagn Mol Pathol 1996;5:260-4.
10
Keohavong P, Zhu D, Whiteside TL, et al. Detection of infrequent and
multiple K-ras mutations in human tumors and tumor-adjacent tissues.
Anal Biochem 1997;247:394-403.
11
Ward R, Hawkins N, O’Grady R, et al. Restriction endonucleasemediated selective polymerase chain reaction: a novel assay for the
detection of K-ras mutations in clinical samples. Am J Pathol 1998,
153:373-9.
12
Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for
panitumumab efficacy in patients with metastatic colorectal cancer. J
Clin Oncol 2008;26(10):1626-34.
13
Lièvre A, Bachet JB, Boige V, et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer
treated with cetuximab. J Clin Oncol 2008;26(3):374-9.
14
Petersen I, Schewe C, Schlüns K, et al. Inter-laboratory validation of
PCR-based HPV detection in pathology specimens. Virchows Arch
2007;451:701-16.
15
Weichert W, Schewe C, Lehmann A, et al. KRAS Genotyping of
Paraffin-Embedded Colorectal Cancer Tissue in Routine: Diagnostics Comparison of Methods and Impact of Histology. J Mol Diagn
2010;12:35-42.
16
Neumann J, Zeindl-Eberhart E, Kirchner T, et al. Frequency and
type of KRAS mutations in routine diagnostic analysis of metastatic
colorectal cancer. Pathol Res Pract. 2009;205(12):858-62.
17
http://www.dgp-berlin.de
7
Transplantation pathology
Moderators: F.W. Grigioni (Bologna), M. Rugge (Padova)
The role of the pathologist in the assessment of
kidney adequacy
G. Monga, G. Mazzucco *
Dipartimento di Scienze Mediche. Facoltà di Medicina e Chirurgia.
Università del Piemonte Orientale, Amedeo Avogadro. Novara; * Dipartimento di Scienze Biomediche e Oncologia Umana. Facoltà di
Medicina e Chirurgia. Università di Torino
Every year, no more than 1/3-1/4 of patients awaiting kidney
transplant can receive the graft. This shortage of grafts has
led to an ever increasing use of kidneys from marginal deceased donors (subjects aged ≥ 55 years or < 55 years with
a history of hypertension and/or diabetes, or deceased after
a cerebrovascular incident). At present, pretransplant renal
biopsy (PTRB) is the most reliable method available to assess
the kidney state.
However, there are several problems connected to this diagnostic procedure:
1. Morphologic evaluation of fixed and paraffin embedded
samples vs frozen tissue. The former procedure is greatly
favoured. Indeed, the frozen sections technique allows for a
faster evaluation, offering briefer diagnostic times. However,
the price is paid by the quality of the material, which is less
satisfactory than that available after fixation and paraffin
embedding.
2. Semiquantitative vs morphometric evaluation of the morphologic changes. The latter procedure is more accurate, but
time consuming and, therefore, unsuitable in an emergency
diagnostic setting. It follows that semiquantitative evaluation
must be used.
3. Bioptical procedure: needle (NB) vs wedge (WB) biopsy.
Opinions as to the primacy are conflicting among both surgeons and pathologists. WB offers larger amounts of tissue,
but, according to several authors, increases the risk of an overestimation of glomerular sclerosis (GS) and interstitial fibrosis
and allows for only limited sampling of the deeper renal tissue
where larger arteries are present.
4. The choice of the morphological parameters for the grading of the kidney damage. Global GS alone has been used, but
opinions on this procedure are conflicting. At present, besides
GS, three other parameters (interstitial fibrosis, tubular atrophy and vascular arterio-arteriolosclerotic damage) are usually included in different scoring systems.
PTRB was considered useful in this setting in the prediction of
short- and long-term graft outcome, in supplying a reference
frame in the interpretation of subsequent graft biopsies and
mandatory in the assessment of kidney adequacy for single
and/or double transplant or its being discarded 1.
Since PTRB is justified on the assumption that it represents
the real state of the whole kidney, it follows that the critical
issue is its reliability, i.e. how accurately it represents the true
histology of the whole kidney. This question prompted a study
which has already been published 2 dealing with a comparative
evaluation, according to the Karpinski et al. scoring system 3
159
Lectures
of 154 PTRB (118 NB and 36 WB) and the matched 154 kidneys, subsequently untransplanted for different reasons and
used as gold standard.
The concordance between the total score at biopsy and
that on the kidney was fairly good, with higher values for
NB (k 0.73) than for WB (k 0.57). When the individual
parameters (global GS, interstitial fibrosis, tubular atrophy
and vascular damage) were considered, agreement between
biopsy and matched kidneys was found to be low for GS,
mainly in the NB (k 0.18), intermediate for tubular atrophy
and interstitial fibrosis and high for vascular changes (k 0.75
and 0.74 for NB and WB respectively). It is worth stressing
that agreement for each parameter (including GS) consistently increased when the biopsy contained more tissue. If
the biopsies were subdivided into three categories, according
to the number of the glomeruli (A: 6-10 glomeruli; B: 11-24
glomeruli; C ≤ 25 glomeruli) considered as the index of the
tissue amount, reliable results were achieved in those with
more than 11 glomeruli.
As to the judgement of the fitness for the transplant of the kid-
neys, biopsy and whole kidney evaluation agreed in 100/118
cases of the NB group and in 29/36 cases of the WB group.
Conclusive remarks
1. PTRB supplies reliable information as to the actual kidney
state, with slightly better results with NB than with WB.
2. Although the biopsy size does play a role, samples with
over 10 glomeruli suffice for a satisfactory evaluation.
3. Vascular damage is the most faithful single parameter,
whereas it is not the case of global GS, the estimation of
which requires some caution. A multiparametric evaluation is
strongly recommended.
References
1
Remuzzi G, Ruggenenti P. Renal transplantation: single or dual for
donors aging = 60 years? Analyses & Commentaries. Transplantation
2000;69:2000-4.
2
Mazzucco G, Magnani C, Fortunato M, et al. Nephrology, Dialysis
and Transplantation (in press).
3
Karpinski J, Lajoie G, Cattran D, et al. Outcome of kidney transplantation from high-risk donors is determined by both structure and fuction.
Transplantation 1999;67:1162-7.
Pharmacogenetics in cancer: transfer of translational research
into clinical practice
Moderators: A. Scarpa (Verona), P.P. Piccaluga (Bologna)
Pharmacogenetics in cancer care: transferring
translational research into clinical practice
G. Toffoli
Director Experimental and Clinical Pharmacology Unit, CRO National Cancer Institute (Aviano)
Antitumoral drugs are characterized by a low therapeutic
index, with a modest difference between toxic and efficient
dose: this could determine in some individuals a number of
toxic effects, to which a real benefit in terms of antitumoral
activity does not always correspond. These compounds are
also characterized by a sometimes very evident interindividual
variability, and the same drug can determine in different individuals a different toxic effect or a different antitumoral
activity. It is then important to personalize the antitumoral
activity, that is to give each patient the right drug at the right
dose. Therapy personalization represents one of the most
important future challenges in the therapy of oncological
patients. On this ground it is crucial to identify the biomolecular specificities of the neoplastic cells towards which
the antitumoral drugs must be vehiculated, or to identify the
genetic specificities of the patient that can explain the reason
for the differential effect of antitumoral drugs. Pharmacogenetics, the study of the genetic basis of the interindividual
differences in response to drugs results from the intersection
between genetics and pharmacology is a notably interesting
field for the possible implications in the clinical practice.
Genetic polymorphisms, that is the structural alterations of
genes involved in the metabolism, transport or drug interaction with the cellular target, have been described both for the
drugs traditionally employed in the oncological patient, and
for the so-called targeted molecules recently employed in
clinic. Pharmacogenetics investigates the individual genetic
specificities which are relatively common among the population and that are involved in antitumoral drugs action. The
most common form of genetic polymorphism is represented
by the SNPs (single nucleotide polymorphisms). Other forms
of polymorphisms are represented by abnormal nucleotide sequence repetitions (microsatellites or midisatellites), genomic
duplications, pseudogenes etc. At present, more than 100 million SNPs of human genome have been described: it is then
important to individuate those which have an effective impact
on drug action, modulating their pharmacogenetics and pharmacogenomics (toxicity and response) and that can influence
the ultimate result of the antitumoral therapy, influencing the
patients survival. Sometimes the pharmacologic effect cannot
be explained by the action of a single polymorphism. The
complex metabolic or intercellular transport ways that often
characterize antitumoral drugs require the involvement of
more genes and only the precise definition of polymorphisms
of all these genes results of some utility for prognosis. In the
study of interactions between genome and drug, we can aim
at a single polymorphism, applying in this case a pharmacogenetic approach or we can analyze the combined effect of
more polymorphisms, employing in that case a pharmacogenomic approach. Basically, the strategies adopted at present
in pharmacogenetics/omic studies are three: “candidate gene
approach”, “gene pathway approach” and “genome wide
analyses”. In the so-called “candidate gene approach” the
polymorphisms of a single gene considered critical for the
action of the drug are analyzed. This strategy presupposes
that the action of the gene in question is actually important
for the pharmacologic action and that the polymorphism or
the polymorphisms of interest have an effective prognostic
power in the clinical practice. It is also important to establish
what is the combined effect of the polymorphisms present
in a single gene and the modality of their distribution, that
is if the contemporary presence of more polymorphisms in a
single gene is casual or not (linkage disequilibrium). At present, the kits recommended by Food and Drug Administration
160
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
(FDA) in the oncological field for single polymorphisms are
relatively few (UGT1A1*28 (irinotecan) (invader assay) and
TPMT (6-MP, azatioprine) (pro-Predict Py), nevertheless, the
data recently produced in literature are encouraging for further
clinical development.
In the so-called “gene pathway approach”, the polymorphism
of the genes involved in the complex metabolic ways of antitumoral drugs are analyzed. The rationale of this approach is
that the pharmacologic effect is a multifactorial event and that
the pleiotropic effect could depend on polymorphisms located
in different genes. In this context, it is important to define
for each antitumoral drug all polymorphisms with prognostic
potentiality, and the attention of pharmaceutical companies is
to design pharmacogenetic kits including all polymorphisms
that are relevant for the action of a specific drug.
The “genome wide analysis” represents an innovative strategy in the field of pharmacogenomics obtained by the recent
advances in the technological field. With this approach, the
entire human genome is analyzed, in an attempt to define
clusters of polymorphisms predictive of the pharmacological
effect. At present, this strategy is employed principally in the
research field, to individuate those polymorphisms whose real
meaning will be successively validated, but the possibility to
adopt this strategy in the clinical practice represents a stimulating perspective, analogous to what is being done with the
microarrays of gene expression in human tumors.
Gene polymorphisms that could influence the biochemistry of
the most common tumoral drugs have been described. Among
the polymorphisms of enzymes involved in the metabolism
of phase I of antitumoral compounds, the attention is pointed
at the polymorphic variants of cytochrome isoforms, in particular 3A4 and 3A5 (irinotecan, taxanes, alkilant agents).
For what concerns phase II enzymes, we particularly focus
on the polymorphisms in uridindifosfoglucoronosiltransferasi
and in particular on UGT1A1*28 polymorphism in the gene
that inactivates SN38, the active principle of irinotecan. We
have recently published (Toffoli et al. J. Clin. Oncol. 2010)
a phase I pharmacogenetic study to define the maximum
tolerated dose of irinotecan associated with FOLFIRI regimen based on patients genetic characteristics. The study has
demonstrated that the patients carrying the wild type genotype
(UGT1A1) can tolerate higher drug dose with respect to patients carrying UGT1A1*28 polymorphism. We observed an
increased response rate in patients receiving the higher dose
of irinotecan.
Other polymorphic variants of phase II enzymes concern the
isoforms of glutathione S-transferase gene (GST). Numerous
polymorphic variants of these genes that include allelic deletion or point mutations have been described. Recently, it was
found how polymorphic variants of isoforms of GST gene can
influence toxicity, and in particular neurotoxicity to chemotherapeutic regimens with oxaliplatin.
Numerous polymorphisms have been described in genes
involved in the action of fluoropyrimidines (thymidylate
sintetasi, methylenetetrahydrofolate reductase and dihydropyrimidine dehydrogenase). In particular, IVS 14 + 1G > A
variant dihydropyrimidine dehydrogenase can be associated
with severe toxicity, even lethal, by fluorouracil. This polymorphism is extremely rare in our population (< 1%) but it
should be attentively evaluated before starting a therapy with
fluoropyrimidines.
Finally, polymorphic variants have been described for the
genes involved in the transmembrane transport of antitumoral
drugs and in particular in MDR1 gene that codes for Pgp and
for other “multidrug resistant proteins” MRPs. The real im-
pact of these polymorphisms on toxicity and tumor response
as well as on drug pharmacokinetics of these transporters
needs further investigation.
In conclusion, pharmacogenetic/omics represents a potential
instrument for personalizing cancer therapy. This approach
seems extremely interesting to reduce toxic effects of antitumoral drugs and for increasing their efficacy. Nevertheless, it
is still necessary to validate this innovative perspective, both
in analytical terms (sensibility, specificity and reproducibility
of tests) and for what concerns the precise relations among
genotype, pharmacokinetics and pharmacodynamics (toxicity
and response). Finally, it is crucial to define the clinical utility
of this approach. In particular, if based on a pharmacogenetic
test the therapeutic regimen could be modified, what pharmacologic alternatives can be used and especially what will
be the effect of these modifications in terms of toxicity and
response to therapy.
Pharmacogenomics of brain tumors
L. Morandi, D. de Biase, G. Marucci, A Pession *, G Tallini
Dipartimento di Ematologia e Scienze Oncologiche, Sezione di Anatomia Istologia e Citologia Patologica “M. Malpighi” UniversitàASL Ospedale Bellaria; * Dipartimento di Patologia Sperimentale
Università di Bologna
Background. Epigenetic silencing of the MGMT gene by
promoter methylation is associated with loss of MGMT expression, diminished DNA-repair activity and longer overall
survival in patients with glioblastoma who, in addition to
radiotherapy, received alkylating chemotherapy with carmustine or temozolomide (TMZ) 1-4. The MGMT gene is located
on chromosome 10q26 and encodes a DNA-repair protein
that removes alkyl groups from the O6 position of guanine, an
important site of DNA alkylation. The restoration of the DNA
consumes the MGMT protein, which the cell must replenish.
Left unrepaired, chemotherapy-induced lesions, especially
O6-methylguanine, trigger cytotoxicity and apoptosis 4 5. High
levels of MGMT activity in cancer cells create a resistant phenotype by blunting the therapeutic effect of alkylating agents
and may be an important determinant of treatment failure 4-6.
Patients with glioblastoma containing a methylated MGMT
promoter showed a major benefit from temozolomide 3. Given
the key roles of cytosine methylation, there has been a wide
interest in the development of procedures for DNA methylation analyses 7-10. Here we present a novel methylation sensitive quantitative real time PCR assay (MS-qLNAPCR) which
permits high throughput quantification of the methylation
status of the MGMT promoter in an accurate, very sensitive
and cost-effective manner. High specificity was achieved
recognizing methylated and unmethylated CpGs by 3’-locked
nucleic acid (LNA) primers and molecular beacon probes. In
order to calculate the ratio between methylated and unmethylated MGMT allele, the CpG islands of SNURF were selected
as a reference gene. SNURF belongs to the 15q imprinted
center mapped on 15q12. The maternal allele is usually methylated, while the paternal one is unmethylated 11. In theory in
a homogeneous population of cells of the same individual,
the methylated maternal alleles should be balanced with the
unmethylated paternal alleles if the tumor cells did not acquire
any deletion for this locus or aberrant methylation of the paternal allele (loss of imprinting). This feature allows an easy
and precise calculation of the ratio between the methylated
and unmethylated alleles of MGMT following the method
described by Ginzinger et al. 12.
Lectures
Methods. 159 GBM patients followed prospectively between
April 2004 and October 2008 were included in this study.
After bisulfite treatment, methylated and unmethylated CpGs
were detected by previously described MS-PCR 2 3 and by
MS-qLNAPCR using LNA primers and molecular beacon
probes 23. SNURF was used as a reference for normalization
allowing calculation of the percentage of MGMT methylation
using the ∆∆Ct method 11 12. Two SNPs adjacent to MGMT
(rs8473; rs3740427) were used for loss of heterozygosity
(LOH) analysis. They were interrogated by allele specific
quantitative PCR using LNA at 3’- end of the discriminating
primer as previously described 13 14.
Results. Concordance between already described nested MSPCR and MS-qLNAPCR was found in 158 of 159 samples
(99.4%). MGMT promoter was found to be methylated using
MS-qLNAPCR in 70 patients (44.02%), and completely unmethylated in 89 samples (55.97%). Median overall survival
was of 24 months, being 20 months and 36 months, in patients
with MGMT unmethylated and methylated, respectively. Considering MGMT methylation data provided by MSqLNAPCR
as a binary variable, overall survival was different between
patients with GBM samples harboring MGMT promoter unmethylated and other patients with any percentage of MGMT
methylation (p = 0.003). This difference was retained using
other cut off values for MGMT methylation rate (i.e. 10%
and 20% of methylated allele), while the difference was lost
when 50% of MGMT methylated allele was used as cut-off.
LNA modified primers for mMGMT showed higher PCR
efficiency (slope: -3.271; efficiency: 102%,) than unmodified primers (slope: -4.339; efficiency: 69%). The analytical
detection limit of 0.01% was reached only for LNA modified
primers, while conventional primers showed a detection limit
of 0.1%. To the best of our knowledge, we are the first to
describe and validate a method to quantitate DNA methylation using LNA modified primers and an imprinted gene as
a reference, instead of a methylation independent calibrator
such as ACTB 15. In our opinion ACTB does not represent the
best reference gene for normalization because it is located at
7p15-p12, a chromosomal site subject to copy number variations in gliomas 16 17, and because it is close EGFR (located at
7p12) that is amplified in about 40% of GBMs 18. We use of
an imprinted gene (SNURF) as an internal control, because it
may check the efficiency of the assay from DNA purification,
through bisulfite treatment to PCR. Additionally it should be
used as a reference because mSNURF and uSNURF mimic the
biallelic MGMT status. In fact, in normal cells the maternal allele of SNURF is methylated at the promoter locus, while the
paternal allele of the same gene is usually unmethylated and
expressed 11. This condition is thus similar to a tumor population of cells in which the MGMT is methylated at one of the
two alleles. In order to consider SNURF as an ideal reference,
the ratio between methylated and unmethylated SNURF alleles might not be disturbed by copy number changes, or by
loss of imprinting, both of which are common in cancer. However, several references demonstrate that SNURF, which has
been mapped at 15q12, is hardly ever altered in gliomas 19 20.
These data were confirmed by our study because the methylated and unmethylated SNURF ∆Ct of the most part of cases
(91.2%) was nearly always very close to 0. The SNURF methylation values outside the normality range in 14 of the 159
GBMs (8.8%) may be due to a distinct CpG methylation pattern among tumor cell population, to partial loss of one allele
(LOI: loss of imprinting), or to a methylation machinery disorder that methylates the paternal allele (GOI: gain of imprinting). The requirement for using SNURF as a reference is that
161
methylated and unmethylated SNURF alleles are at a ratio of
1:1, and in our series in only one of these 14 cases did the ∆Ct
of SNURF have a negative impact on the final calculation for
the mMGMT/uMGMT ratio. In these circumstances we avoid
using SNURF as a reference loosing relative quantification data. Additionally in cases with balanced ratio, the ∆Ct between
uMGMT and uSNURF or mSNURF contributed to check
for allelic imbalance status, as the PCR efficiencies of each
marker are very closed to each other. These data were verified interrogating by ASqPCR the following SNPs adjacent to
MGMT gene: rs8473 and rs3740427. Concordant results were
obtained and 28.2% of cases showed AI. Among them 75.6%
revealed MGMT methylation. AI alone and AI associated with
MGMT methylation were not correlated with longer overall
survival after TMZ treatment respect to cases with balanced
copy number at this locus. Quantitative analysis showed a
bimodal distribution of ratio values between methylated and
unmethylated MGMT alleles, with two prevalent groups with
ratios between 0.001-0.33 and 0.67-1, respectively. This bimodal distribution is similar to that found by Vlassenbroeck
et al. 21 In summary, we report and clinically validate an accurate, robust, and cost effective MS-qLNAPCR protocol for
the detection and quantification of methylated MGMT alleles.
Using MS-qLNAPCR we demonstrate that even low levels of
MGMT promoter methylation have to be taken into account to
predict response to temozolomide-chemotherapy 22-24.
References
1
Gerson SL. MGMT: its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 2004;4(4):296-307.
2
Esteller M, Garcia-Foncillas J, Andion E, et al. Inactivation of the
DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000;343:1350-4.
3
Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing
and benefit from temozolomide in glioblastoma. N Engl J Med
2005;352(10):997-1003.
4
Liu L, Markowitz S, Gerson SL. Mismatch repair mutations override
alkyltransferase in conferring resistance to temozolomide but not to
1,3-bis(2-chloroethyl)nitrosourea. Cancer Res 1996;56(23):5375-9.
5
Gerson SL. MGMT: its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 2004;4(4):296-307.
6
Komine C, Watanabe T, Katayama Y, et al. Promoter hypermethylation of the DNA repair gene O6-methylguanine-DNA methyltransferase is an independent predictor of shortened progression free
survival in patients with low-grade diffuse astrocytomas. Brain Pathol
2003;13(2):176-84.
7
Liu ZJ, Maekawa M. Polymerase chain reaction-based methods of
DNA methylation analysis. Anal Biochem 2003;317(2):259-65.
8
Cottrell SE, Distler J, Goodman NS, et al. A real-time PCR assay for
DNA-methylation using methylation-specific blockers. Nucleic Acids
Res 2004;32(1):e10.
9
Esteller M, Garcia-Foncillas J, Andion E, et al. Inactivation of the
DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000;343:1350-4.
10
Jeuken JW, Cornelissen SJ, Vriezen M, et al. MS-MLPA: an attractive
alternative laboratory assay for robust, reliable, and semiquantitative
detection of MGMT promoter hypermethylation in gliomas. Lab Invest
2007;87(10):1055-65.
11
El-Maarri O, Buiting K, Peery EG, et al. Maternal methylation
imprints on human chromosome 15 are established during or after
fertilization. Nat Genet 2001;27(3):341-4.
12
Ginzinger DG, Godfrey TE, Nigro J, et al. Measurement of DNA copy
number at microsatellite loci using quantitative PCR analysis. Cancer
Res 2000;60(19):5405-9.
13
Latorra D, Campbell K, Wolter A, et al. Enhanced allele-specific PCR
discrimination in SNP genotyping using 3’ locked nucleic acid (LNA)
primers. Hum Mutat 2003;22(1):79-85.
14
Marucci G, Morandi L, Bartolomei I, et al. Amyotrophic lateral sclerosis with mutation of the Cu/Zn superoxide dismutase gene (SOD1)
in a patient with Down syndrome. Neuromuscul Disord 2007;17(910):673-6.
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15
16
17
18
19
20
21
22
23
24
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Vlassenbroeck I, Califice S, Diserens AC, et al. Validation of realtime methylation-specific PCR to determine O6-methylguanine-DNA
methyltransferase gene promoter methylation in glioma. J Mol Diagn
2008;10(4):332-7.
Roversi G, Pfundt R, Moroni RF, et al. Identification of novel genomic
markers related to progression to glioblastoma through genomic profiling of 25 primary glioma cell lines. Oncogene 2006;25(10):157183.
Yin D, Ogawa S, Kawamata N, et al. High-resolution genomic copy
number profiling of glioblastoma multiforme by single nucleotide
polymorphism DNA microarray. Mol Cancer Res 2009;7(5):665-77.
von Deimling A, Louis DN, von Ammon K, et al. Association of epidermal growth factor receptor gene amplification with loss of chromosome 10 in human glioblastoma multiforme. J Neurosurg 1992;77:295301.
Freire P, Vilela M, Deus H, et al. Exploratory analysis of the copy
number alterations in glioblastoma multiforme. PLoS One 2008;3(12):
e4076.
Lo KC, Bailey D, Burkhardt T, et al. Comprehensive analysis of loss
of heterozygosity events in glioblastoma using the 100K SNP mapping
arrays and comparison with copy number abnormalities defined by
BAC array comparative genomic hybridization. Genes Chromosomes
Cancer 2008;47(3):221-37.
Vlassenbroeck I, Califice S, Diserens AC, et al. Validation of realtime methylation-specific PCR to determine O6-methylguanine-DNA
methyltransferase gene promoter methylation in glioma. J Mol Diagn
2008;10(4):332-7.
Brandes AA, Tosoni A, Franceschi E, et al. Recurrence pattern after
temozolomide concomitant with and adjuvant to radiotherapy in newly
diagnosed patients with glioblastoma: correlation With MGMT promoter methylation status. J Clin Oncol 2009;27(8):1275-9.
Morandi L, Franceschi E, De Biase D, et al. Promoter methylation
analysis of O6-methylguanine-DNA methyltransferase in glioblastoma: detection by locked nucleic acid based quantitative PCR
using an imprinted gene (SNURF) as a reference. BMC Cancer
2010;10:48
Brandes AA, Franceschi E, Tosoni A, et al. O(6)-methylguanine DNAmethyltransferase methylation status can change between first surgery
for newly diagnosed glioblastoma and second surgery for recurrence:
clinical implications. Neuro Oncol 2010;12(3):283-8.
Pharmacogenetics in hematologic neoplasia
S. Rasi, A. Bruscaggin, S. Franceschetti, R. Bruna, D. Rossi,
G. Gaidano
Division of Hematology, Department of Clinical and Experimental
Medicine, Amedeo avogadro University of Eastern Piedmont, 28100
Novara
The field of pharmacogenetics investigates how genetic inheritance might influence the patients’ individual response to
drugs. In fact, the majority of drugs exhibit large interindividual variability in their efficacy and toxicity, and this individual host response is influenced by genetic polymorphisms,
in particular single nucleotide polymorphisms (SNPs).
The main aim of pharmacogenetics is to optimize therapy
based on the patient’s genotype, in order to maximize the
therapeutic index of a given drug or regimen. Indeed, several
pharmacogenetic studies have documented that host SNPs
affecting genes involved in drug metabolism, detoxification,
transport and targeting are in fact responsible, at least in part,
for the interindividual variability in efficacy and toxicity of
a given pharmacological treatment. Moreover, several drugs
utilized in therapeutic treatment of hematologic neoplasms
rely on DNA damage as part of their mechanisms of tumor
cell killing. On these bases, treatment benefit and/or toxicities
in patients may be modulated by the host DNA repair capacity. Also in this context, pharmacogenetic studies have shown
that SNPs within genes of DNA repair pathways alter the host
DNA repair capacity, thus affecting the individual response to
drugs and the prognosis of the patients.
One of the most consolidated pharmacogenetic model in
hematological-oncology is represented by the case of acute
lymphoblastic leukemia (ALL) of childhood. Among hematological neoplasms, ALL of childhood is the sole disease
whose pharmacogenetics has been characterized in detail,
and has entered clinical practice. The enzyme thiopurine Smethyltransferase (TPMT), classified as a phase II enzyme,
metabolizes chemotherapeutic agents, and in particular is
responsible for the metabolism of 6-mercaptopurine. The
activity of the TPMT enzyme displays marked variability in
the population, being influenced by SNPs of the TPMT gene
that determine a reduction of the cellular content of TPMT.
In particular, three allelic variants (TPMT*2, TPMT*3A,
and TPMT*3C) are responsible for more than 90% of cases
with an intermediate- or low-enzyme activity. ALL patients
with a wild-type TPMT allele (TPMT*1 or TPMT*1S) and
a non functional variant allele (TPMT*2, TPMT*3A, and
TPMT*3C) have an intermediate activity of TPMT, while
patients with two non functional variant alleles are TPMT
deficient. In the context of ALL of childhood, the TPMT
genotype identifies patients who are at risk of hematopoietic
toxicity after thiopurine therapy. In fact, patients who are
homozygous carriers of these SNPs and are therefore devoid
of TPMT activity, are at higher risk of hematological toxicity
when treated with 6-mercaptopurine. Instead, patients who
are heterozygous carriers of these SNPs have an intermediate
risk of dose-limiting toxicity. A 6-mercaptopurine dose reduction of 90% is generally required for homozygous-TPMT
deficiency patients, whereas the TPMT heterozygous carriers
require a mean dose reduction only of 35%.
Instead, scant information is available about the impact of
pharmacogenetics in predicting outcome and toxicity in the
context of non-Hodgkin lymphoma (NHL) and available information is restricted to a limited number of studies. Studies
in follicular lymphoma and diffuse large B cell lymphoma
(DLBCL) aimed at assessing the association between antilymphoma efficacy of rituximab and SNPs of Fcγ receptors
have reported conflicting results. Fcγ receptors are are involved in rituximab antibody-dependent cellular toxicity. A
few studies have identified SNPs located in the Fcγ receptors
(FcγRIIIa 158V/F and FcγRIIa 131H/R) as being associated
with tumor response in patients treated with rituximab as
first-line therapy. On the other hand, both in the context of
follicular lymphoma and in DLBCL, other studies failed to
identify an association between prognosis and SNPs of Fcγ
receptors.
In the context of DLBCL, the host pharmacogenetic background predicts efficacy of R-CHOP21 (R=rituximab,
C=cyclophosphamide, H=doxorubicin, O=vincristine and
P=prednisone) chemotherapy program, that is considered the
standard treatment for this disease. In fact, SNPs modulating gene expression and/or function of enzymes involved
in R-CHOP pharmacogenetics may contribute to prognostic
stratification and prediction of toxicity in DLBCL patients
treated with R-CHOP21. In particular, host SNPs affecting alkylating agent detoxification (GSTA1 rs3957357) and
doxorubicin pharmacodynamics (CYBA rs4673) may predict
survival in DLBCL treated with R-CHOP21. GSTA1 encodes
a glutathione S-transferase that catalyses the conjugation of
cyclophosphamide with glutathione to facilitate excretion.
In particular, the GSTA1 rs3957357T minor allele associates
with reduced levels of GSTA1 enzyme in healthy individuals,
and predicts for reduced detoxification of alkylating agents,
thus increasing tumor cell exposure to drug. In fact, DLBCL patients carrying GSTA1 rs3957357 CT/TT genotypes
Lectures
displayed a better outcome compared to patients who carry
the GSTA1 rs3957357 CC genotype. CYBA is a gene that
encodes the p22phox subunit of the NAD(P)H oxidase complex. Individuals carrying the CYBA rs4673T minor allele
have a substantial reduction in ROS (reactive oxygen species)
generation by NAD(P)H oxidase. Because ROS generation is
one of the antitumor mechanisms of doxorubicin, the CYBA
rs4673T minor allele is expected to reduce the tumor cytotoxicity of doxorubicin based regimens. According to this
model, DLBCL patients carrying CYBA rs4673 TT genotype
displayed a poorer outcome compared to patients who carried
the CYBA rs4673 CT/TT genotypes.
Only few studies have shown that SNPs affecting genes involved in R-CHOP pharmacogenetics, in particular NAD(P)H
oxidase subunit genes and ATP binding transporter genes,
may predict toxicities of the CHOP regimen in DLBCL. In
fact, in addition to outcome, the pharmacogenetic background
of the host appears to be relevant for predicting R-CHOP21
toxicity in DLBCL patients. In the context of DLBCL treated
with R-CHOP21, a SNP of the NCF4 gene (rs1883112), that
encodes the p40phox subunit of the NAD(P)H oxidase, recurs
as a protective genotype against both hematologic and nonhematologic toxicities of R-CHOP21. In fact, carriers of the
NCF4 rs1883112 G minor allele experience less frequently
hematologic, infective and cardiac toxicity. NCF4 rs1883112
affects the gene promoter with potential consequences on
NCF4 expression and ROS generation, that may have a relevant function in several R-CHOP21 toxicities. Increased
exposure to anthracycline-derived ROS is a widely accepted
mechanisms of doxorubicin cardiotoxicity, and moreover
ROS are involved in neutrophil death upon exposure to
chemotherapy. In an other study, CHOP-induced cardiotoxicity has been shown to associate with selected SNPs of the
NAD(P)H oxidase complex and with SNPs of ATP-binding
cassette genes.
In the context of DLBCL, SNPs modulating gene expression
and/or function of enzymes involved in DNA repair pathways
may contribute to the prognostic stratification in DLBCL
patients treated with R-CHOP21. DNA damage is one of the
163
mainstay of cancer treatment. Cells can repair DNA damage
induced by chemotherapeutic agents through several major
repair pathways. Several drugs utilized in DLBCL treatment,
including drugs of R-CHOP and second line regimens, rely on
DNA damage for tumor killing. On these bases, DNA repair
capacity may modulate treatment benefit and/or toxicities
in DLBCL patients. In particular, a SNP of the MLH1 gene
(rs1799977) is a predictor of survival in DLBCL treated with
R-CHOP21. MLH1 rs1799977 is a nonsynonymous SNP
causing the I219V amino acidic substitution on MLH1, a gene
that encodes the mutL homolog 1 protein of the mismatch
repair (MMR) pathway. In silico, MLH1 rs1799977 is predicted as a pathological SNP. In vitro, the G variant allele of
MLH1 rs1799977 is known to associate with a reduction of
MLH1 protein expression and function, and loss of MLH1 in
tumor cells is known to induce refractoriness to doxorubicin
and platinum compounds. According to these observations,
DLBCL patients who carried the MLH1 rs1799977 AG/GG
genotype displayed an increased risk of death compared to
patients who carried the MLH1 rs1799977 AA genotypes.
The poor prognosis heralded by MLH1 rs1799977 AG/GG
genotype in DLBCL is due to an increased risk of failing both
first and second line treatment. Consistently, DLBCL carriers
of the MLH1 rs1799977 AG/GG genotypes displayed poor
outcome possibly due to lack of MLH1 function.
These studies of pharmacogenetics may contribute to increase
knowledge in the field of advanced genomics applied to
hematologic tumors. In particular, the expected results concern the possibility of identifying a priori, e.g. at the time of
diagnosis, specific host pharmacogenetic profiles that may
predict efficacy and toxicity of a given treatment. Overall,
studies of host pharmacogenetics can therefore enable: i) the
identification of novel markers for prognostic stratification
and for toxicity prediction of pharmacological treatments in
the context of hematological neoplasms; ii) the construction
of a model for dose adjustement of drugs in order to maximize
therapeutic benefit and minimize treatment toxicity; iii) the
design of a personalized drug treatment for specific types of
hematological neoplasms.
164
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Thursday, September 23rd, 2010
Colon neoplasms
Moderators: G. Lanza (Ferrara), M. Risio (Torino)
Role of endoscopy for polypoid
and non-polypoid colonic lesions
L.H. Eusebi, F. Bazzoli
Department of Internal Medicine and Gastroenterology, University
of Bologna, Italy
Colorectal cancer (CRC) is the third most common cancer
diagnosed in men and women and a leading cause cancer-related death worldwide. Declining rates in CRC incidence and
mortality have been revealed by recent trends, due to reduced
exposure to risk factors, screening’s effect on early detection
and prevention of neoplastic and pre-neoplastic lesions, and
improved treatment.
Most colorectal cancers are believed to evolve through adenoma-carcinoma sequence; therefore, the goal of CRC screening
is to reduce mortality through a reduction in incidence of advanced disease. Indeed, CRC screening can achieve this goal
through the detection of adenomatous polyps and of earlystage adenocarcinomas, followed by endoscopic resection of
such lesions.
Screening programs are based on patients risk stratification,
evaluating their personal, familial and clinical history. Indeed,
identifying high risk patients, such as those with a family or
personal history of CRC or adenomatous polyps, inflammatory bowel disease or of genetic syndromes such as Hereditary
Non Polyposic Colorectal Cancer (HNPCC) and Familial
Adenomatous Polyposis (FAP), allows to determine the most
adequate screening strategy.
Among the available screening techniques, several aspects
underline the advantage of endoscopy; indeed, colonoscopy
is the only single-stage strategy not requiring pretesting and
polyps can be removed immediately during the screening
procedure; besides, all other forms of screening, if positive,
require colonoscopy as a second procedure 1.
The long term colorectal cancer incidence and mortality reduction provided by endoscopic polypectomy has been confirmed
by the findings of the National Polyps Study. Indeed, in this
study, patients with adenomas who had undergone endoscopic
resection experienced a 76-90% reduction in CRC incidence
compared with the expected general population incidence 2.
Other case-control and cohort studies have reported lower risk
reduction rates of CRC after therapeutic colonoscopy than the
National Polyp Study. Indeed, although endoscopy has a major
protective role against colorectal cancer, colonoscopy is not an
infallible “gold standard” and colonic lesions might still develop despite surveillance screening; detection miss-rates vary
from 27% for adenomas < 5 mm to about 2% for CRC 3 4.
Several reasons might explain the imperfect colonoscopy
protection such as the presence of rapidly growing tumours
(HNPCC, increased risk of microsatellite instability in “interval” cancers), ineffective polypectomy, incomplete bowel
preparation or ineffective application of current colonoscopic
detection technologies 5.
Therefore, quality indicators and targets for colonoscopy,
such as bowel preparation quality, cecal intubation rate, mean
colonoscopic withdrawal time, polyp detection rate and adverse or unplanned events occurring within 24 hours of colonoscopy, have been suggested to improve the effectiveness of
the endoscopic inspection 6.
Furthermore, an underlying principle of quality improvement in colonoscopy is that such quality indicators must be
recorded and monitored during examination.
In Emilia-Romagna, CRC screening started in 2005 and since
then more than 1000000 people have been involved, about one
third of the adult population. Among people that were positive
at the Faecal Occult Blood Test, 79% underwent colonoscopy. During endoscopic examination, 16% of patients were
diagnosed with non-advanced adenomas, 32% with advanced
adenomas and in 6% of cases CRC was found.
Finally, although recent trend confirm increasing rates of patients applying to the screening programs and distribution of
colorectal cancer stages has shifted towards earlier stages, in
the near term, even greater incidence and mortality reductions
could be achieved if a greater proportion of adults received
regular CRC screening.
References
1
Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous
polyps, 2008: a joint guideline from the American Cancer Society, the
US Multi-Society Task Force on Colorectal Cancer, and the American
College of Radiology. CA Cancer J Clin 2008;58:130-60.
2
Winawer SJ, Zauber AG, Ho MN, et al. Prevention of colorectal
cancer by colonoscopic polypectomy. The National Polyp Study Workgroup. N Engl J Med 1993;329:1977-81.
3
Rex DK, Cutler CS, Lemmel GT, et al. Colonoscopic miss rates of adenomas determined by back-to-back colonoscopies. Gastroenterology
1997;112:24-8.
4
Bressler B, Paszat LF, Vinden C, et al. Colonoscopic miss rates for
right-sided colon cancer: a population-based analysis. Gastroenterology 2004;127:452-6.
5
Rex DK, Eid E. Considerations regarding the present and future roles
of colonoscopy in colorectal cancer prevention. Clin Gastroenterol
Hepatol 2008;6:506-14.
6
Lieberman D, Nadel M, Smith RA, et al. Standardized colonoscopy reporting and data system: report of the Quality Assurance Task Group
of the National Colorectal Cancer Roundtable. Gastrointest Endosc
2007;65:757-66.
Histology quality assurance of neoplastic
colorectal lesions
P. Cassoni, A. Cassenti, P. Cardone, I. Castellano, M. Risio *
Department of Biological Sciences and Human Oncology, University
of Turin; * IRCC, Candiolo, Italy
The diagnostic agreement on the neoplastic lesions of the colorectum may be affected by several factors, including lack of
standardization in terminology, and objective diagnostic “grey
areas”. The recent publication of the European guidelines for
pathology quality assurance in colorectal cancer screening
represented a robust attempt in limiting diagnostic discordance, and outlined the major criteria which should be adopted in order to achieve a good diagnostic agreement in key
165
Lectures
features crucial for screening patient management, such as,
among others, grading of dysplasia, villousness, recognition
of invasion. In fact, these are controversial histology features
not only in the field of screen-detected adenomatous polyps,
but in general account for a limited inter and intra-observer
reproducibility within the routinary histopathology diagnoses
of colorectal lesions.
Moreover, in colorectal carcinomas, some recently introduced
additional diagnostic criteria (i.e. evaluation of tumour budding, or definition of the entity of regression in chemo-radiotreated advanced rectum cancers) requires to pathologists
a continuous resetting of previously acquired diagnostic
categories. When dealing with either controversial or newly
introduced entities, a satisfactory diagnostic standard can be
achieved by verifying the diagnostic concordance to a second
opinion. We recently verified, in a large series of screendetected polyps, that telepathology can be a reliable tool to
diffuse microscopic images in the context of quality control
efforts in screening, given its cost-effectiveness advantage
in regard to preparation, distribution, and circulation of glass
slides. In addition, improvement in image digitization technology have led to significant progress in manageability, and actually pathologists can interact with the slide image acquired
on the pc through digital virtual microscopy, which organizes
the acquisition process scanning the entire histologic slide at
the selected resolutions, and provides the observer, through
real-time image compression, with either the actual overview
image and series of microscopic images derived from zooming of well defined histologic areas. The concordance between
optic and virtual microscopy on the screen-detected cases was
good (k-statistics = 0,8), and therefore the digital approach
could be considerate an accurate tool for histology quality
assurance in this context and deserves consideration in the
validation of controversial and/or new diagnostic topics in the
field of colorectal tumors.
Morphological and immunohistochemical
factors with prognostic and predictive roles
in colorectal carcinoma
L. Terracciano
Department of Patology, University Hospital, Basel, Switzerland
Colorectal cancer (CRC) is one of the most common malignancies in the Western world. Despite improvements in
surgical techniques, dosing and scheduling of adjuvant and
neoadjuvant therapy, the 5 year survival rate for patients with
CRC decreases significantly from 93.2% to8.1% with tumour
progression. The TNM stage remains the “gold standard”
prognostic factor although patients within the same stage can
demonstrate considerable variation in terms of prognosis.
TNM stage is also used to help guide the clinical management
of CRC. Patients with stage III disease may be candidates for
postoperative chemotherapy, while those with stage II typically undergo surgery only. In addition to TNM stage, several
other tumour related features have been identified as essential
or important prognostic factors. Venous and lymphatic invasion represent a crucial step in the formation of micrometastases and eventually macroscopic tumour growth at a secondary
site. Tumour budding is associated with an infiltrative tumour
border, and shown to be an independent risk factor of local
spread, lymph node and distant metastasis, recurrence and
worse survival following curative surgery. Molecular characterisation is expected to improve the identification of patients
with more aggressive tumours there by leading to individualised treatment protocols.
Despite promising results using genotyping, mutation analysis
and allelic imbalance, the applicability of these methods to
routine practice is likely to have limited impact. By contrast,
immunohistochemistry is frequently employed as a routine
diagnostic test and is relatively inexpensive. Nevertheless,
immuno-histochemical markers have yet to find routine application as prognostic factors in CRC in which TNM stage and
other morphologically defined features continue to provide
the clinical gold standard.
Promising studies on potential prognostic markers are often
followed up by subsequent reports contradicting these initial
results. Several sources of discrepancy between different
reports have been acknowledged including methodological differences, poor study design, non-standardised assays
which lack reproducibility and inappropriate or misleading
statistical analyses often performed on underpowered patient
samples that are too small to enable meaningful conclusions
to be drawn.
The wide range of scoring methods employed to evaluate immunoreactivity as well as the use of pre-determined and often
unvalidated or unjustified cut-off scores for tumour marker
“positivity” is a major contributor to the conflicting results
reported in the literature on the same tumour marker. We have
recently proposed the use of receiver operating characteristic
(ROC) curve analysis as an alternative method for determining the threshold values for tumour marker “positivity” and
have applied this method to several well-established and novel
tumour markers in CRC for a range of clinical endpoints.
Recent data, produced by our group as by other Authors,
regarding immunohistochemical biomarkers, as RHAMM,
TOP-K, PTEN, RKIP, seems to indicate them as new promising prognostic markers in CRC.
Cardiac pathology
Moderators: G. Thiene (Padova), P. Gallo (Roma)
Endomyocardial biopsy
O. Leone
Azienda Ospedaliero-Universitaria “S. Orsola-Malpighi”, Anatomia
ed Istologia Patologica, Bologna, Italy
The endomyocardial biopsy (EMB) is now routinely used in
diagnostic strategies for cardiac diseases in the main Centres
specializing in diagnosis and treatment of cardiac failure,
although there is a considerable and debatable diversity in
recourse to this technique, for two main reasons:
• the shortage of specifically trained cardiovascular pathologists 1, able to deal appropriately with cardiac disease diagnostics and to apply suitable protocols and diagnostic criteria to
obtain all necessary information in these complex diseases;
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
• the absence in many Centers of a team approach integrating
the competences of pathologists and clinicians.
Important prerequisited in using appropriately EMB 2 are:
• perfoming EMB in Centers with a high volume of cases and
expert operators, in order to minimize procedural risks;
• improving pathological diagnostic reproducibility and reducing the percentage of nonspecific pathological diagnoses, referring patients to Centers with an expert cardiovascular pathologist and making use of adequate protocols and
standardized diagnostic criteria;
• optimizing EMB diagnostic sensitivity limits in multi-microfocal diseases, in common with all other non-targeted
bioptic techniques, applying when warranted imaging techniques able to focus on the sampling site.
Since the 1970’s, when EMB was beginning to come into
diagnostic use for heart transplanted patients, the indications
for diagnostic EMB have become increasingly more targeted
and the protocols more elaborate resulting in an increased
potential for information.
Recently, in evidence of the renewed interest in EMB, major
Guidelines have been published:
• the joint clinical Guidelines of the American Heart Association, the American College of Cardiology and the Europen
Society of Cardiology 3;
• the Position Paper on Endomyocardial biopsy promoted
by the “Association for Italian Cardiovascular Pathology”,
a jointly document produced by Italian cardiovascular
pathologists and the representatives of the main Italian
cardiologic scientific societies 2. Part II of the document
specifically addresses everyday diagnostic practice, dealing with the diagnostic role, particular technical notes,
protocols and diagnostic criteria for each cardiac disease
requiring EMB.
The principal clinical conditions that require EMB are cardiac failure 4 5, rhythm disorders 6, cardiac masses 7 and heart
transplantation 8.
Here let us confine ourselves to cardiomyopathies 9 10, the specific topic of the Symposium, whose complexity is very much
stresses by the most recent classifications.
The diagnostic iter in cardiomyopathies starts when a cardiologist identifies some clinical, functional and morphological
phenotypes, frequently aspecific and potentially caused by
numerous different diseases, whose course and therapies are
very different.
Here the role of pathologist 2 is:
• to give a definite diagnosis, when possible;
• to exclude some diseases, guiding forwards a diagnostic
program;
• to provide useful information for therapeutic choice and
prognosis;
• to contribute to monitoring the clinical evolution of the
disease and therapeutic program efficacy;
• to help decrease diagnostic errors 11;
• to guide genetic tests, when appropriate.
It is noteworthy that, even if the main target of a diagnostic
test is to identify a specific disease, excluding certain diseases
is equally important, especially when the clinical picture is
aspecific.
The diagnostic potential of EMB in various cardiac diseases
may be very different, so the level of its diagnostic contribution in a specific disease may vary from a definite diagnosis
to a probable diagnosi, to a possible diagnosis, or even an
aspecific picture 2.
The most significant contribution of EMB is in the diagnosis
of secondary myocardial diseases 10, either involving only
or mainly the heart or as a part of a multi-organ systemic
disease.
It is possible to optimize EMB diagnostic accuracy by taking
certain precautions, which may be considered general rules:
• careful selection of EMB candidates 4 and the evaluation
of EMB effects on the overall clinical management of
the patient. EMB is performed only after the other basic
clinical-instrumental tests have already excluded various
diseases and focused more closely on a possible diagnosis.
An appropriate indication for EMB is the first step towards
decreasing nonspecific diagnoses.
• Appropriate EMB timing and adequate bioptic sampling 12
with multiple specimens, from different sites 13 (possibly
guided by imaging techniques) in various cardiac diseases.
• The knowledge of diagnostic potential in various cardiac
diseases.
• The use of protocols in which the traditional histological
examination should be supported by other tissue investigation techniques (histochemical, histoenzymatic, immunohistochemical, molecular, ultrastructural), opportunely
selected on the basis of the histological picture or of clinical
suspicion.
By way of example, I will describe the EMB diagnostic role
and the tissue investigations required to increase information
in some cardiomyopathies.
Inflammatory cardiomyopathies 2. EMB, integrating the information from the histological picture, immunohistochemical
tests, molecular tests checking of possible viral genomes 14,
may rapidly provide:
• a definite diagnosis of myocardial involvement;
• the etiology ot the disease in many cases;
• the degree of activity of the disease;
• monitoring of the disease course and the efficacy of therapy;
• cardiac localization in inflammatory systemic autoimmune
diseases.
Appropriate EMB timing and adequate sampling are essential.
Amyloidotic Cardiomyopathy 2. EMB is the only test able to
reach a definite diagnosis of myocardial involvement.
Moreover, when it is included in a complete clinical-instrumental program, it may:
• contribute to etiological diagnosis using immunohistochemical tests on both histological and ultrastructural specimens,
to identify the main fibrillar component;
• provide further morphological data as to location, amount
and type of distribution of deposits, myocardial damage and
any associated inflammatory reactions;
• guide genetic analysis in familial forms.
Definite diagnosis of cardiac involvement and identification
of type of amyloidosis is essential for therapy.
Arrythmogenic right ventricle cardiomyopathy 2. EMB is a
major diagnostic standard in the score system for the diagnosis of ARVC and it may provide:
• probable diagnosis of cardiac involvement showing myocardial atrophy with fibrosis or fibro-fatty replacement and
differential diagnosis with myocarditis, sarcoidosis, dilated
cardiomyopathy and idiopathic forms;
• evaluation of the extent of myocite morphologic compromise.
Diagnostic accuracy increases if the site of bioptic samples is
selected using imaging- or electroanatomic voltage mappingguided techniques
Cardiomyopathies in storage diseases 2. (Glycogenoses,
Anderson-Fabry disease, Desmin related cardiomyopathy).
167
Lectures
EMB with the help of ultrastructural and immunohistochemical tests may:
• provide a definite diagnosis of myocardial involvement;
• grade the extent of cardiac disease and, in Fabry’s disease,
check the effects of enzymatic substitutive therapy on intramyocite accumulation;
• raise diagnostic suspicions in absence of a clinical suspicion
or in cases with generic clinic diagnosis of hypertrophic
cardiomyopathy, suggesting subsequent molecular and
biochemical tests;
• guide genetic tests.
Hemochromatosis/hemosiderosis 2. EMB using only Perls’
staining may:
• provide a definite diagnosis of myocardial involvement;
• grade myocardial involvement;
• evaluate the extent of morphologic myocyte involvement.
Dystrophin-related cardiomyopathies, Lamin-related cardiomyopathies, Emery-Dreifus disease 2. In these diseases,
the role of immunohistochemistry has recently been gaining
ground in:
• providing a definite diagnosis of cardiac involvement in
Duchenne MD (a disease whose diagnosis does not normally require a EMB) and a definite/probable diagnosis in
Becker MD and in X-linked cardiomyopathy X21.2 MIM
302045. In these latter forms EMB may be the sole diagnostic tool able to raise the question of a dystrophin gene linked
disease;
• raising diagnostic suspicion in laminopathies;
• guiding genetic analysis;
In all cases, EMB can exclude other diseases.
References
1
Thiene G, Veinot JP, Angelini A, et al. AECVP and SCVP 2009 recommendations for training in cardiovascular pathology. Association
for European Cardiovascular Pathology and Society for cardiovascular Pathology Task Force on Training in Cardiovascular Pathology.
Cardiovasc Pathol 2010;19:129-35.
2
Leone O, Rapezzi C, Sinagra G, et al. Documento di consenso sulla
biopsia endomiocardica promosso dall’Associazione per la Patologia
Cardiovascolare Italiana. G Ital Cardiol 2009;10(Suppl 1-9):1S-50.
3
Cooper LT, Baughman KL, Feldman AM, et al. The Role of Endomyocardial Biopsy in the Management of Cardiovascular Disease. A
Scientific Statement from the American Heart Association, the American College of Cardiology, and the European Society of Cardiology.
Circulation 2007;116:2216-33.
4
Perkan A, Di Lenarda A, Sinagra G. Dilated cardiomyopathy: indication and role of endomyocardial biopsy. Ital Heart J Suppl 2002;3:41925.
5
Ardehali H, Qasim A, Cappola T, et al. Endomyocardial biopsy plays
a role in diagnosing patients with unexplained cardiomyopathy. Am
Heart J 2004;147:919-23.
6
Basso C, Corrado D, Marcus FL, et al. Arrhythmogenic right ventricular cardiomyopathy. Lancet 2009;373:1289-300.
7
Flipse TR, Tazelaar HD, Holmes DR Jr. Diagnosis of malignant cardiac disease by endomyocardial biopsy. Mayo Clin Proc 1990;65:141522.
8
Stewart S, Winters GL, Fishbein MC, et al. Revision of the 1990
Working Formulation for the standardization of nomenclature in the
diagnosis of heart rejection. J Heart Lung Transplant 2005;24:171020.
9
Veinot JP. Diagnostic endomyocardial biopsy pathology–general biopsy considerations, and its use for myocarditis and cardiomyopathy:
a review. Can J Cardiol 2002;18:55-65.
10
Veinot JP. Diagnostic endomyocardial biopsy pathology: secondary
myocardial diseases and other clinical indications–a review. Can J
Cardiol 2002;18:287-96.
11
Luk A, Metawee M, Ahn E, et al. Do clinical diagnoses correlate with
pathological diagnoses in cardiac transplant patients? The importance of endomyocardial biopsy. Can J Cardiol 2009;25:e48-54.
12
Billingham ME. Acute myocarditis: is sampling error a controindication for diagnostic biopsies? J Am Coll Cardiol 1989;14:921-2.
13
14
Burke AP, Farb A, Robinowitz M, et al. Serial sectioning and multiple
level examination of endomyocardial biopsies for the diagnosis of
myocarditis. Mod Pathol 1991;4:690-3.
Calabrese F, Thiene G. Myocarditis and inflammatory cardiomyopathy: microbiological and molecular biological aspects. Cardiovasc
Res 2003;60:11-25.
Molecular diagnosis of myocarditis
A. Angelini
Dept of Medical-Diagnostic Sciences and Special Therapies, University of Padua, Padua, Italy
Myocarditis is a non-ischemic inflammatory disease of the
myocardium associated with cardiac dysfunction 1. It most often results from infectious agents, hypersensitivity responses,
or immune related injury. In spite of the development of
various diagnostic modalities, early and definite diagnosis of
myocarditis still depends on the detection of inflammatory
infiltrates in endomyocardial biopsy specimens according to
Dallas criteria 2. Routine application of immunohistochemestry (for characterization of inflammatory cell infiltration) and
molecular analysis (PCR for identification of infective agents)
have become an essential part of diagnostic armamentarium
for a more precise biopsy report 3-6.
Three different forms of the diseases can be recognized:
a) non-infectious disease due to allergic/immune causes (giant cell-myocarditis; rheumatic and eosinophilic myocardities; forms associated with immune systemic diseases; virusnegative myocardities with or without circulating anti-heart
antibodies), b) infectious (bacterial, protozoan and viral)
and c) myocardities in which the immune mechanism starts
or is supported by an infection. Unfortunately, the clinical
diagnosis of myocarditis still remains a challenge owing to
the non-specific pattern of the clinical presentation and to
the lack of universally accepted and standardized diagnostic
criteria. Since the spectrum of clinical presentation is broad,
including asymptomatic electrocardiographic abnormalities
reported during enterovirus epidemic, vague symptoms of
flu-like syndrome, congestive heart failure of recent onset,
cardiogenic shock and sudden death, many false-positive
and false-negative clinical diagnoses may be expected 7.
EMB,despite the low sensitivity due to the frequent sampling
errors and to the lack of quantitative diagnostic criteria still
represents the main diagnostic tool for myocarditis. Viral
myocarditis usually lacks specific cytopathic effects and can
cause different magnitude in the inflammatory response both
as consequence of the virulence of the causative agents as
well as the host status. As suggested in the consensus document 8 on EMB published by the Associazione per la Patologia Cardiovascolare Italiana endomyocardial biopsy (EMB)
still represent the gold standard for the diagnosis even though
myocardial scintigraphy and MRI may help in diagnosis.
EMB is mandatory:
• to reach a definite diagnosis of myocardial involvement;
• to contribute to etiological diagnosis (infectious microorganism, immune aetiology);
• to indicate the degree of activity of the disease.
Pathological diagnosis. In diagnosing myocarditis important
points include:
• timing of EMB in relation to the onset of symptoms;
• number and size of bioptic fragments;
• contemporary checks for heart auto-antibodies in serum7.
Histological examination: In hematoxylin-eosin stained sections 1) inflammatory infiltrate, 2) myocellular damage and
3) fibrosis.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Further elements to look for are:
a)inflammatory infiltrate types (lymphocytic, polymorphous,
granulomatous, eosinophilic) and extention using semiquantitative or quantitative evaluation;
b)myocellular damage (not only necrosis or myocytolysis but
also other alterations, such as cytoplasmic vacuolization,
apoptosis, and atrophy);
c)pattern of fibrosis (interstitial, perivascular, subendocardial)
and its extent using semi-quantitative or quantitative evaluation.
Histo-morphological stains:
• Azan-Mallory trichrome is useful to highlight and quantify
fibrosis.
• Weigert-Van Gieson, which highlights elastic fibres in
brown/black, and allows for evaluation of vessel wall structure and endocardial fibroelastosis.
Immunohistochemical tests: immunohistochemical analysis
represents a fundamental corollary to traditional histology
enabling the characterization of inflammatory infiltrate and,
when this is present in very small quantities, its identification.
Antibody panel to use: CD45, CD68/PGM1, CD3, CD4, CD8,
CD20, HLA-ABC, HLA-DR.
Morphometric quantification: it is desirable to quantify
inflammatory infiltrate (currently a lymphocytic infiltrate
> 7 T lymphocytes/mm2 is considered as pathologic) using computerized morphometry on immunohistochemical
sections stained with anti-CD3 antibody. Morphometry on
Azan-Mallory trichrome stained sections may allow precise
quantification of fibrosis.
Molecular tests and in particular techniques of gene amplification, such as polymerase chain reaction (PCR) or nestedPCR, because of their high sensitivity, allow the amplification of viral DNA or RNA, thus detecting any viral genome
present in the small samples of EMB tissue. Nowadays,
sequence analysis and the identification of replicating virus
forms are increasingly utilized to characterize infective agents
precisely 5-9. If myocarditis is clinically suspected, at least the
following most frequent cardiotropic virus genomes must be
checked for in myocardial tissue: enterovirus, adenovirus,
cytomegalovirus, Epstein Barr virus, herpes simplex virus, Influenza A and B viruses; B19 parvovirus and C hepatitis virus.
In the setting of positive PCR results blood samples collected
at the time of the biopsy should be tested: if positivity for the
same virus is present in both myocardial tissue and blood, it is
necessary to quantify its load with quantitative PCR analysis
to exclude any haematic contamination of the myocardial
specimen. Gene sequencing is a more sophisticated technique
allowing the characterization of the infective agents as well as
its virulence and cardiotropism. Different serotypes can bear
a different virulence and cardiotropism and guide prognosis
and therapeutic interventions. However the detection of viral
genome does not necessarily imply a direct pathogenetic role,
since it could be an innocent by stander. Also a negative PCR
does not exclude viral disease. Final diagnosis of myocarditis
must be the results of an integrated clinical, instrumental,
morphological and molecular approach.
Key points
• The accurate diagnosis of myocarditis requires a representative number of specimens to be subjected to complete
traditional histopathological, and immunohistochemical
(lymphocyte types, HLA, C3-C4) and molecular virological
(a study of the presence/persistence of RNA and DNA virus
genome) analysis.
• The application of immunohistochemical methodologies
(which allows the identification of inflammatory infiltrates,
their more adequate quantification and the evaluation of
myocardial expression of immunological activation markers) increases interpretative capacity, especially in cases
of prevalently autoimmune mechanism responsible for
“chronic myocarditis”.
• It is mandatory to apply molecular tests, especially gene
amplification techniques such as the Polymerase Chain
Reaction (PCR), quantitative (real time-PCR) or qualitative (nested-PCR), which, nowadays, because of their high
sensitivity, allow the identification even of a small number
of copies of any viral genome present in the EMB.
• The exclusion of a viral aetiology is an essential requirement in considering a myocarditis as immuno-mediated
(both antibody- and cell- mediated) and choosing the most
appropriate therapeutic strategy 8 9.
• Etiological characterization is important also in assessing
prognosis.
References
1
Maron BJ, Towbin JA, Thiene G, et al. Contemporary definitions and
classification of the cardiomyopathies. An American Heart Association Scientific statement from the Council on clinical cardiology, heart
failure and transplantation Committee; quality of care and outcomes
research and functional genomics and translational biology interdisciplinary working Groups; and Council on epidemiology and prevention. Circulation 2006;113:1807-16.
2
Aretz HT, Billingham ME, Edwards WD, et al. Myocarditis: a histopathological definition and classification. Am J Cardiovasc Pathol
1987;1:3-14.
3
Angelini A, Crosato M, Boffa GM, et al. Active vs borderline myocarditis: clinicopathological correlates and prognostic implications.
Heart 2002;87:210-5.
4
Calabrese F, Rigo E, Milanesi O, et al. Molecular diagnosis of myocarditis and dilated cardiomyopathy in children. Clinico-pathologic
features and prognostic implications. Diagn Mol Pathol 2002;11:21221.
5
Calabrese F, Thiene G. Myocarditis and inflammatory cardiomyopathy: microbiological and molecular biological aspects. Cardiovasc
Res 2003;60:11-25.
6
Calabrese F, Carturan E, Thiene G. Cardiac infections: focus on molecular diagnosis. Cardiovascular Pathology 2010;19:171-182.
7
Caforio AL, Calabrese F, Angelini A et al. A prospective study of
biopsy-proven myocarditis: prognostic relevance of clinical and aetiopathogenetic features at diagnosis. Eur Heart J 2007;28:1326-33.
8
Documento di consenso sulla biopsia endomiocardica promosso
dall’Associazione per la Patologia Cardiovascolare Italiana. G. Ital
Cardiol 2009;10(Suppl):1-9.
9
Kuhl U, Pauschinger M, Noutsias M, et al. High prevalence of
viral genomes and multiple viral infections in the myocardium of
adults with “idiopathic” left ventricular dysfunction. Circulation
2005;111(7):887-93.
Diagnostic and terapeuetic work-up for
cardiovascular diseases: the role of pathologists
E. Arbustini, M. Grasso, M. Diegoli, A. Agozzino, M. Concardi
Centre for Inherited Cardiovascular Diseases, Transplant Research
Area, IRCCS Foundation Policlinico San Matteo, Pavia, Italy
Background. The progression of knowledge on the pathologic
basis of cardiovascular diseases and the development of biotechnological tools for pathological and molecular studies are
progressively increasing the number of specific vs. descriptive
diagnoses. Disease-specific diagnostic work-up tailored on
phenotypes (percorsi diagnostico-terapeutici assistanziali:
PDTA) constitute the tool; in this scenario pathology may play
a fundamental role for diagnosis, prognostic stratification,
Lectures
treatment guide, and evaluation of the benefits of treatments.
This short review concentrates on cardiovascular diseases
and discusses a few examples, but similar considerations can
extend to other disease settings.
Methods. To evaluate the clinical impact of disease-specific work-up in cardiovascular diseases, we generated an
outpatient centre where cardiovascular pathology, clinics and
genetics are integrated and other multidisciplinary contributions are herein organised according to the type of disease.
The centre policy is to offer patients and families tailored care
strategies, starting from the specific diagnosis, contrapposed
to the concept of descriptive diagnosis. The activity started
in early 80ies with cardiovascular pathology; then it progressively incorporated genetics and clinics and finally evolved to
the organisation of multidisciplinary teams of specialists that
are now active on precise disease-specific protocols (PDTA).
The centre participates to national and international quality
controls and is equipped with instruments that can provide
accurate, precise, and high-quality data for patient care and
for research; it consists of three integrated units: 1) the cardiovascular pathology lab that is equipped with automated
instruments for tissue and blood processing, light, electron
and confocal microscopes, cell culture and cell sorter facilities and slide scanner; 2) the molecular genetic lab that is fully
equipped for genomics and transcriptomics/gene expression
profiling, as well as for very basic protein studies (Western
Blot) for immunohostichemistry/immunoblotting comparative studies; 3) the clinical out-patient centre that permanently
hosts cardiologists and geneticists, as well as nurses and administrative personnel to organise and run the disease specific
diagnostic work-up for patients and families; on scheduled
programs, specialists of disciplines that are included in the
different diagnostic work-up have regular access to the centre. Personnel active in the three units includes 24 between
pathologists, cardiologists, geneticists, engineers, biologists,
technicians, nurses and a person responsible for international
liaisons.
The pathology provided the basic knowledge and tools for
the entire organisation: from pathology to clinics and genetics
first (1984-2000) and now from clinics to pathology and genetics (2001-2010). Each disease-specific diagnostic work-up
is organised as follows: 1. The centre is contacted by medical doctors, specialists of different disciplines, according to
the disease, or directly by patients or voluntary associations.
2. All clinical/pathological reports are evaluated before the
access of patients to the centre and tailored work-up is then
planned, including clinical multidisciplinary evaluations,
biopsy procedures, genetic testing and counselling (when
necessary).
Results. The Centre now offers multidisciplinary diagnostic
work-up for acquired (inflammatory and autoimmune) and
heritable cardiovascular diseases manifesting with vascular
life-threatening events, heart failure and arrhythmias. Major
groups of diseases are: genetic aneurismal syndromes [Marfan
Syndrome, Loeys-Dietz Syndromes, Elhers Danlos Syndrome
type IV, Thoracic Aortic Aneurysm and Dissections (TAAD),
familial and non-familial Bicuspid Aorta (BAV)], heritable
an acquired cardiomyopathies [hypertrophic sarcomeric and
non-sarcomeric], dilated, restrictive and arrhythmogenic right
ventricular cardiomyopathies and phenocopies), complex syndromes with cardiovascular involvement [such as Noonan, Alstrom, Holt-Oram and Barth syndromes, MELAS, MERFF],
lysosomal diseases such as Danon Disease, Pompe Disease,
Anderson Fabry Disease] as well as other more rare conditions. The centre currently follows more than 2500 families.
169
Based on the prevalence of each of the above diseases, more
than 180.000 individuals are affected in our country.
Acute myocardial diseases: examples of acute non-ischemic
myocardial illnesses. Excluding the most prevalent ischemic
heart disease, a setting in which novel diagnostic and treatments dramatically decreased mortality and improved survival, acute myocardial diseases also include myocarditis of
viral origin (PVB19 or EV), idiopathic giant cell myocarditis
(possible phenotype: ”fulminant myocarditis”), acute rheumatic carditis, pheochromocytoma (contraction band necrosis
without inflammation), acute hypercalcaemia (myocardial
microcalcifications), neoplastic infiltration [such as thymoma), septic emboli, drug toxicity (eosinophilic infiltrates in
the absence of hypereosinophilc syndromes associated with
FIP1L1/PDGFR alpha fusion transcript), as well as other
more rare conditions. In the PDTA for acute heart failure, the
correct diagnosis of the above rare causes may be life saving.
The probability of specific pathologic diagnosis depends on
the participation of the pathologists to the overall multidisciplinary evaluation/discussion. Most descriptive diagnoses can
be done on H&E stain of endomyocardial biopsies (EMB).
The myocardium however has a limited spectrum of morphologic responses to different types of insults; a clinically
oriented hypothesis may guide the choice of appropriate immunohistochemical and molecular studies.
Chronic myocardial diseases: the example of cardiomyopathies. The modern diagnostic strategies for cardiomyopathies require that pathologists know the clinical phenotypes,
the imaging features and the genetic bases of these diseases.
The intergation of this knowledge is the basis for providing a
useful contribution of tissue studies in cardiomyopathies.
Dilated cardiomyopathy (1:2500) (DCM) is a clinical descriptive diagnosis; pathology and genetics may provide a specific
diagnosis. Up to 50% DCM are heritable diseases with genetic
heterogeneity (up to 35 disease genes). A DCM phenotype
may result from defects of genes that code for nuclear envelope proteins (Lamins and Emerin), desomosome proteins,
myocyte membrane proteins (Dystrophin and Dystrophin-associated glycoproteins), mitochondrial proteins (Tafazzin).
The appropriate immunostain may provide diagnostic information. Genetic test is the gold standard, when available. Both
pathology and genetics integrate to document the functional
impact of the mutation on the myocardial tissue.
Hypertrophic phenotypes (1:500, or more). As DCM, Hypertrophic Cardiomyopathy (HCM) is a descriptive diagnosis: it
simply indicates idiopathic increased left ventricular thickness. HCMs are grouped in sarcomeric (13 disease-genes that
code for sarcomeric proteins) and non-sarcomeric, mostly
lysosomal diseases such as glycogenosis (ex. Pompe disease), sphingolipidoses (ex. Anderson Fabry Disease), Danon
Disease) or Protein receptor Kinase AG2 (PRKAG2)-related
cardiomyopathy. The non-sarcomeric group also includes mitochondrial DNA-related cardiomyopathes, as well as pathologic phenocopies of lysosomal diseases such as cloroquine
myocardial toxicity. Depending of the available facilities, the
diagnosis can be obtained by genetic testing; when genetic
tests are not available, the EMB uniquely contributes to the
diagnosis. A novel role for EMB is therefore emerging in the
scenario of non-sarcomeric HCM phenotypes.
Restrictive cardiomyopathy (RCM) is characterised by functional restrictive pattern without hypertrophy (prevalence
< 1:100,000). There are several genetic, systemic autoimmune, neuromuscular and inflammatory diseases that may
cause a RCM phenotype. The starting point for establishing a
specific diagnosis is the evaluation of cardiac and non-cardiac
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
markers associated with RCM and the family phenotype. In
this context EMB may play a fundamental role for the diagnosis of Desminopathies in patients with pure RCM associated
with AVB. When cataract is also present, CRYAB gene should
be considered as more likely candidate gene. Troponinopathies (defects of troponin T and I) may manifest as pure RCM
w/o hypertrophy with high risk of sudden death: in this case,
they are not associated with AVB and/or myopathy: a potentially useful maker is the mismatch between hypertrophy
(minimal, if any) and disarray (present).
Cardiac Amyloidoses: thickened hearts with restrictive pattern. With more than 20 different amyloidogenic proteins
potentially causing systemic deposits, tissue studies document
the presence of amyloid deposits in myocardium, valves,
vessels, epicardial fat and provides the characterisation of
the amyloidogenic protein by immunoelectron microscopy.
In systemic amyloidoses, this result can be easily achieved
by sampling periumbelical fat, but in non-systemic cardiac
amyloidoses EMB is the only diagnostic option. Pathologists
are also involved in monitoring treatment effects as well as
in detecting possible recurrence of amyloid in transplanted
organs; no imaging tool is equally informative.
Arrhythmogenic right ventricular cardiomyopathy (ARVC)
(1:5000). Pathology played a key role in the disease characteri-
sation. ARVC/D typically affects the right ventricle; it is clinically characterised by life-threatening ventricular arrhythmias.
The pathologic markers are fibro-fatty replacement of RV
myocardium with myocyte degeneration. ARVC/D is a desmosomal cardiomyopathy: defects of one of the 5 major proteins
of the desmosome, plakophillin, plakoglobin, desmoglein,
desmocollin and desmoplakin cause loss of continuity between
myocytes caused by failing desmosomes, progressive degeneration and death of the myocytes leading to fibro-fatty repair.
Although genetic testing is now available in tertiary centres
that perform specific cardiogenetic programs, new directions
for pathologic diagnosis include immuno-staining with antiplakoglobin and gap-junction protein Cx 43 antibodies, that
should be markedly decreased. Cardiac sarcoidosis is included
in the list of phenocopies that may mimic ARVC/D, as does
myocarditis: EMB is the gold standard fro the diagnosis.
Conclusion. As owners of a systematic approach to the pathologic bases of the diseases in general, and cardiovascular in
particular, pathologists can either coordinate or deeply integrate in disease-specific clinical work-ups that represent the
frontiers of sustainable health programs (the right diagnosis
and care‡ right patients vs. descriptive diagnoses and care‡
all patients). The model is expanding in our country as well as
throughout Europe.
Gastrointestinal inflammatory diseases
Moderators: C. Capella (Varese), E. Tavani (Rho)
Eosinophilic Esophagitis:clinical aspects
M. Marini
Pediatric Gastrointestinal Endoscopy, University Hospital Santa Maria alle Scotte, Siena (Italy)
Introduction. Eosinophilic esophagitis (EE) is an increasingly recognized disorder previously described in children,
and emerged as a clinical disorder that afflicts adults, presents
with dysphagia and food impaction and characterized by a
dense eosinophilic infiltration of the surface lining of the
esophagus 1-3.
EE is a primary esophageal disorder without associated eosinophilic infiltration of the stomach or intestine.
There are many other diseases that can cause eosinophils
in the tissue of the esophagus, including gastroesophageal
reflux disease(GERD), parasitic infections, fungal infections,
inflammatory bowel diseases
(Crohn), certain cancers, recurrent vomiting,collagen vascular disorders, eosinophilic gastroenteritis. and others. These
diseases need to be ruled out before primary EE can be diagnosed.
Symptoms. EE has many different presentations. In the
majority of studies to date, individuals affected by EE have
been predominantly male children and adolescents; patients
commonly have difficulty eating, failure to thrive, vomiting,
epigastric or chest pain, dysphagia, and food impaction 4 5.
Adult patients typically have recurrent dysphagia and food
impactions that are refractory to anti-GERD therapy; in fact,
recent studies indicate that 10%–50% of adult male patients
with these symptoms have EE 6 7.
Although a fixed stricture could account for the esophageal dysphagia and food impaction observed in some patients with EE 8,
evidence is mounting that the esophagus displays impaired
smooth muscle function, likely from asynchrony of circular and
longitudinal muscle contraction during swallowing 9.
A variety of motor disturbances that are reversible with
therapy have been reported in patients with EE 10 11.
Patients often have personal and family histories of asthma, allergic rhinitis,atopic dermatitis, food and drug allergies, eosinophilia, elevated serum levels of immunoglobulin E (IgE), and
positive allergic skin and radio allergo sorbent tests (RAST).
Although EE was originally recognized in pediatric patients, it
has similar characteristics(including atopic sensitization) and
occurrence rates in adults. The disease has also been recognized in patients older than 90 years. EE is a chronic disorder
that has no significant evidence of spontaneous remission,
even over a 14-year period,but some patients have seasonal
variations in symptoms, consistent with an etiology related to
airborne allergen exposure.
Diagnosis. A number of endoscopic features have been described, including strictures (frequently proximal), mucosal
rings (often multiple), mucosal ulceration, linear furrows,
small-caliber esophagus, and multiple white papules (eosinophilic microabscesses) 12-18.
Vasilopoulos have proposed a classification system for EE
that includes three types. Type 1 is called the early small
caliber esophagus, type 2 is the advanced small caliber
esophagus and type 3 is the ringed esophagus. It is not clear
whether these types represent isolated variants of this disorder
or whether they are sequential stages in its evolution 19.
The whitish exudates seen in EE can be fine, pinpoint and
scattered and are often mistaken for Candida or debris. The
exudates actually represent collections of eosinophils, which
can also appear as mucosal nodules or plaques.
Lectures
The most dramatic endoscopic finding of EE is the appearance of a long and deep mucosal tear (with visualization of
the submucosa and muscle fibers) following passage of a
dilator. These tears or mucosal rents can also occur simply
with insertion of the endoscope, particularly when there is an
unrecognized diffusely narrowed esophagus.
Endoscopic ultrasound findings in EE include thickening of
the mucosa, submucosa and muscularis propria 20.
Endoscopy is used to obtain biopsy samples from patients
with proton pump inhibitor (PPI)-refractory upper gastrointestinal symptoms. These tissue samples are analyzed by
microscopy;a minimum of 15 eosinophils/hpf indicates that
a patient has EE.
However, it is now recognized that there can be significant
overlap between GERD and allergic EE. Eosinophil counts
greater than 100 per hpf can be seen in some patients with
GERD, even at multiple levels of the esophagus 21.
In facts a diagnosis of allergic EE cannot be made until GERD
is ruled out either by ambulatory pH testing or by an eight
week trial of a PPI taken twice daily, followed by repeat endoscopy with biopsies.
Recently analysis of transcription profiles has indicate dysregulated expression of 1% of the human genome, including overexpression of eotaxin-3, is found in samples from patients with
EE. This gene expression profile can be used to distinguish between biopsy specimens from patients with EE, patients with
reflux esophagitis (RE), and normal individuals (NL).
In patients successfully treated with dietary modification and/
or glucocorticoids, eosinophil numbers in biopsy samples are
reduced to 1/hpf and the transcriptome more closely resembles
that of NL, although there are residual gene expression differences between patients treated for EE and RE and NL 22.
Managment. Optimal treatment for EE has not been defined.
Management has been better evaluated in children where
dietary restrictions and treatment with corticosteroids have
proven effective, but compliance and toxicity, respectively,
have limited usefulness. Fluticasone propionate (FP) applied
topically appears to be equally effective and better tolerated 23 24.
Strictures in adults have been managed by dilation. Approaches include use of antihistamines, sodium cromoglycate,and
systemic and topical corticosteroids; andleukotriene receptor
antagonists.
Mepolizumab, an anti-interleukin-5 monoclonal antibody, recently has been reported to have histologic and clinical benefit
in an adult case of EE.
Esophageal dilation has been associated with deep mucosal
tears, severe pain, and perforation 25.
Experience with (FP), an inhaled corticosteroid routinely used
in the management of asthma, has shown benefit in a pediatric
population with EE and, more recently, in adults 26-28.
FP has not always been efficacious, particularly in the allergic
EE subgroup.
The likelihood of response to anti-GER therapy decreases with
increased severity of eosinophilic inflammation of the esophageal mucosa. The eosinophil density within the esophageal
mucosa required for the diagnosis of AEE is often defined by
R15 eosinophils per high power field (eos/hpf).
An eosinophil density of %5 eos/hpf is thought to represent
gastric-acid–mediated injury.
Consequently, the diagnosis and treatment of patients with
moderate tissue eosinophilia, ie, 6 to 14 eos/hpf, is unclear.
Because AEE may take months to years to evolve, moderate
esophageal eosinophilia may represent a mild or evolving
form of EE 29.
171
References
1
Fox VL, Nurko S, Furuta GT. Eosinophilic esophagitis: itsjust not kids
stuff. Gastrointest Endosc 2002;36:260-70.
2
Arora AS, Perrault J, Smyrk TC. Topical corticosteroid treatment of
dysphagia due to eosinophilic esophagitis inadults. Mayo Clin Proc
2003;78:830-5.
3
Croese J, Fairley SK, Masson JW, et al. Clinical and endoscopic
features of eosinophilic esophagitis in adults. Gastrointest Endosc
2003;58:516-22
4
Noel RJ, Putnam PE, Rothenberg ME. Eosinophilic esophagitis. N
Engl J Med 2004;351:940-1.
5
Noel RJ, Rothenberg ME. Eosinophilic esophagitis. Curr Opin Pediatr
2005;17:690-4.
6
Desai TK, Stecevic V, Chang CH, et al. Association of eosinophilic
inflammation with esophageal food impaction in adults. Gastrointest
Endosc 2005;61:795-801.
7
Mackenzie SH, Go M, Chadwick B, et al. Eosinophilic oesophagitis in
patients presenting with dysphagia – a prospective analysis. Aliment
Pharmacol Ther 2008;28:1140-6.
8
Bassett J, Maydonovitch C, Perry J, et al. Prevalence of esophageal
dysmotility in a cohort of patients with esophageal biopsies consistent
with eosinophilic esophagitis. Dis Esophagus 2009;6:543-8.
9
Korsapati HR, Babaei A, Bhargava V, et al. Dysfunction of the longitudinal muscles of the oesophagus in eosinophilic esophagitis. Gut
2009;58:1056-62.
10
Lucendo AJ, Castillo P, Martin-Chavarri S, et al. Manometric findings
in adult eosinophilic oesophagitis: a study of 12 cases. Eur J Gastroenterol Hepatol 2007;19:417-24.
11
Nurko S, Rosen R. Esophageal dysmotility in patients who have eosinophilic esophagitis. Gastrointest Endosc Clin North Am 2008;18:7389, ix.
12
Langdon DE. “Congenital” esophageal stenosis, corrugated ringed
esophagus, and eosinophilic esophagitis. Am J Gastroenterol
2000;95:2123-4.
13
Langdon DE. Corrugated ringed and too small esophagi. Am J Gastroenterol 1999;94:542-3.
14
Bousvaros A, Antonioli DA, Winter HS. Ringed esophagus: an association with esophagitis. Am J Gastroenterol 1992;87:1187-90.
15
Vasilopoulos S, Murphy P, Auerbach A, et al. The small-caliber
esophagus: an unappreciated cause of dysphagia for solids in patients
with eosinophilic esophagitis. Gastrointest Endosc 2002;55:99-106.
16
Croese J, Fairley SK, Masson JW, et al. Clinical and endoscopic
features of eosinophilic esophagitis in adults. Gastrointest Endosc
2003;58:516-22.
17
Straumann A, Spichtin H-P, Bucher KA, et al. Eosinophilic esophagitis: red on microscopy, white on endoscopy. Digestion 2004;70:10916.
18
Potter JW, Saeian K, Staff D, et al. Eosinophilic esophagitis in adults:
an emerging problem with unique esophageal features. Gastrointest
Endosc 2004;59:355-61.
19
Vasilipoulos S, Shaker R. Defiant dysphagia: small-caliber esophagus
and refractory benign esophageal strictures. Current Gastroenterology
Reports 2001;3:225-230.
20
Fox VL, Mirko S, Teitelbaum JE. High resolution EUS in children with eosinophilic allergic esophagitis. Gastrointest Endosc
2003;57:30-6.
21
Rodrigo S, Abboud G, Oh D, et al. High intraepithelial Eosinophil
counts in esophageal squamous epithelium are not specific for eosinophilic esophagitis in adults Am J Gastroenterol 2008;103:435-42.
22
Blanchard C, Mingler MK, Vicario M, et al. IL-13 involvement in
eosinophilic esophagitis: transcriptome analysis and reversibility with
glucocorticoids. J Allergy Clin Immunol 2007;120:204-14.
23
Faubion WA, Perrault J, Burgart LJ, et al. Treatment of eosinophilic
esophagitis with inhaled corticosteroids. J Pediatr Gastroenterol Nutr
1998;27:90-3.
24
Langdon DE. Fluticasone in eosinophilic corrugated ringed esophagus. Am J Gastroenterol 2001;96:926-7.
25
Faubion WA Jr, Perrault J, Burgart LJ, et al. Treatment of eosinophilic esophagitis with inhaled corticosteroids. J Pediatr Gastroenterol
Nutr1998;27:90-3.
26
Attwood SE, Lewis CJ, Bronder CS, et al. Eosinophilic esophagitis: a
novel treatment using Montelukast. Gut 2003;52:181-5.
27
Liacouras CA, Wenner WJ, Brown K, et al. Primary eosinophilic
esophagitis in children: successful treatment with oral corticosteroids.
J Pediatr Gastroenterol Nutr 1998;26:380-5.
172
28
29
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Arora AS, Perrault J, Smyrk TC. Topical corticosteroid treatment of
dysphagia due to eosinophilic esophagitis in adults. Mayo Clin Proc
2003;78:830-5.
Noel RJ, Putnam PE, Collins MH, et al. Clinical and immunopathologic effects of swallowed fluticasone for eosinophilic esophagitis.
Clin GastroenterolHepatol 2004;2:568-75.
Le esofagiti eosinofile: aspetti istopatologici
C. Vindigni
Division of Pathological Anatomy, AOUS, Siena, Italy
Eosinophilic esophagitis (EE) is a primary disease of the
esophagus characterized by esophageal and/or upper gastrointestinal tract symptoms and by dense esophageal eosinophilia
associated with a normal gastric and duodenal mucosa and absence of pathologic gastroesophageal reflux disease (GERD)
as evidenced by a normal pH monitoring study or lack of
response to high dose PPI medication 1.
EE is more predominant in males, young adults and children,
and is often associated with a history of allergic disease 2 3.
Many reports suggest familial clustering of the disease but it
is difficult to determine whether this represents genetic predisposition or similar environmental exposure 4.
Recent studies suggest an increase in the prevalence of EE,
but it is unclear whether this is due to a true escalation in
incidence or to a better awareness of the disease by both gastroenterologists and pathologists 5.
The diagnosis of EE is based on the clinical presentation,
endoscopic features and histopathological findings. Failure to
respond to anti-reflux therapy and dense eosinophilic infiltration in oesophageal biopsies are essential.
At endoscopy, several mucosal abnormalities have been identified, including friability, white specks, whitish exudates,
“crepe paper mucosa”, narrow caliber esophagus, longitudinal
furrows, and transient or fixed rings; some studies have also
reported a normal mucosa 6. It has been reported that multiple
biopsy specimens from different areas, including the distal,
mid and proximal oesophagus, improve the diagnostic ability
because of the heterogeneous distribution of eosinophilic infiltration. Biopsies should also be obtained from the stomach
and duodenum to rule out eosinophilic gastroenteritis 7.
The diagnostic criterion for the diagnosis of EE is an intense
eosinophil infiltration in oesophageal squamous epithelium
but the number and the method used varies among studies. It
has been recommended that intraepithelial eosinophils should
be counted in the most intensely inflamed HPF of the biopsy
at x400 magnification 7. A consensus opinion as to the histological diagnostic criteria is still lacking but most of the pathologists believe that the presence of > 20 eosinophils in one
HPF or > 15 eosinophils in multiple HPFs is diagnostic for
EE 5. This is based on studies that showed that GERD is often
associated with less than seven eosinophils per HPF 8. Other
associated histopathologic features have been observed in EE
as eosinophils degranulation, eosinophilic microabscesses,
preferential superficial distribution of eosinophilic inflammation, basal zone hyperplasia and papillary lengthening, lamina
propria fibrosis. These features may be helpful to the pathologist for the diagnosis of EE and should be included in the pathology report in addition to the number of eosinophils 7.
The relationship between GERD and EE is not clear, and it
must be kept in mind that these entities may sometimes coexis 9. Absolute eosinophil counts cannot be used to establish
a definitive diagnosis of EE. Consensus recommendations
state that the diagnosis of EE should only be made in the
proper clinical context and therefore close communication
between the pathologist and gastroenterologist is necessary
for the diagnosis 10.
Recognition of EE and the differential diagnosis from GERD
is critical for appropiate patient care.
References
1
Landres RT., Kuster GG, Strum WB. Eosinophilic esophagitis in a
patient with vigorous achalasia. Gastroenterology 1978;74:1298-301.
2
Liacouras CA, Spergel JM, Ruchelli E, et al. Eosinophilic esophagitis: a 10-year experience in 381 children. Clin Gastroenterol Hepatol
2005;3:1198-206.
3
Parfitt JR, Gregor JC, Suskin NG, Jawa HA, Driman DK. Eosinophilic
esophagitis in adults: distinguishing features from gastroesophageal
reflux disease: a study of 41 patients. Mod Pathol 2006;19:90-6.
4
Patel SM, Falchuk KR. Three brothers with dysphagia caused by eosinophilic esophagitis. Gastrointest Endosc 2003;61:165-7.
5
Chang F, Anderson S. Clinical and pathological features of eosinophilic oesophagitis: a review. Pathology 2008;40:3-8.
6
Dellon ES, Gibbs WB, Fritchie KJ, et al. Clinical, Endoscopic and
histologic findings distinguish eosinophilic esophagitis from gastroesophageal reflux disease. Clin Gastroenterol Hepatol 2009;7:130513.
7
Furuta GT, Liacouras CA, Collins MH. Eosinophilic esophagitis in
children and adults: a systematic review and consensus recommendations for diagnosis and treatment. Gastroenterology 2007;133:134263.
8
Remedios M, Campbell C, Jones DM, et al. Eosinophilic esophagitis
in adults: clinical, endoscopic, histologic findings, and response to
treatment with fluticasone propionate. Gastrointest Endosc 2006;63:312.
9
Spechler SJ, Genta RM, Souza RF. Thoughts on the complex relationship between gastroesophageal reflux disease and eosinophilic
esophagitis. Am J Gastroenterol 2007;102:1301-6.
10
Noffsinger AE. Update on esophagitis. Controversial and underdiagnosed causes. Arch Pathol Lab Med 2009;133:1087-95.
Diagnosis of infectiuos enterocolitis
S. Antinori, C. Parravicini, A.L. Ridolfo, L. Fociani,
G.L.Vago
Department of Clinical Sciences “L. Sacco”, Section of Infectious Diseases and Immunopathology, University of Milano, Italy
Background. Infectious eneterocolitis (IE) is frequently a
short course and self-limiting disease in otherwise healthy
adults; however, severe and potential life threatening course
may be observed in particular in infants, elderly and immunocompromised individuals. IE are most often caused by
ingestion of contaminated food or water. In addiction, it may
arise from reactivation of quiescent infectious agents (i.e., cytomegalovirus, CMV), in particular in immunocompromised
hosts.
The characteristics of the illness and the epidemiologic setting
are essential for differential diagnosis and evaluation 1. Moreover, there is general agreement that a distinct diagnostic approach is required in three different epidemiologic setting of
IE, i.e., community-acquired (including traveller’s diarrhea),
nosocomial and in immunocompromised hosts 2.
Methods. Four case vignettes have been used to addressed
the challenges in diagnosing severe IE in three different epidemiological scenarios.
Results.
Case 1. A 23 years old women presented with fever and chills
lasting two weeks after returning from a vacation in Bali. On
admission she was febrile (39°C) and physical examination
revealed mild hepatomegaly. Blood analysis showed anaemia
(Hb 9.6 g/dl, MCV 69 fl), leukocytosis (WBC 12.000/µl),
and elevated liver enzymes (ALT 104 U/l, AST 91 U/l).
After 3 days she complained of diffuse abdominal pain and
bloody diarrhea. Abdominal radiography and ultrasound
Lectures
were negative. Cultures of blood and stool for Salmonella,
Shigella, Campylobacter and Clostridium difficile toxins were
negative. She underwent a colonoscopy which showed several
discrete purulent ulcers along the sigmoid colon and rectum,
and disseminated haemorrhagic suffusions with an intense
hyperaemic and oedematous mucosa in the transversal and
descending colon. Multiple colonic biopsies showed marked
chronic inflammation of the lamina propria extending into
the submucosa with scattered crypt abscesses, erosions, and
several CMV inclusions in endothelial cells. CMV pp65antigenemia (11 positive cells/slide) and CMV serology (IgM
11.9, IgG 0.9) were compatible with acute CMV infection.
Intravenous therapy with ganciclovir was started with a rapid
clinical response.
Case 2. A 71-year-old man with underlying diabetes mellitus,
hypertension and hypertriglyceridemia was admitted to our
hospital with bloody diarrhea and a three-day history of severe
watery diarrhea, vomiting and cramping abdominal pain. The
patient had just returned from a trip in Madagascar where he
had eaten raw meat of zebu and had drunk tap water.
On physical examination he was alert, dehydrated with sick
appearance, and showed abdominal distention with tenderness
and guarding to deep palpation in the lower quadrants. Laboratory studies revealed leukocytosis (10,720/µl), hyperglicemia (284 mg/dl), and acute renal failure (creatinine 6.5 mg/
dl) with hyperkaliemia (7.3 mmol/l) and severe metabolic
acidosis (pH 7.16, HCO3- 9.3 mmol/l, lactate 14.2 mmol/l).
The patient underwent hemodyalisis, parenteral hydratation
and correction of acidosis and was put on empirical antibiotic therapy (levofloxacin and metronidazole). Stool culture
for Salmonella, Shigella, Campylobatcer, Yersinia as well
as C. difficile toxin resulted negative; examination of fresh
and stained samples of stool for ova and parasite was negative. An abdominal CT demonstrated ascites and colon-wall
thickening. Due to the development of toxic megacolon the
patient underwent explorative laparotomy which showed diffuse ascitic fluid and thickening of intestinal loops. A biopsy
of rectal mucosa showed an acute proctosigmoiditis without
demonstrable microorganisms. A blood culture obtained at
admission grew Shigella sonnei that was sensitive to the ongoing antimicrobial therapy. The patient was discharged after
one month hospital stay.
Case 3. A 83-year-old man with multiple comorbidities
(gastroresection, COPD, coronary artery disease, lower limb
Kaposi’s sarcoma, psoriasis with psoriatic arthritis and hypertension) was admitted to our hospital with fever and vomiting.
During the previous two months he had repeated hospitalizations and antibiotic treatments for urinary tract infection. On
admission he had anaemia (Hb 8.3 g/dl; HT 28%), normal
leukocytes (5380/µl), increased creatinine (1.7 mg/dl); a chest
X-ray showed reticulo-nodular infiltrates. He was started on
piperacillin-tazobactam after blood and urine cultures had
been taken. Three days later he developed watery diarrhea
with 4 to 5 loose stools daily along with poor appetite and
vomiting. Stool culture for Salmonella, Shigella and C. difficile were negative whereas two stool samples were positive
for C. difficile toxins. He subsequently developed stypsis,
worsening abdominal pain and marked leukocytosis (27,320/
µl). A plain abdominal X-ray and abdominal CT demonstrated
signs of ileus with marked thickening of the wall of rectum
and sigma. Despite treatment with vancomycin plus intravenous metronidazole the patient died 15 days later.
Case 4. A 39-year-old HIV-infected woman not taking antiretroviral therapy presented with a 4-month history of watery
and bloody diarrhea, cramping abdominal pain and weight
173
loss. Her last CD4 cell count was 400/µl and plasma HIV load
11,726 copies/ml. Previous stool examinations for bacteria,
mycobacteria and protozoa gave negative results; antigliadin
and transglutaminase antibodies were absent, while fecal occult blood tests were persistently positive. On admission physical examination was remarkable for fever (39°C), tenderness
in left lower abdomen and external haemorrhoid. Laboratory
tests showed anemia (9.5 g/dl), high CRP (211 mg/l), hypokaliemia (2.5 mmol/l), hypoalbuminemia (2.4 g/dl). Abdominal
CT-scan showed moderate hepato-splenomegaly, without
other relevant findings. Colonoscopy revealed the presence
of multiple ulcerations in the left colon that were biopsied.
The ulcers where characterized by a complete loss of the
lamina propria, of the muscolaris mucosae and of part of the
submucosal tissues, with a dense lymphoplasmocytic infiltrate. By immunohistochemistry, most of the lymphoid cells
in the ulcerative lesions were a mixture of CD3/CD4+ and
CD3/CD8+ T lymphocytes, intermingled with CD20+ B cells,
CD138+ plasma cells and CD30+/CD15- blastic cells. Immunohistochemistry for CMV and in-situ hybridization for EBV/
EBER were negative. Extensive microbiologic and serologic
investigations in faeces and peripheral blood were uniformly
negative. A complete autoantibody panel was also negative.
Empiric antimicrobial therapy with gancyclovir, ciprofloxacin
and metronidazole was started but profuse diarrhoea persisted
unchanged. After a massive rectal bleeding a new colonoscopy demonstrated multiple ulcers and a recto-vaginal fistula.
Methylprednisolone (20.mg bid) was empirically added to the
antibiotic regimen and antiretroviral treatment was started.
After 15 days a follow-up colonoscopy was perfomed but the
procedure was complicated by perforation of the sigma and
subsequent emergency laparotomy with left hemicolectomy
and ileostomy. The patient was then treated with a prolonged
course of tapering steroids along with combined antiretroviral
therapy. She is now without diarrhoea or rectal bleeding for
6 months.
Discussion. The first two vignettes, which respectively
describe a case of CMV colitis and a case of shigellosis,
highlight the diagnostic work-up of acute bloody diarrhoea
(ABD) with special emphasis on travellers. Initial microbiological work-up should include bacteria (Salmonella, Shigella, Campylobacter, Yersinia, E. coli O157:H7) and parasites
(Entamoeba histolytica; Schistosoma), which are the most
frequent agents involved in ABD. Routine microbiologic
cultures usually target the first three microorganisms whereas
special requests are needed for Yersinia and E. coli O157:
H7. A test for C. difficile should be included as it may cause
infections running a severe course also in non-traditional risk
groups including healthy persons in the community without
antimicrobial exposure 3. Freshly passed stool examination is
required to search for trophozoites of E. histolytica and for
ova of Schistosoma; moreover staining of fixed faecal smears
with iron-hematoxylin or Ziehl-Neelsen can determine the
presence of the E. histolytica/E. dispar complex. Imaging
studies, in particular abdominal CT scan with oral or intravenous contrast, are useful to assess anatomic localization and
extent of bowel involvement and complications such as perforation. Colonoscopy permits to identify and characterize mucosal lesions, and obtain biopsies that would allow differential
diagnosis with non infectious diseases (e.g. inflammatory
bowel disease, colon cancer or ischemic colitis) and detect
unexpected infections (e.g. schistosomiasis) and infections
that cannot be diagnosed otherwise (e.g. CMV colitis). Bowel
infections with CMV is typical of immunocompromised patients; it has been also increasingly observed in IBD with most
174
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
studies supporting a role for active CMV infection in causing
exacerbations of the disease. Although rare, CMV colitis may
occur in the immunocompetent-host during primary infection
and may be potentially severe erosive disease with significant
morbidity.
The third vignette is exemplificative of nosocomial IE that is
commonly defined as a diarrhoeic illness that occurs after 3
days of hospitalization. C. difficile is the most important cause
of nosocomial diarrhea in adults; the infection causes a toxin
mediated intestinal disease that may range from mild watery
diarrhea to life-threatening colitis. Its diagnosis is based primarily on the detection of C. difficile toxin A or toxin B.
The last vignette underscore the diagnostic challenge of diarrhoeic syndrome in the setting of HIV/AIDS; the diagnostic
work-up is largely influenced by the stage of the disease with
classic intestinal opportunistic infections (e.g. microsporidiosis; cryptosporidiosis; isosporiasis; CMV colitis; intestinal
mycobacterioses) occuring in patient with less than 100 CD4+
lymphocytes/µl. If after exhaustive stool studies no pathogen
is isolated there is clearly a role for invasive endoscopic evaluations, particularly in patients with severe refractory diarrhoea 4. Active idiopathic ulcerative colitis has been described
in HIV-positive patients independently of the depression of
peripheral CD4 cells count.
References
1
Thielman NM, Guerrant RL. Acute infectious diarrhea. N Engl J Med
2004;350:38-47.
2
Pawlowski SW, Warren CA, Guerrant R. Diagnosis and treatment of
acute or persistent diarrhea. Gastroenterology 2009;136:1874-86.
3
Rupnik M, Wilcox MH, Gerding DN. Clostridium difficile infection:
new developments in epidemiology and pathogenesis. Nature Rev
Microbiol 2009;7:526-36.
4
Cello JP, Day LW. Idiopathic AIDS enteropathy and treatment
of gastrointestinal opportunistic pathogens. Gastroenterology
2009;136:1952-65.
Coeliac disease
Initial lesions – Differential diagnosis – Refractory forms
Moderators: V. Villanacci (Brescia), A. D’Errico (Bologna)
Gluten- and non-gluten-dependent
non-atrophic lesions: the clinician
and the pahologist viewpoint
U. Volta, G. Caio, E. Tavani *, A. Andorno *
Dipartimento di Malattie dell’Apparato Digerente e Medicina Interna, Policlinico “S. Orsola-Malpighi”, Bologna, Italia, * U.O Anatomia Patologica, A.O. “G. Salvini” – Ospedale di Rho, Italia
Non-atrophic lesions of the small bowel mucosa are characterised by minimal intestinal lesions with an increased
number of intraepithelial lymphocytes (IEL), with or without crypt hyperplasia and in presence of a normal architecture of intestinal villi. Minimal intestinal lesions, though
non-specific for coeliac disease (CD), are included in the
wide spectrum of gluten-dependent histological damage and
they can be an expression of potential CD. However, they
can be a sign of many disorders other than gluten-sensitive
enteropathy. Therefore, it is important to differentiate patients with gluten-dependent intestinal damage from those
with non gluten- dependent intestinal lesions in order to
plan the correct treatment and follow-up. On the basis of
the up-to-date histological classifications small intestinal
non-atrophic lesions correspond to type 1 (IEL increase)
and type 2 lesions (IEL increase with crypt hyperplasia),
according to Marsh classification, modified by Oberhuber 1
or to grade A, according to the most recent Corazza-Villanacci classification, which gathers Marsh-Oberhuber type
1 and 2 lesions 2.
IEL are normally present in the intestinal mucosa of healthy
people where they play a pivotal role in the surveillance and
activation of the immune system. The majority of these lymphocytes is represented by T cells expressing α/β receptor. In
the normal mucosa only 3% of IEL express γ/δ T-cell receptor. The upper normal limit of IEL in the duodenum, once
fixed in 40/100 epithelial cells, is generally acknowledged
to be 25/100, evaluating the mean value of the lymphocyte
counts in five different points.
Non-atrophic lesions of the intestinal mucosa have been observed in 2.2%-24% of patients undergoing duodenal biopsy
due to the clinical suspect of small bowel disease 3-5. As well
known, the typical histological picture of CD is based on more
o less severe villous atrophy with a significant decrease of
villous/crypt ratio, an increased number of IEL and crypts hyperplasia. When the villous architecture and the villous/crypt
ratio are normal, the increased number of IEL and crypt hyperplasia show a very low predictive value for CD, since only
in a small percentage of these patients (about 10%) the aetiology of the intestinal damage is attributable to a developing
gluten-sensitive enteropathy 6. In the remaining 90% of cases
the finding of non-atrophic lesions of small intestinal mucosa
is an expression of a wide spectrum of non-gluten-dependent
disorders such as food allergy, Crohn disease, lymphocytic
colitis, bacterial and parasitic infections (giardiasis is one of
the most frequent one), common variable immunodeficiency
(CVID), autoimmune disorders (Hashimoto thyroiditis, diabetes mellitus type 1, rheumatoid arthritis, systemic lupus
erythematosus), small bowel bacterial overgrowth, non-steroidal anti-inflammatory drug treatment and helicobacter pylori
infection. Moreover, it must be remembered that an increased
number of IEL is present in 10-38% of 1st degree relatives
of coeliac patients without a pathological significance in the
majority of cases.
Although the diagnostic criteria for gluten-sensitive enteropathy clearly establish that non-atrophic lesions of small bowel
mucosa are compatible, but not specific for CD, one of the
emerging problems encountered in the clinical practice is the
over-diagnosis of CD, improperly performed on the basis of
these minimal changes in the intestinal mucosa, without reference to the other predictive factors for the identification of the
gluten-sensitivity 7. The importance of these wrong diagnoses
of gluten-sensitive enteropathy is still much more relevant,
if we consider that, following these diagnostic mistakes, a
lifelong, expensive and socially limiting gluten-free diet and a
periodical follow-up are recommended for an inexistent disor-
Lectures
der. On the other hand, the identification of the small percentage of patients, in whom non-atrophic intestinal lesions can
predict the development of CD, is mandatory since this allows
to confine the follow-up for confirming the gluten-dependent
origin of intestinal damage to this small subgroup, avoiding
to monitor uselessly the majority of other patients. To achieve
this goal, it is relevant that:
– small intestinal mucosal lesions are evaluated in the clinical, serological and genetic context;
– histological evaluation of intestinal damage must be performed in the respect of well-defined technical rules.
Since the clinical-serological context is extremely variable,
the interpretation of the morphological changes performed by
the pathologist should be flexible, above all at the beginning
of the diagnostic work-up, drawing up the conclusion only
when the whole “scenario” will be assessed together with the
clinician.
As for the technical rules concerning small intestinal histology, first of all it is relevant to underline that mucosal lesions
in CD are not always continuous, but they can be patchy and
irregular. Therefore, at least four biopsy samples from the
second-third portion of duodenum should be picked up.
It is essential that biopsy samples are correctly oriented. This
is important not only for the evaluation of atrophic lesions,
but also for a correct interpretation of minimal changes of
small intestinal mucosa. A well-oriented intestinal biopsy allows a good evaluation of villous/crypt ratio (≥ 3:1 in a normal
mucosa) and above all an accurate count of IEL, which is very
difficult to obtain with transversal sections and/or convoluted
villi.
When an increase of IEL is suspected as the sole marker of
intestinal mucosa damage, the use of immunohistochemistry
represents a mandatory adjunctive technique to their counting,
allowing to stain CD3+ and CD8+ T lymphocytes. An associated evaluation of CD4+ T lymphocytes, though suggested
in the past, seems to be useless in the histological work-up
of intestinal mucosa. IEL counts should be done taking into
consideration five villi; moving from villous tips, lymphocytes present in 20 enterocytes (10 on the right and 10 on
the left part of the villi) must be enumerated, establishing the
mean value. The mean value is 9.2 lymphocytes/20 epithelial
cells (range 5.8-21.8) in subjects with an abnormal mucosa
(gluten-sensitivity or other pathological conditions) vs 4.6
lymphocytes/20 epithelial cells (range 1.4-7.8) in subjects
with a normal mucosa. The lymphocyte distribution along
villi is substantially homogeneous; a higher lymphocytic
concentration in the villous tip helps to identify patients with
gluten-sensitivity 8. Immunohistochemical characterization of
lymphocyte populations in the intraepithelial compartment
by using duodenal biopsy frozen sections may be useful in
identifying gluten-sensitive patients. It is well established
that a high density of T-cells with γ/δ receptors in he surface
epithelium is a characteristic feature of gluten sensitivity.
The mean proportion of γ/δ T cells in gluten-dependent nonatrophic lesions varies from 20% to 30%, whereas, when
non-atrophic lesions are non-gluten-dependent, γ/δ T cells are
about 2%-3%. However, this modality has limited diagnostic
utility due to the non-availability of an assay for identifying γ/δ T cells in formalin-fixed, paraffin-embedded tissue.
The characterization of crypt mitotic index by measuring the
number of K67+ cells by immunohistochemistry can help to
differentiate between gluten-dependent and non gluten-dependent intestinal damage. A value of K67+ cells higher than
60-65% is suggestive for a condition of gluten sensitivity.
On the contrary, many attempts to evaluate variations of the
175
immunohistochemical expression of tissue transglutaminase
activity did not produce interesting results.
Non-atrophic intestinal lesions should be evaluated in the
context of clinical, serological and genetic data 9. Although
the diagnosis of CD on the basis of clinical data is an utopia,
among symptoms, that can rise the suspect of gluten-sensitve
enteropathy, there are bowel abnormalities, including both severe diarrhoea and marked constipation, weight loss, recurrent
abdominal pain, iron-deficiency anaemia, hypertransaminasaemia of unknown origin, unexpected osteoporosis, recurrent
miscarriages and CD-related autoimmune disorders.
In order to establish if non-atrophic lesions are or not
are gluten-dependent the detection of CD-related serological markers is much more relevant 10. Many subjects with
minimal changes of small bowel mucosa are classified as
potential coeliacs on the basis of an antibody pattern that is
not specific for CD. It is well-known that anti endomysial
antibodies (EmA) of IgA class are the immunologic marker
with a nearly always absolute specificity for CD. Anti tissue
transglutaminase antibodies (anti-tTG) of IgA class display a
very high predictive value for identifying CD when positive
at a very high titer (> 5x the cut-off), whereas they show at
least 10% of false positives when their antibody titer is very
low (< 2x the cut-off). Therefore, their positivity, particularly
at a low titer, should be always confirmed by EmA finding.
Antibodies to deamidated gliadin peptides (DGP-AGA) of
IgG class are another immunological marker which proved to
be particularly useful in differentiating between gluten- and
non-gluten-dependent intestinal lesions. Their specificity for
gluten sensitivity is far higher than that of IgA anti-tTG and
very close to that of IgA EmA. Moreover, DGP-AGA display
a higher diagnostic accuracy than the traditional and obsolete
AGA test. After the introduction of DGP-AGA in the workup of CD, the traditional AGA lost their last indication for
CD screening, that remained the identification of CD in the
infancy (children aged less than 2 years).
Another immunological sign predictive of gluten-sensitive intestinal damage is the finding of IgA anti-tTG in small bowel
biopsies. Indeed, these antibodies can appear at intestinal level
when they are still negative in patients’ sera and when the intestinal barrier is quite normal or shows only mild abnormalities 11. Most of these patients, left on a gluten containing diet,
display after a less or more lasting follow-up the development
of villous atrophy associated with the appearance of serum
antibodies.
The puzzle of gluten sensitivity comprises another relevant
element represented by genetic testing. As generally acknowledged, CD is closely related to a well-defined HLA pattern,
characterized by positivity for HLA-DQ2 and -DQ8. The
positivity of the test (finding of DQ2 or DQ8 or DQB1*02) is
never diagnostic for CD by itself since about 30% of the general population displays the same HLA pattern of CD patients.
The most important clinical message of the test comes from
its negativity since the absence of DQ2, DQ8 and DQB1*02
allows to exclude CD (negative predictive value 100%) 9. In
patients with a suspect of potential CD (positive serology
with mild or absent histological lesions) HLA genotyping
for DQ2 and DQ8 is useful to reinforce (when positive) or
exclude (when negative) the suspect of a gluten-dependent
intestinal damage. In patients with non atrophic intestinal lesions and negative serology the HLA negativity should alert
us to search for another cause of small bowel abnormalities
(CVID, giardiasis, helicobacter pylori infection, etc.). In patients with positivity at low titer for IgA anti-tTG, with IgA
EmA and IgG DGP-AGA negativity, the absence of HLA-
176
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
DQ2 and -DQ8 gives evidence that IgA anti-tTG are likely
“false positives”.
The erroneous interpretation of clinical, serological and genetic data significantly contributes to the plethora of false
CD diagnoses performed in patients with minimal intestinal
changes. The most frequent pitfalls leading to an over-diagnosis of CD in patients with non-atrophic intestinal lesions
are the positivity for IgG anti-tTG in absence of selective IgA
deficiency, the isolated finding of HLA-DQ2 or -DQ8 (that
are only expression of a genetic predisposition for CD), the
low titer positivity for IgA anti-tTG associated with negativity
for IgA EmA, the isolated positivty for IgA AGA in children
older than 2 years and in adults, and a gluten hypersensitivity
on a clinical ground, often caused by irritable bowel syndrome
or wheat allergy
To sum up, the finding of non-atrophic intestinal lesions is
a non-specific immunopathological phenomenon, that has a
large number of possible causes, and by itself is never synonymous of gluten-sensitive enteropathy. A correct histological
evaluation is recommended in order to avoid false CD diagnoses, caused by artifacts due to a not well-oriented biopsy. The
evaluation of IEL count in villous tip as well as of γ/δ T-cell
receptor lymphocytes can be of help in distinguishing between
non-celiac patients and patients at risk of developing CD.
A careful evaluation of the clinical, serological and genetic
aspects must be carried out in all patients with non-atrophic
lesions in order to identify the minority of cases affected by
potential CD or who must undergo a periodic follow-up for
the possible development of gluten-sensitive enteropathy,
and to eliminate this suspect in the majority of cases, who
should be studied for pathological conditions other than gluten-sensitivity. Awareness of the wide spectrum of disorders
associated with non-atrophic lesions of small bowel mucosa
is important to guide the clinician and the pathologist toward
a correct diagnosis.
References
1
Oberhuber G, Granditsch G, Vogelsang H. The histopathology of celiac disease: time for a standardized report scheme for pathologists.
Eur J Gastroenterol Hepatol 1999;11:1185-94.
2
Corazza GR, Villanacci V. Coeliac Disease. J Clin Pathol 2005;58;5734.
3
Brown I, Mino-Kenudson M, Deshpande V, et al. Intraepithelial
lymphocytosis in architecturally preserved proximal small intestinal
mucosa. Arch Pathol Lab Med 2006;130:1020-5.
4
Biagi F, Bianchi PI, Campanella J, et al. The prevalence and the
causes of minimal intestinal lesions in patients complaining of symptoms suggestive of enteropathy. A follow-up study. J Clin Pathol
2008;61:1116-8.
5
Lahdeaho ML, Kaukinen K, Collin P, et al. Celiac disease: from
inflammation to atrophy, a long-term follow-up study. J Pediatr Gastroenterol Nutr 2005;41:44-8.
6
Kakar S, Nehra V, Murray JA, et al. Significance of intraepithelial
lymphocytosis in small bowel biopsy samples with normal mucosa
architecture. Am J Gastroenterol 2003;98:2027-33.
7
Upton MP. “Give us this day our daily bread”. Evolving concepts in
celiac sprue. Arch Pathol Lab Med 2008;132:1594-9.
8
Biagi F, Luinetti O, Campanella J, et al. Intraepithelial lymphocytes in
the villous tip do they indicate potential coeliac disease? J Clin Pathol
2004;57:835-9.
9
Volta U, Villanacci V, Tavani E, et al. La diagnosi di malattia celiaca.
Pathologica 2007;99:412-4.
10
Volta U, Granito A, Fiorini E, et al. Usefulness of antibodies to deamidated gliadin peptides in celiac disease diagnosis and follow-up. Dig
Dis Sci 2008;53:1582-8.
11
Salmi TT, Collin P, Järvinen O, et al. Immunoglobulin A autoantibodies against transglutaminase 2 in the small intestinal mucosa predict
forthcoming coeliac disease. Aliment Pharmacol Ther. 2006;24:54152.
Thyroid cytology
Moderators: A. Fassina (Padova), M. Papotti (Torino)
Immunohistochemical markers in cytology
of neoplastic thyroid disease
M. Volante, L. Daniele, M. Papotti.
Department of Clinical and Biological Sciences, University of Turin,
Turin, Italy
Thyroid nodules represent a common clinical problem.
The prevalence of palpable thyroid proliferations in adults
increases with age, with an average of 4-7% for the United
States population but higher in iodine-deficient areas where
sub-clinical nodules are frequently incidentally discovered
following thyroid ultrasound-scan. More than 90% of these
thyroid proliferations are benign and for this reason a reliable and systematic approach to their evaluation represents
an important task to be pursued for avoiding a surgical overtreatment. Ultrasound-guided fine-needle aspiration biopsy
(FNAB) is the gold standard for thyroid nodule evaluation,
but it is widely known that this method has some intrinsic
limitations and immunohistochemistry represents the most
reliable technique to assess the cytomorphological diagnosis
in difficult cases. The application of immunohistochemistry
in thyroid cytology may have as a first aim the identification
the cell type of origin of the lesion, including thyroglobulin or TTF-1 for follicular cell-derived lesions, calcitonin,
chromogranin A or CEA for medullary carcinoma, parathyroid hormone for parathyroid lesions, pan-cytokeratin for
anaplastic carcinoma, lymphoid markers for lymphomas,
among others. However, the major applicative field is represented by the distinction between benign (i.e. microfollicular
nodular hyperplasia and follicular adenoma) and malignant
follicular lesions (i.e. follicular thyroid carcinoma and follicular variant of papillary carcinoma). In fact, these follicular
thyroid nodules, that remain indeterminate at thyroid FNAB
cytology (classified TIR3 according to recently proposed
SIAPEC guidelines), are referred to surgery more for diagnosis than for therapeutic purposes, and less that 10-20%
of such cases will prove to be malignant at final histology.
To reduce the number of follicular proliferations referred
to surgery, and therefore to reduce costs for public health,
several immunocytochemical markers have been proposed
to distinguish malignant from benign follicular proliferations. The use of immunocytochemical markers on FNAB
material may be generally employed on smears, although
cell block preparations seem to be more reliable in this
specific setting. The markers proposed are mainly related to
tumor-associated abnormal expression of cellular antigens,
such as cell surface mesothelial antigen HBME-1 (HBME1),
cytokeratin-19 (CK19), thyreoperoxidase (TPO), keratan-
177
Lectures
sulfate (KS), or to the specific expression of cell-cycle or
apoptosis related molecules, such as galectin-3 (GAL-3), or
oncogenes, such as RET. As a general comment, it is generally advisable to rely not on a single marker but rather on a
combination, to achieve the best specificity and sensitivity 13
. The role of one of the most employed markers, GAL-3, has
been recently validated in a large prospective multicentric
study from an Italian population 4 and confirmed an overall
sensitivity and specificity of this immunocytochemical test
of 85% and 93%, respectively, with estimated positive and
negative predictive values of 83% and 94% respectively.
More than 91% of indeterminate (TIR3) follicular thyroid
nodules enrolled in this study were considered correctly
classified preoperatively.
References
1
Maruta J, Hashimoto H, Yamashita H, et al. Immunostaining of galectin-3 and CD44v6 using fine-needle aspiration for distinguishing follicular carcinoma from adenoma. Diagn Cytopathol 2004;31:392-6.
2
Rossi ED, Raffaelli M, Minimo C, et al. Immunocytochemical evaluation of thyroid neoplasms on thin-layer smears from fine-needle
aspiration biopsies. Cancer 2005;105:87-95.
3
Saggiorato E, De Pompa R, Volante M, et al. Characterization of
thyroid ‘follicular neoplasms’ in fine-needle aspiration cytological
specimens using a panel of immunohistochemical markers: a proposal
for clinical application. Endocr Relat Cancer 2005;12:305-317.
4
Bartolazzi A, Orlandi F, Saggiorato E, et al., Italian Thyroid Cancer
Study Group (ITCSG). Galectin-3-expression analysis in the surgical
selection of follicular thyroid nodules with indeterminate fine-needle
aspiration cytology: a prospective multicentre study. Lancet Oncol
2008;9:543-9.
Breast cytology
Moderators: A. Bellomi (Mantova), A. Leotta (Lamezia Terme)
Breast cytology: reporting
L Di Bonito, F Martellani, D Bonifacio, S Dudine, M Di Napoli, E Isidoro, E Ober, E Leonardo, A Romano, A Zacchi,,
T Al Omoush, D Bonazza, A De Pellegrin, O Haxhijmeri, M.
Petris, L Zandonà, V Bandiera *, F Giudici *, L Torelli *, M
Bortul ***, M Tonutti **, F Zanconati
U.C.O. Anatomia e Istologia Patologica Azienda Ospedaliero Universitaria Ospedali Riuniti Università di Trieste; * Dipartimento di
Matematica e Informatica Università di Trieste; ** U.C.O. Radiologia
Universitaria Azienda Ospedaliero Universitaria Ospedali Riuniti
Trieste; *** U.C.O. Clinica Chirurgica Azienda Ospedaliero Universitaria Ospedali Riuniti Università di Trieste
appearance, although highly suggestive for malignancy, is not
conclusive. This category includes the cases with few highly
atypical cells and some very well-differentiated tumors. These
lesions must undergo biopsy to obtain a conclusive diagnosis
or, in cases with low cellularity, FNAC can be repeated.
C5 = MALIGNANT; cytological features are diagnostic for
malignancy. Sometimes, through FNAC the histotype of malignancy can be determined.
Trieste’s Breast Unit has been using FNAC as first morphological investigation for many years, in particular, in 20082009, it was used as first diagnostic approach for 1835 cases
(88.6%) out of 2091. Thanks to FNAC, 742 lesions were
diagnosed as benign (C2) (with clinical follow-up confirmation) avoiding more invasive histological investigations (i.e.
microbiopsy and surgical biopsies). Besides that, thanks to
the FNAC it was possible, for cases with surgical indication,
to plan a targeted intervention: excisional nodulectomy for
large benign (C2) or likely benign (C3) lesions, conservative
treatment for malignant monofocal or small lesions (quadrantectomy) or mastectomy for malignant multifocal or locally
advanced tumors. For suspicious lesions (C4) surgical approach (nodulectomy vs. diagnostic quadrantectomy with or
without Sentinel Lymph Node) was decided considering the
type of radiological suspicion.
The advantage of using of the diagnostic categories is the possibility to correlate with the final outcome of the histology or
follow-up. In Table I cyto-histological correlations of FNAC
of breast nodules only (excluding the sampling of lymph
nodes and chest wall’s nodules).
Fine needle aspiration cytology (FNAC) is widely used as
first choice approach for the definition of the radiologically
dubious or suspicious cases or to confirm their benign origin.
In experienced hands, FNAC represents a reliable technique
that has many advantages: it is a simple and fast exam with
minimal invasiveness and low costs.
Regarding FNAC reporting we refer to the guidelines proposed
by the English Screening Program 1 subsequently adopted by
the European guidelines 2. The use of these categories by the
Breast Unit of Trieste since 2002 provides a standardization
of the diagnostic report. According to this classification’s system each lesion is placed in one of the five categories (C1-5)
shortly described below.
C1 = INADEQUATE; includes all those cases which do not
provide the possibility to solve a specific diagnostic problem
(poor cellularity, bad technical preparation, excessive inflammatory or blood’s elements,);
The table shows a prevalence of C5 and C2 lesions: C5 were
C2 = benign; there’s no evidence of malignancy. It includes
626 (29.9%) and C2 were 790 (37.8%) accounting for 67,7%
all cases characterized by absence of nuclear or morphological
of the total lesions investigated.
alterations.
C3 = probably benign; Tab. I
includes all cases in which
Data
C5 cytology C4 cytology C3 cytology C2 cytology C1 cytology
the smear’s cells are not
2008-2009
malignant suspicious
atypical
benign
inadequate
Total
certainly interpretable as beHistology
591
72
14
1
10
688
nign. Management of such
malignant
cases requires correlation of
Histology
1
19
76
47
13
156
cytology with clinical and /
benign
or radiological aspect.
No histology
34
4
75
742
57
912
C4 = suspicious for Total C
626
95
165
790
80
1756
malignancy; the cellular
178
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Cyto-histological correlation allows continuous monitoring
of quality’s indicators of breast diagnostic cytology provided
by the laboratory, with the possibility to compare them with
the standard suggested by the guidelines; the results are summarized in Table II.
Tab. II
Quality
Indicators
As
Cs
Sbx
Spe
C5 Ppv
C4 Ppv
C3 Ppv
FisFis+
Ina
Inca
Sus
Two-years period
2008-2009: 1756
86.6%
98.5%
30.1%
76.6%
99.8%
79.1%
8.5%
0.14%
0.14%
4.5%
1.4%
14.7%
Standard (%)
> 60%
> 80%
/
> 60%
> 95%
70-80%
< 20%
< 5%
< 1%
< 25%
< 10%
< 20%
As: absolute sensitivity; Cs: full sensitivity;
SBX: specificity (only biopsied cases);
Spe: full specificity;
C5 Ppv: positive predictive value of C5; C4 Ppv: positive predictive
value of C4; C3 Ppv: positive predictive value of C3; Fis-: rate of false
negatives; Fis+: rate of false positives; Ina: inadequate’s rate; Inca:
inadequate rate with final diagnosis of cancer; Sus: rate of suspects.
The direct participation of the cytopathologist in all sampling
sessions has allowed to get optimal smears without artifacts
with an immediate adequacy assessment. This has allowed
the immediate repetition – in the same session – of the cases
with suboptimal material, helping to minimize the number of
inadequates (4.5%), well below the maximum allowed value
(25%). This activity also allows the pathologist a constant
dialogue with the radiologist, providing a chance to choose
collectively the most appropriate method for solving the
single diagnostic doubt.
The positive predictive value of C5 has been constantly
maintaining at high levels: 99.8% (standard required: > 95%):
respect of this parameter is necessary to omit frozen sections
in all C5 cases, allowing to plan conservative surgery without
further confirmation.
The positive predictive value of C4 (95 lesions, 5.4%) was
79.1% (standard value 70-80%). The positive predictive value
of C3 (165 lesions, 9.3%), that according to the guidelines
must remain below 20%, was 8.5%. Overall inconclusive lesions (C3 and C4) were 14.7% with a rate of suspicious cases
well below 20%.
The use of diagnostic categories in cytology reporting has
found wide acceptance among radiologists and surgeons
because it allows to apply to each lesion, a precise diagnostic/
therapeutic pathway and it represents, in our experience, an
essential element of the report itself.
References
1
Guidelines for Cytology Procedures and Reporting in Breast Cancer
Screening - Cytology Sub-Group of the National Coordinating Commitee for Breast Screening Pathology. NHS-BSP 1993;22.
2
European guidelines for quality assurance in breast cancer screening
and diagnosis, fourth edition, 2006.
Management and standardization in anatomic pathology
Analysis of the SIAPEC-IAP study and future perspectives
Moderators: C. Angeli (Vercelli), F. Crivelli (Gallarate)
Analysis of the results of the research/study
SIAPEC-IAP
E. Trinchero
Public Management and Policy Department, SDA Bocconi School of
Management, Milan, Italy
Background. The possible aims related to the definition
of standard times for the execution of the proper and typical activities of a Pathology Unit basically consists of the
staff definition/identification, the personnel planning and
management, the eventual restructuring and reorganisation
of the Unit, the rationalisation in the employment of human
resources, the definition of a rational and quantitative base for
the budgeting negotiation. The methodologies for the determination of the standard times of health services are several.
The possible alternatives, on which the research done for (and
in cooperation with) Italian Society of Anatomic Pathology
and Cytopathology (SIAPEC) is based, can be schematised
as follows.
1. Methodologies based on a TOP-DOWN or “synthetic”
APPROACH. Following this approach, the calculation of
the workloads is made on the final output. The standard
work time per unit needed for the production of each typol-
ogy of output is calculated by dividing the total time which
each professional profile actually works by the output which
is actually produced. The TOP-DOWN approach typically
allows for the definition of time standards through synthetic
surveys, which are simple and quick and allow for the determination of a good overview of the situation. This approach
is focused on the service: it produces standards which can be
compared between Organisation Units or hospitals applying
the same method, although it consider neither the health organization processes nor the quality of the services offered.
The validity of the result is strongly affected by the way in
which estimations are introduced, by the method of data
collection and by the survey sources used, although, being a
synthetic approach, it implies a diffused and non-answerable
involvement.
2. Methodologies which follow a BOTTOM-UP or “analytical” APPROACH. Following this approach, the workload
calculation is made on the procedures subdivided into microphases. The standard unit work time needed for the production of each typology of output is calculated by the analytical
measurement of the time necessary for the execution of each
procedural micro-phase in terms of man-time. The determination of standard loads requires analytical surveys, which
179
Lectures
absorbs time to the business applying it: for this reason this
methodology involves a high motivation and participation, but
once implemented, it can surely be a useful tool for the management of Organisation or Department Units. It is focused on
the business processes and produces results that are very little
comparable between different Units or hospitals.
Actually, the most diffused approach among the existing
methodologies for the determination of workloads, which is
applied by different businesses to comply with law obligations, is the top-down approach. The reason for this choice
can be mainly identified in the fact that the complexity of
the health system does not allow for analytical surveys about
the procedures (which are very often not explicit) within the
deadline set by the legislation. Furthermore, this approach
makes it possible to obtain a global overview on the use of
human resources, thus on the efficiency levels both at a business and inter-business level (regional or national), by comparing similar structures, and it allows for the determination
of standard loads which are common to different businesses.
It overcomes the problem of the determination of standard
loads by comparing production times of similar realities.
Last but not least, we can affirm that this method respects
more the professionalism of the operator, who is not considered as a mere executor of tasks, but is made responsible for
a determined set of objectives.
Methods. The project on standard load measurement for
Pathology Unit has been developed and implemented into the
following phases: i) creation of the work team and definition
of the hospitals sample; ii) choice of the methodology; iii) creation of the reference activities list; iv) attribution of activity
to the different professional profiles involved; v) creation of
the informatics support for the collection and analysis of the
information; vi) data collection; vii) data elaboration and
simulation.
The analysis of the national and regional legislation carried
out by the hospital reference persons has not evidenced anything particular that would have driven the choice towards a
particular methodological approach. The choice of the methodology has been influenced by the cost of the information
collection, on one side, and by the necessity to obtain a standard that would have made possible the comparison among
different hospitals realities (limitation of the standard variability), on the other. Therefore, the work team has decided
for the application of the methodology with TOP DOWN
APPROACH for the calculation of the standard execution
time of the activities, together with observations based on the
BOTTOM UP APPROACH to determine the standard load
of some specific and particularly critical activities (Exfoliative cervico-vaginal cytology; exfoliative cervico-vaginal
cytology on thin-layer preparation; cervical-vaginal drawing;
autopsy with histology).
The work team has decided to test such method on data concerning human resources and activities in a limited number of
Italian Pathology Units (8) over the three-year period 20032005 and to extend the observation to a larger number of
Italian Pathology Units (27) over the period 2005-2007. The
27 Italian Pathology Units are distributed as follows: 20 in the
North Area; 6 in the Central Area and 1 in the South Area.
Concerning the typology of health organizations to which the
experimenting Pathology Units belongs, 5 Units belong to
Teaching Hospitals, 1 Unit belong to a Cancer Institute, the
others belong to General Hospitals.
As far as the observation of the activities is concerned, the
work team has decided to adopt the 2002 SIAPEC activities list (“Nomenclatore tariffario” – second revision) which
already included a weight system defined by SIAPEC itself,
and which is also used to attribute the activities to the different
professional profiles.
Results. Pathologist, Biologist and Pathology Technician
are the key professional profiles on which the analysis
is focused. The average amount of hours over the period
2005-2007 of the whole sample varies very much both in
case of the same professional profile and in case of different
professional profiles. This can be explained by a different
labour organisation. Also the average amount of activities
of the whole sample over the three years 2005-2007 varies
concerning both the total amount produced, and the weight
of the activities observed with the BOTTOM UP approach
(Pap test and autopsies) on the total of the activity produced.
In order to limit the variability phenomenon, the work team
has decided to exclude the following from the data analysis:
i) the centres with values strongly above average and ii) the
centres with values well below average. The work team has
decided not to consider outlier the centres with values below
average for some professional profiles and above average
for others. Therefore the analysis has been carried out on
the data of 22 centres. The Average Time per weight unit
(total hours/total points) of the sample excluded the outlier
centres, both per year and aggregated for the three-years
period (Tab. I) shows anyway a great variety among the
centres, probably due to a different distribution of the activities among the professional profiles, thus due to a different
labour organisation.
Tab. I
Avarage Time
Avarage Time
Liguria
Avarage Time
Piemonte+VDA
Avarage Time
Lombardia
Avarage Time
North
Avarage Time
Centre
Nurses
min/point
1.37
0.00
Path/Bio
OTA
min/ point min/ point
9.76
3.21
11.21
4.23
Administrative PathTechn.
min/ point
min/ point
3.45
14.54
1.69
17.03
0.46
10.86
2.76
3.64
17.33
0.73
6.92
3.78
3.31
14.42
0.43
8.59
3.37
3.06
15.29
2.53
12.36
2.30
1.68
11.45
180
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
The appropriateness of pathological reports
M. Pavesi
s.o.c. Anatomia e Istologia Patologica, ASLAL Casale Monferrato
Italia
Pathologists are faced with two different kinds of relationships: one with the patient (from whom the specimen was
taken) and one with the patient’s doctor, either a specialist or
a general practitioner.
Therefore, a pathologist’s report should be intelligible to
addressees differing significantly from a cultural and a linguistic point of view but having the same needs: discovering
pathologies quickly and accurately in order to programme a
well-timed therapy.
In the light of these facts, pathologists’ reports should be
concise (stating the final diagnosis briefly), clear (easily interpretable, though not giving up the usually necessary scientific
terminology) and have a univocal interpretation (understood
by both specialist and family doctors despite their different
training).
In any case, the appropriateness of pathological reports
cannot do without the evolution of etiological and pathogenetical findings of illnesses, together with a complete and
complex evaluation of the prognostic factors of cancers.
These aspects are of paramount importance to oncologists,
who are to stadiate the illness and may use new drugs in the
target therapy, which has also been legislatively approved
for certain neoplastic diseases (breast, colonic and lung
carcinoma).
Therefore, an appropriate report should be complete and include, where possible, information about the biological and
clinical course of the illness and all the factors that define its
pathological stadiation.
Moreover, pathologists’ reports should be precise and unequivocally specify the received and examined tissue, any
technical and special stains integration into normal procedures
for diagnostic reasons, and the clinical and anamnestic data
which are believed useful to pathological diagnoses.
In the light of this, pathologists organise their final report in a
complex way, again with the aim of giving more complete and
clearer information about the pathology in question.
Sometimes an attitude as such may turn out to be counterproductive, insofar as pathologists are unconsciously led to
make deductions on a purely unscientific basis: the need for a
complete consultation on pathologies should not confuse the
objectivity of the observation.
Observations should never be considered of minor importance
as pathology is mainly a morphological study. As such, it
cannot do without a descriptive observation of the extracted
tissue. Ancillary colouring techniques are an essential aid to
the final diagnosis, but should not lead to conclusions based
on their interpretation only.
The final diagnosis of reports should result from an integration
of morphological data and biological information obtained
from the tissue in question (immunophenotype, molecular biology) together with clinical, serological and anamnestic data
of the patient from whom the specimen for the pathological
diagnosis was taken.
Pathologists’ proper behaviour while writing the final report
safeguards themselves and their whole staff from legal measures in case of patients’ complaints against inappropriate
medical treatments.
Pigmented skin lesions
Moderators: G. Massi (Roma), G. Collina (Bologna)
Dermoscopy and histopathology of nevi and
melanomas: pitfalls and diagnostic correlations
3
Bauer J, Metzler G, Rassner G, et al. Dermatoscopy turns histopathologists’s attention to the suspicious area in melanocytic lesions. Arch
Dermatol 2001;137:1338-40.
G. Ferrara
Anatomic Pathology Unit, Gaetano Rummo General Hospital, Benevento, Italy, (E-mail [email protected])
The increasing use of dermoscopy in preoperative diagnosis
of melanocytic skin neoplasms (MSN) is impacting on routine histopathology to a relevant extent. We herein present
the dermoscopic-pathologic features of some cases of histopathologically controversial MSN. By illustrating these cases,
we would like to emphasize at least three different fields of
interest for a combined (clinico-)dermoscopic-pathologic
diagnostic approach, namely: information about the evolution
of lesions; detection of gross sampling errors; definition of
peculiar clinicopathologic entities. The theoretical and practical aspects of a close interaction among dermoscopists and
histopathologists are itemized in detail.
References
1
Ferrara G, Argenziano G, Soyer HP, et al. Dermoscopic and histopathologic diagnosis of equivocal melanocytic skin lesions. An interdisciplinary study on 107 cases. Cancer 2002;95:1094-100.
2
Ferrara G, Argenyi Z, Argenziano G, et al. The influence of the clinical
information in the histopathologic diagnosis of melanocytic skin neoplasms. PLoS ONE 4(4):e5375. doi:10.1371/journal.pone.0005375.
Spitz nevi, atypical spitz tumors and spitzoid melanomas: diagnostic application of fluorescence in situ hybridization and
p16 immunohistochemical stain
C. Clemente, F. Cetti Serbelloni, S. Pagliarini, L. Scopsi *
Pathology and * Cancer Genetics Services, Casa di Cura S. Pio
X, Milan, Italy
Background. Recently, Abbott Molecular commercialized a
multi-color FISH probe mixture to assist pathologists in the
differential diagnosis of difficult melanocytic lesions. The
probe mixture includes a centromeric probe for chromosome 6
and unique sequence probes for the RREB1 gene (6p25), MYB
gene (6q23-q23), and CCND1 gene (11q13). The centromeric
probe (CEP6) was included as a control for the ploidy level
of chromosome 6, while the other three were chosen because
their respective chromosomal regions have most frequently
shown amplifications or deletions in melanoma. After a preliminary study aimed at evaluating the technical application of
the kit 1, we wanted to test this new tool on an array of spitzoid
lesions including: Spitz/Reed nevi, Spitz/Reed tumors with
atypical features, spitzoid melanomas, and other more complex lesions with features simulating the previous ones. The
181
Lectures
p16 immunohistochemical stain developed by mtm Laboratories AG was also tested. The spitzoid lesions represent the
most difficult area in the differential diagnosis of melanocytic
tumors and in our second opinion experience Reed nevus is
the most frequent entity misdiagnosed for melanoma (Clemente, unpublished).
Methods. Sections from 112 archival paraffin blocks corresponding to 109 patients were obtained (four cases had
two different specimens each). Eighty of the 112 specimens
were from consultation files and came from a wide array
of Italian health institutions. The selection criteria adopted
included those listed in the background section. H&E slides
were used to accurately identify the area(s) of interest, which
were marked with a glass pen on the back of the slides to be
used in the FISH procedure. The samples for FISH analysis
were treated strictly following the manufacturer’s protocol as
specified in the kit’s instructions (SP). The evaluation was
carried out using an Olympus BX 51 fluorescent microscope
equipped with a filter set including DAPI, spectrum aqua,
spectrum green, spectrum yellow and spectrum red. Scoring
was restricted to cells from the areas previously identified on
the matched H&E sections and was carried out by two observers separately, without prior knowledge of the diagnosis
(LS and FCS). Whenever feasible, scoring was done on 150
(75 + 75) non-overlapping intact nuclei. A specimen was considered positive if at least one of the following criteria was
met: CCND1 % gain > 38, RREB1 % gain > 29, percent loss
of MYB against CEP6 > 40%, percent gain of RREB1 against
CEP6 > 55%. A specimen was labeled as FISH-negative if
none of the above criteria were met.
P16 immunostaining was tested on a large series of 342 cases:
68 Spitz/Reed nevi, 22 atypical Spitz/Reed tumors, 56 dysplastic nevi, 47 common nevi and 130 melanomas.
Results. Seven FISH samples were not assessable because
of technical reasons. For practical purposes, specimens were
divided into four main categories: benign nevi, atypical
melanocytic tumors, dysplastic nevi, malignant melanomas.
Positivity ensued mainly from loss of MYB, followed by gain
in RREB1 and gain in CCND1. Forty out of 46 histologically
benign nevi scored negative: among these were all Reed nevi
and 90% of Spitz nevi. The six positive nevi included two
Spitz nevi, three Spitz-like compound nevi and one epithelioid blue nevus. Of the 21 atypical tumors, 16 scored negative; the five scoring positive included three atypical Spitz
tumors, one atypical cellular blue nevus and one atypical
epithelioid melanocytic (Spitz-like) tumor. Only one out of
seven dysplastic nevi scored positive. 10 out of 31 melanoma
specimens scored positive, the remaining resulting negative.
Among these latter: one is a vulvar lesion of a 9-year girl;
two others have features of nevoid and one of desmoplastic
melanoma, two types of melanoma we found negative also
in a previous study performed with this FISH tool 1. One is
a melanoma arising in (and mixed with) a nevus and it cannot be excluded that a portion of the nuclei evaluated during
the scoring procedure belonged to normal melanocytes. This
drawback, which is also present in specimens where the lesion is represented by small nests or even single cells in close
contact with the epithelial cells or lymphocytes, calls for caution in interpreting the results. Interestingly, a lymph node
metastasis from this same patient was FISH-processed too
and resulted strongly positive. Five other negative melanomas
belonged to the superficial spreading category and five to the
spitzoid type. A preliminary look at possible links between
FISH results and prognostic factors in melanomas showed
that positivity was mainly associated with the worst presenting signs and that the strongest positivity was restricted to the
three metastasis.
A p16 positive moderate to intense stain was present in 78%
of Spitz/Reed nevi, 41% of melanomas, 36% of atypical
Spitz/Reed tumor, 68% of dysplastic nevi and 64% of common nevi.
Reference
1
Clemente C, Bettio D, Venci A, et al. A fluorescence in situ hybridization (FISH) procedure to assist in differentiating benign from malignant melanocytic lesions. Pathologica 2009;101(5):169-74.
Nevoid Melanoma
G. Collina
Bologna
Nevoid melanoma is one of the most deceptive lesions in
dermatopathology. Probably it is the
hottest issue in the area of pigmented lesions at the moment,
given that atypical Spitz/nevi tumors
are well studied and more often approached in practical work
with a defensive attitude.
The term nevoid melanoma was used by Schmoeckel, Castro
and Braun-Falco in 1985 to describe primary cutaneous malignant melanomas with histological features suggestive of
benign nevocytic nevi 1. They stated that some of the following histological characteristics were always observed: cellular
atypia, mitoses, adnexa infiltration in the deeper dermis, infiltrative growth, pigmented tumor cells, sharply demarcated
tumor nests, and the absence of maturation.
The clinical behavior of nevoid melanoma does not differ significantly from ordinary melanoma, and tumor thickness was
the most important prognostic criterion. Lesions which share
similar histological findings were called borderline melanoma
by Reed, Clark and Mimh in 1975 2. In 1985 the Mhim group 3
initially described this subset of lesions with the term minimal
deviation melanoma, but in 1995 they decided to use the more
committal and popular term of nevoid melanoma, suggesting
that proper attention to cytological detail and subtle architectural features will aid in recognizing this unusual variant of
malignant melanoma 4 5. Similar observations were made by
Zembowicz et al. 5
Three paradigmatic cases are discussed:
1)a nevoid melanoma which turned out to be a benign nevus;
2)a previous benign nevus which behaved as a melanoma;
3)I don’t know-case.
References
1
Schmoeckel C, Castro CE, Braun-Falco O. Nevoid malignant melanoma. Arch Dermatol Res 1985;9:362-9.
2
Reed RJ, Ichinose H, Clark WH Jr, et al. Common and uncommon melanocytic nevi and borderline melanomas. Sem Oncol 1975;2:119-47.
3
Mérot Y, Mihm MC Jr. Unusual and unknown aspect of cutaneous
malignant melanoma: minimal deviation malignant melanoma. Retrospective study of 4 cases. Ann Dermatol Venereol 1985;112:3256.
4
Wong TY, Duncan LM, Mihm MC Jr. Melanoma mimicking dermal
Spitz’s nevus (“nevoid” melanoma). Semin Surg Oncol 1993;9:18893.
5
Wong TY, Suster S, Duncan LM, et al. Nevoid melanoma: a clinicopathological study of seven cases of malignant melanoma mimicking
spindle and epithelioid cell nevus and verrucous dermal nevus. Hum
Pathol 1995;26:171-9.
6
Zembowicz A, McCusker M, Chiarelli C, et al. Morphological
analysis of nevoid melanoma: a study of 20 cases with a review of the
literature. Am J Dermatopathol 2001;23:167-75.
182
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Slide seminar: Non-neoplastic skin diseases
Moderators: C. Angeli (Vercelli), D. Massi (Firenze)
The granulomatous pattern in skin diseases
A.M. Cesinaro
Department of Anatomic Pathology, Azienda Ospedaliero-Universitaria, Policlinico di Modena, Italia
Background. The granulomatous reaction pattern is characterized by the presence of granulomata, i.e. collections of
histiocytes or epithelioid histiocytes, in the dermis, with or
without admixed multinucleated giant cells and other types
of inflammatory cells. The granulomata can show peculiar
arrangements, accessory features such as necrosis, suppuration, or necrobiosis, and the presence of organisms or foreign
material. Based on the histological features, granulomata can
be sub-classified in the following types: sarcoidal, with the
classic “naked” appearance; tuberculoid, characterized by
central “caseation” necrosis; necrobiotic, showing more loose
arrangement and necrobiosis (collagenolysis); suppurative,
featuring central collections of neutrophils; foreign body-type,
in which foreign material, either exogenous or endogenous,
is identifiable; xanthogranulomatous, characterized by histiocytes with foamy or pale cytoplasm and admixture of other
inflammatory cells; a miscellanea of other conditions 1.
Case report nr. 1. A 28-years old woman, born in Philippines, presented a solitary, annular plaque on the lower leg,
asymptomatic and slowly enlarging in the last few months. A
4-mm punch biopsy was performed on the border of the lesion
and sent for histological examination with a clinical diagnosis
of granuloma annulare. The haematoxylin-eosin stained slide
showed a granulomatous inflammatory infiltrate in the superficial and deep dermis, coupled to a moderate lymphocytic
component admixed with few plasma cells. The granulomata
were arranged mostly around vessels and encased a nerve,
as highlighted by immunostaining for S-100 protein. Special
stains (PAS, Grocott, Ziehl-Neelsen, Fite) failed to show microorganisms. PCR studies were not performed. A diagnosis
suspicious for leprosy, tuberculoid type, was rendered. The
patient was referred to a national centre for infectious diseases
(Genoa) for further investigations. The diagnosis of leprosy,
tuberculoid type, was confirmed.
The presence of granulomata should always suggest to look
for an infectious agent. It is also recommended to perform
special stains on multiple sections. Despite exhaustive search,
these stains can fail to demonstrate the presence of microorganisms. The presence of perineural granulomatous inflammation in this case strongly addressed toward the diagnosis
of leprosy.
Case report nr. 2. A 55-years old woman, living in Sicily,
complained of erythematous plaques on face and trunk for
2 years. Histological examination of a punch biopsy from
the dorsum showed a granulomatous inflammatory infiltrate
throughout the dermis, around vessels and also surrounding
nerves. Special stains were negative. The pattern of distribution suggested an infectious disease, i.e. leprosy, but this possibility was excluded by further investigations. The clinical
history of the patient allowed to achieve the right diagnosis:
the woman suffered from hypogammaglobulinemia and biliary cirrhosis and had had a diagnosis of common variable immunodeficiency. Patients with immunodeficiency disorders
can develop cutaneous lesions with a granulomatous reaction
pattern 2. Moreover, a patient with congenital combined immunodeficiency has been reported, whose cutaneous lesions
featured granulomata with perineural distribution 3, analogously to the present case.
Besides the two stereotypical cutaneous granulomatous diseases, i.e. granuloma annulare and necrobiosis lipoidica, the
granulomatous reaction pattern in the skin can have several
causes and associations. It can be due to the deposition of
foreign material, or to prolonged sun-light exposure leading to
actinic changes, such as the group of so-called elastolytic granulomata. It can be observed in a large number of infectious
diseases (TBC and non tuberculous mycobacteriosis, leprosy,
leishmaniasis, fungal infections). It can be related to systemic
conditions, such as sarcoidosis, Crohn’s disease, Rosai-Dorfman disease, haematological disorders, immunologic disorders, and also to the use of certain drugs. On the other hand,
it is known also that a skin disease characterized by a peculiar
granulomatous pattern at histology, such as granuloma annulare, can show protean clinical manifestations 4. Infrequently,
mycosis fungoides features a granulomatous pattern that
overlaps the histological characters of granuloma annulare.
Only few subtle clues allow to make the differential diagnosis
between the two diseases, and sometimes the differentiation is
almost impossible and can only rely on molecular biology 5.
Moreover, granuloma annulare-like features can be observed
in certain drug reactions, again with only subtle histological
differences 6. Finally, pathologists should be aware of the possibility that an apparently innocent granulomatous reaction can
hide a life-threatening condition, such as lymphoma 7.
All these observations underline the importance of the clinicopathological correlations when one is dealing with a granulomatous reaction in the skin, since histology alone could not
be sufficient and sometimes can also lead toward the wrong
diagnosis.
References
1
Weedon D. Skin Pathology. 3rd Ed.
2
Mitra A, Pollock B, Gooi J, et al. Cutaneous granulomas associated with primary immunodeficiency disorders. Br J Dermatol
2005;153:194-9.
3
Krupnick AI, Shim H, Phelps RG, et al. Cutaneous granulomas
masquerading as tuberculoid leprosy in a patient with congenital
combined immunodeficiency. Mt Sinai J Med 2001;68:326-30.
4
McKee PH, Calonje E, Granter SR. Pathology of the skin with clinical
correlations. Vol. 1. 3rd Ed.
5
Su LD, Kim YH, LeBoit PE, et al. Interstitial mycosis fungoides, a
variant of mycosis fungoides resembling granuloma annulare and
inflammatory morphea. J Cutan Pathol 2002;29:135-41.
6
Magro CM, Crowson AN, Shapiro BL. The interstitial granulomatous
drug reaction: a distinctive clinical and pathological entity. J Cutan
Pathol 1998;25:72-8.
7
Scarabello A, Leinweber B, Ardigò M, et al. Cutaneous lymphomas
with prominent granulomatous reaction: a potential pitfall in the histopathologic diagnosis of cutaneous T- and B-cell lymphomas. Am J
Surg Pathol 2002;26:1259-68.
Non-neoplastic skin diseases: Case n. 2
G. Collina
Bologna
Clinical History. 30-year-old man showed coppery-red papules localized in the trunk and limbs. Previous clinical history
183
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was unremarkable and the patients was in good health. A
papule present in the leg was biopsied.
Histopathology. A sparse, mostly superficial, perivascular
infiltrate made up overwhelmingly of lymphocytes arranged
also in patchy lichenoid fashion that obscures focally the base
of unevenly hyperplastic epidermis topped by parakeratosis in
mounds staggered in the lower half of a stratum corneum is
characteristic of secondary syphilis. Among the lymphocytes,
especially in the immediate vicinity of venules of the superficial plexus, are numerous plasma cells. Vacuolar alteration in
company with a sprinkling of lymphocytes along the dermoepidermal junction and a tad of spongiosis in loci within surface epidermis are also present.
Diagnosis. Secondary syphilis
Discussion. The case presented is an example of secondary
syphilis occurring in a 30-year-old patient. Clinically, this
could, conceivably, be a drug reaction, but the possibility can
be excluded by the assessment of the rest of the integument,
the results of the histology and of studies serologically. The
lesion are papules mostly because of somewhat lichenoid arrangement of the infiltrate of lymphocyte and plasma cells
and the peculiar orange hue is consequence, in part, of the
combination of widely dilated venules which in vivo housed
countless erythrocytes and the peculiar distribution of inflammatory cells, those two findings are present in the upper part
of the dermis.
The histological findings were those of a lichenoid-psoriasiform dermatitis in which plasma cells predominate. This was
strongly suggestive of secondary syphilis. The diagnosis was
confirmed by serology. We could not demonstrate Treponema
pallidum (TP) on histological sections using Warthin Starry
stain.
The correlation between clinical and histopathological findings were crucial for achieving the correct diagnosis.
Acquired syphilis caused by TP has affected humanity since at
least the fifteen century, but the advent of penicillin reduced
the incidence of the disease in the rich world so that many clinicians are nowadays unfamiliar with its signs and symptoms.
Recently the incidence of syphilis is rising because is linked
to the immunodeficiency virus infection.
TP is generally spread to contact between infectious lesions
ad disrupted epithelium at the site of minor trauma during the
intercourse. The transmission rate is between 10% and 60%.
The disease shows four clinical detectable phase. Primary
syphilis is defined as the typical chancre that appear clinically as a regular edge, regular based, hard and bottom-like
ulceration measuring up to one centimetre in diameter. Unless
secondary infected, chancre is not painful. Multiple lesion
may be present and 25% of patients (predominately woman)
diagnosed at second-stage syphilis had no history of primary
infection. As a rule, the chancre heals spontaneously in 3-8
weeks and rarely persist for more than three months. Histologically, fully developed lesions are constituted by dense
inflammatory infiltrate composed of lymphocytes, histiocytes
and plasma cells. The blood vessels are increased in number
and are bordered by plump endothelial cells. Oedema is a
features in the upper dermis along with the ulcerations of the
epidermis; this latter covered by fibrin and crust.
Secondary syphilis results from the haematogenous dissemination of the TP, resulting in more widespread clinical signs
accompanied by fever, malaise and generalized lymphoadenopathy. A generalized eruption can occur comprising orange
maculae, papules and papulosquamous lesions resembling
guttate psoriasis. Rarely pustules are present. The three most
common histopatological patterns of secondary syphilis are
psoriasiform, lichenoid and psoriasiform-lichenoid and they
occur in conjunction with superficial and deep perivascular
infiltrate in which plasma cells predominate. Exocytosis of
lymphocytes spongiform pustulations (which harbour TP)
and parakeratosis may be seen. Parakeratosis may be broad
or paltry. Granulomatous inflammation may be seen in older
lesions.
Primary and secondary lesions may be unnoticed by the patient who then passes in the latent phase of the disease.
Tertiary syphilis is categorized into nodular tertiary syphilis
confined to the skin; benign gummatous syphilis principally
affecting skin, bone and liver; syphilitic hepatic cirrhosis,
cardiovascular syphilis and neurosyphilis.
The histopathological findings of tertiary syphilis are those of
necrotizing granolumatous reaction.
Secondary syphilis should be differentiated from other inflammatory or neoplastic disease of the skin. When the inflammatory infiltrate is particular heavy and lymphocytes show atypical features the possibility of mycosis fungoides may be suggested. In this latter condition lymphocytes predominate and
plasma cells are usually absent. Mucha- Haberman disease
is composed almost entirely by lymphocytes. Psoriasis lacks
histiocytes and plasma cells and usually the inflammatory
infiltrate is more superficial and does not involve the blood
vessels of mid dermis. In syphilis the present of spongiform
pustules may be observed, but nary attenuation of suprapapillary plate is a feature.
Secondary syphilis should also be differentiated from all
lichenoid dermatitis in which lymphocytes predominate such
as lichenoid drug eruption, lichenoid photodermatitis and
lichenoid discoid lupus erithematosus. In syphilis, except for
the early second phase in which plasma cells may be paltry,
even absent, the infiltrate being made up nearly exclusively of
lymphocytes, the presence of plasma cells may be considered
a signal of this venereal disease.
Pityriasis lichenoides and cutaneous
vasculitides
C. Miracco
Section of Pathological Anatomy- Department of Human Pathology
and Oncology- Siena- Italy
Background. Pityriasis lichenoides (PL) is an uncommon inflammatory skin disorder of unknown histogenesis, that may
occur either in acute (pityriasis lichenoides et varioliformis
acuta, PLEVA; and febrile ulceronecrotic Mucha-Habermann
disease) or chronic (pityriasis lichenoides chronica, PLC)
form. Acute PL is usually a self-limiting polymorphous eruption of macules, papules, and pustulae, which may evolve
into hemorrhagic and necrotic lesions; recurrences over the
years, as well as lethal febrile cases are not infrequent. PLC,
the prolonged form of the disease, has a more indolent clinical
course, with recurrent erythematous, scaling papules, tending
to regress within some weeks. By some authors PL is classified among the cutaneous lymphocytic vasculitides, although
classical signs of blood vessel damage are usually missing. PL
by most is instead included among the interface-dermatitides,
due to the heavy inflammatory infiltrate obscuring the dermalepidermal junction. No invasion of vessel walls by inflammatory cells is in fact observed in PLC; and non-vasculitic vessel
changes are the rule in acute PL, in some lesions, however, a
damage of vessel walls, with fibrinoid necrosis and leukocytoclasis may occur; in these cases, other histological findings
are relevant to exclude a true skin vasculitic process.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Cases and Methods. Two cases of PLEVA occurred in an
8-year-old boy and in a 24-year-old man will be shown. In
the first case, the diagnosis was supported by clinical data and
diagnosis. In the second case, there was no clinical diagnosis,
however a description of an eruption of papules and hemorrhagic lesions was given. The histological diagnosis was of
PLEVA in both cases, based on the observation of a wedgeshaped inflammatory infiltrate, obscuring the dermal-epidermal junction, with variable degrees of keratinocyte damage,
up to necrosis and epidermal ulceration, accompanied by
dermal vessel alterations, with erythrocyte extravasation.
Results and Discussion. Histological findings of PLEVA,
as well as of other true skin vasculitides and other common
forms of cutaneous pseudovasculitides will be shown and
discussed for the differential diagnosis. On the one hand, vas-
cular changes, including engorgement and oscuring of dilated
dermal vessels by lymphocytes, and endothelial proliferation,
as well as extravasation of erythrocytes in the dermis and epidermis, usually seen in acute PL, are helpful diagnostic clues
in the differential diagnosis with other diseases characterized
by an interface dermatitis. On the other hand, the wedgeshaped pattern of the lesion, with a dense, predominantly
lymphocytic infiltrate at the dermal-epidermal junction, with
lymphocyte exocytosis, and vacuolar alteration of the basal
layer, are useful diagnostic criteria for the differential diagnosis with true vasculitides and pseudovasculitides. Clinics will
also be shown and compared with histological findings. An
haematoxylin and eosin stain, supported by clinical findings,
is usually sufficient for a correct diagnosis, which is mandatory for an adequate treatment of PLEVA patients.
185
Lectures
Friday, September 24th, 2010
Symposium on haemolymph pathology: the experience of the WHO
Moderator: S. Pileri (Bologna)
WHO classification of tumours of
haematopoietic and lymphatic tissues:
methods, current issues, future perspectives
S. Pileri
Bologna
As indicated above, there is no one “gold standard”, by which
all diseases are defined in the WHO classification. Morphology is always important, and many diseases have characteristic
or even diagnostic morphologic features. Immunophenotype
and genetic features are an important part of the definition of
these diseases, and the availability of this information makes
arriving at consensus definitions much easier than when only
subjective morphologic criteria were available. Immunophenotyping studies are used in routine diagnosis in the vast
majority of haematologic malignancies, both to determine
lineage in malignant processes and to distinguish benign from
malignant processes. Many diseases have a characteristic
immunophenotype, such that one would hesitate to make the
diagnosis in the absence of the immunophenotype, while in
others the immunophenotype is only part of the diagnosis. In
some lymphoid and in many myeloid neoplasms a specific
genetic abnormality is the key defining criterion, while others lack specific known genetic abnormalities. Some genetic
abnormalities, while characteristic of one disease, are not specific (such as,, or rearrangements or mutations in), and others
are prognostic factors in several diseases (such as mutations
or). The inclusion of immunophenotypic features and genetic
abnormalities to define entities not only provides an objective
criterion for disease recognition but has identified antigens,
genes or pathways that can be targeted for therapy; the success
of rituximab, an anti-CD20 molecule, in the treatment of Bcell
neoplasms, and of imatinib in the treatment of leukemias
associated with ABL1 and other rearrangements involving
tryosine kinase genes are testament to this approach. Finally,
some diseases require knowledge of clinical features – age,
nodal vs extranodal presentation, specific anatomic site, and
history of cytotoxic and other therapies – to make the diagnosis. Most of the diseases described in the WHO classification
are considered to be distinct entities; however, some are not
as clearly defined, and these are listed as provisional entities.
In addition, borderline categories have been created in this
edition for cases that do not clearly fit into one category, so
that well-defined categories can be kept homogeneous, and
the borderline cases can be studied further.
Hepatitis C virus (HCV) related lymphomas: a new
model for lymphomagenesis
M. Paulli, M. Lucioni, G. Fiandrino, M. Nicola, L. Arcaini *
Pathology Section, Department of Human Pathology and * Division of
Hemathology, University of Pavia, Foundation IRCCS Policlinico San
Matteo, Pavia, Italy
Immunodeficiency, autoimmunity and sustained activation of
the lymphoid system, which frequently accompanies chronic
infections, represent a risk factor for subsequent lymphoma
development. Similarly, congenital and acquired immunodeficiencies associated with HIV infection and solid organ
transplantation increase the risk of developing B-cell NHLs.
On the other hand, autoimmune diseases such as Sjögren
syndrome are also associated with an increased risk of lymphomas. The geographic heterogeneity in the incidence of
B-cell non Hodgkin lymphomas (NHLs) suggests that also
environmental factors such as infections might have a role in
lymphomagenesis.
In fact, particular lymphoma subtypes have been associated with specific microbial infections, which may promote
lymphomagenesis by creating a favourable environment for
transformation, with increased proliferation and decreased
apoptosis of lymphoid cells, via direct or indirect mechanisms. Lymphotropic transforming viruses such as Epstein
Barr virus (EBV), human herpes virus 8 (HHV8) and human
T-lymphotropic virus 1 (HTLV-1) directly infect a subset of
lymphoid cells in which they express viral oncogenes, with
transforming abilities. An alternative scenario has emerged
for microbial species that do not directly infect or transform
lymphoid cells, but may persist chronically in host tissues and
trigger a sustained lymphoid proliferation, giving a selective
advantage to lymphoid clones that initially are still dependent
upon antigenic stimulation. In this setting, additional oncogenic events may occur, leading the lymphoid proliferation
to become independent of antigenic stimulation. The best
documented model for indirect lymphomagenesis mediated
by infectious agents is represented by Helicobacter pylori
in gastric marginal zone lymphoma, but similar mechanisms
have been more recently proposed for Borrelia burgdoferi in
cutaneous B-cell lymphomas, Chlamydia psittaci in ocular
adnexal lymphoma of mucosa-associated lymphoid tissue
(MALT) and Hepatitis C virus (HCV) in several B-cell
NHLs.
HCV, a positive single-stranded RNA virus, belongs to the
family of Flaviviruses; it affects millions of patients worldwide
and represents a major burden in term of morbidity, mortality
and social costs. Estimated prevalence in Italy is up to 10%,
representing one of the highest rates among western countries.
It is now well recognized that, in addition to hepatic manifestations, HCV infection is linked to a spectrum of cryoglobulinemic and non-cryoglobulinemic B-cell lymphoproliferative
disorders. A meta-analysis showed that prevalence of HCV
infection in both nodal or extranodal B-cell non-Hodgkin’s
lymphoma (NHL) patients is 15% in comparison with 1.5%
in the general population; this association is more evident in
geographic areas with high HCV seroprevalence. A study in
2004 estimated that for Italy, due to high seroprevalence rates,
the attributable risk for HCV infection in lymphoma development would be up to 10% of all new cases.
Reports in the literature dealing with lymphoproliferative
disorders associated with HCV infection indicate variable incidences for the various lymphoma histotypes. This variability
may reflect alternative classification approaches, differences
in HCV infection rates among geographic areas and variable
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
oncogenic potential and/or lymphoid tropism for different
HCV serotypes, even if the latter hypothesis is most doubtful.
In spite of some discrepancies, HCV-associated B-cell NHL
seem to have distinctive clinicopathological features including a predilection for extranodal localizations and an overrepresentation of the diffuse large B-cell (DLBCL) and marginal
zone (MZL) histological subtypes; some authors also reported
an increased incidence in the female sex.
The association of HCV infection with lymphoma of liver,
salivary glands, and spleen was investigated by many Authors. Among MZL subtypes, we reported a stronger association with HCV infection in the splenic and nodal form as well
as in non-gastric MALT lymphomas. Notably, the association
of HCV with splenic MZL with villous lymphocytes has been
proposed as a paradigmatic example of a HCV-driven lymphoproliferative disorder.
Recently, presence of HCV infection has been also detected in
cases of primary cutaneous B-cell lymphomas, irrespectively
of clinicopathological subtype. Our group has also recently
described a series of HCV-positive patients who developed
a MZL, characterized by unusual lipoma-like subcutaneous
presentation, and a relatively indolent clinical course. Extensive molecular and genetic investigations seem to indicate
that HCV infection may play a causative role in this peculiar
lymphoma subset.
In addition to epidemiological studies, the possible pathogenetic role for HCV in mixed cryoglobulinemia and lymphoproliferative disorders have been confirmed by lymphoma
regression following antiviral therapy, that has been observed
in some patients. Antiviral therapy with interferon-α (IFNα) is known to be highly efficacious in the treatment of
type II mixed cryoglobulinemia. As for NHL developing in
HCV-positive patients, anti-viral therapy with IFN-α with or
without ribavirin was reported to induce complete remission
of the disease in up to 75% of cases irrespective of their histological subtype even if, altogether, the number of analyzed
cases appears disproportionately low. High rates of molecular
remission with disappearance of previously documented of
IgH rearrangements and/or t(14;18) translocation have also
been reported.
From the biological point of view, it is known that HCV can
infect B-cells in vitro; until now, however, there is a lack of
evidence for a direct infection of lymphoid cells by HCV as
a trigger for lymphoma development. In fact, only a fairly
small subset of LNHs arising in HCV positive patients actually host viral genome; on the contrary, the virus itself has
been detected in accompanying stromal cells of affected
lymph nodes. In addition, HCV is a RNA virus lacking DNA
intermediates in its replicative cycle and it seems unlikely that
integration of HCV genome into the host might take place.
Therefore, it appears more plausible that lymphoid antigendriven proliferation represents an early and facilitating event
leading to lymphoma through an indirect pathway. A clue for
this comes from the analysis of B-cell clones obtained from
HCV-positive controls. Among the latter, IgH rearrangements (monoclonal or oligoclonal) are demonstrable in up to
100% of patients with type II mixed cryoglobulinemia; approximately 8 to 10% of all type II mixed cryoglobulinemia
patients actually will develop NHL after a long follow-up.
Additionally, recombinant E2 viral protein exposed on HCV
virion surface was demonstrated as sufficient to induce hypermutations at the immunoglobulin locus when binding to
CD81 coreceptor of B lymphocytes. Finally, a biased usage
of IGH genes was documented in HCV-associate lymphomas
and a certain degree of homology was detected between B-cell
receptors (BCR) and anti-viral antibodies in HCV-positive
NHL patients.
In conclusion epidemiologic data, molecular findings and
clinical evidence of lymphoma regression after antiviral therapy seem to indicate that HCV may play a causative role in
some B-cell NHLs. Some recent clinico-pathological reports
confirm that lymphomas associated with HCV infection may
have peculiar clinical features (such as subcutaneous presentation mimicking lipoma), that may deserve particular attention
in order to be properly recognized.
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Lectures
Burkitt lymphoma, fifty years plus: the
beginning and the future
E. Rogena * **, S. Lazzi *, G. De Falco *, M.R. Ambrosio *,
M. Onorati *, B.J. Rocca *, C. Bellan *, PP. Piccaluga ***, P.
Pileri ***, L. Leoncini *
* Department of Human Pathology and Oncology, Anatomic Pathology Section, University of Siena, Italy; ** UON/KNH, Nairoby, Kenya;
*** Department of Haeematopathology and Oncology “L. & A. Seràgnoli”, University of Bologna, Italy
Introduction. The first clinical records of Burkitt lymphoma
(BL) are found in the Mengo Hospital case-notes of Sir Albert
Cook at the beginning of the 20th century. Sporadic reports
of tumours resembling BL, but often reported as atypical
neuroblastomas, appeared in publications from Africa in the
first half of the century. Denis Burkitt’s seminar publication
in 1957 not only described the clinical features of this tumour
but showed that the jaw tumours and the visceral tumours
were part of the same disease entity.
Burkitt lymphoma (BL) is now listed in the World Health
Organization (WHO) classification of lymphoid tumors as a
B-cell lymphoma with an extremely short doubling time that
often presents in extranodal sites or as an acute leukaemia.
It is composed of monomorphic medium-sized transformed
cells. Translocation involving MYC is the most common
genetic alteration. A combination of several diagnostic techniques (morphology, genetic analysis or immunophenotyping)
is necessary for the diagnosis of BL.
Some clinical aspects of BL. Three clinical variants are recognized: endemic BL (eBL), sporadic BL (sBL), and immunodeficiency-associated BL (ID-BL). Each clinical subset affects different populations and can present in different forms.
Endemic BL occurs in equatorial Africa, representing the
most common childhood malignancy with an incidence peak
at 4 to 7 years and a male:female ratio of 2:1. EBV is present
in 90-95% of the neoplastic cells in most of the patients; the
jaw is the more frequent site of presentation. Sporadic BL
is seen throughout the world, mainly in children and young
adults. The incidence is low, representing only 1-2% of all
lymphomas in Western Europe and in USA. The male: female
ratio is 2 or 3:1. EBV may be detected in 30-40% of cases and
the classical presentation is with a bulky disease involving the
distal ileum/proximal cecum. Immunodeficiency-associated
BL is primarily seen in association with the human immunodeficiency virus (HIV) infection, often occurring as the initial
manifestation of the acquired immunodeficiency syndrome
(AIDS). EBV is identified only in 25-40% of cases. ID-BL
often presents with bulky lymph node involvement.
However, cases with clinical features and presentation typical of sporadic forms, occurring in adults and being HIV and
EBV negative, have been reported also in endemic areas.
BL is an highly aggressive tumour and most patients present with advanced clinical stage (bulky disease with a high
tumour burden, infiltration of the bone marrow, extracerebral
nervous system and liver) but it is potentially curable and
intensive chemotherapy leads to up to 90% cure rates in low
stage disease and 60-80% in patients with advanced disease.
The prognosis is better in children than in adults. Relapse
usually occurs in the first year after diagnosis. Although the
tumour is curable, many patients still die of the disease mainly
in Africa.
Role of infectious agents and environmental factors in
the pathogenesis of BL. In Africa Burkitt’s lymphoma is
endemic in wet regions with mean minimum temperatures
> 15,5°C and annual rainfall. The question as to why Burkitt’s
187
lymphoma (BL) is endemic in equatorial Africa has intrigued
scientists since the first clinical description of this neoplasm
by Denis Burkitt. Investigation of the geographic restriction
demonstrated that BL occurred primarily where there was
sustained and intense exposure to holoendemic malaria. This
gave rise to the hypothesis that the tumour might be caused by
a mosquito borne virus. Following an intensive search for this
virus Antony Epstein and his colleagues later discovered the
Epstein Barr virus in biopsy samples of BL sent from Uganda.
Recent advances in virology, parasitology and immunology
have helped to elucidate the important contributions of malarial infection and Epstein-Barr virus to lymphomagenesis
in African endemic Burkitt’s lymphoma. P. falciparum infection suppresses cytotoxic T cells immunity against EBV. The
importance of cytotoxic T cells in controlling EBV infections
has been well established and it has long been hypothesized
that EBV immunity is suppressed as a consequence of repeated malaria infections. However, the means by which malaria
orchestrate deficiencies in anti-EBV immunity that result in
tumorigenesis have not been so well explained. Recent studies have demonstrated that malaria-induced T cell disfunction
is age-dependent, transient, EBV-specific, and differentially
affects EBV-specific T cell memory subsets. In addition,
through interaction with the Toll like receptor (TLR)9, P. falciparum infection may lead to perturbation of peripheral B
cell subsets and reactivation and expansion of latently infected
memory B cells, where the virus persist in healthy carriers.
In fact, according to recent studies, EBV first infect naïve B
cells and activates a growth program in these cells so they
can differentiate into resting memory B cells or plasma cells
through the process of the germinal center (GC) reaction. The
virus thus gains access to the memory B cell compartment, its
main reservoir during persistence, where no latent viral genes
are expressed. However, when these latently infected memory
cells divide, they express the EBNA-1 protein (latency I), the
same viral latency program as found in BL primary tumours.
Thus, it is then reasonable to argue that the cell of origin of
endemic BL may be the memory B cells. This is in accordance
also with a recent investigation suggesting that EBV-negative tumours derive from early centroblasts, whereas EBV
positive cases derive from memory or late germinal center B.
However, this is in contrast with the GC phenotype shared by
all of the BL variants. This discrepancy may be explained by
the assumption that in EBV-positive BL B cells follow the
normal differentiation process of the GC as they reach the
differentiation stage of memory cells in terms of VH gene
mutation but do not follow the normal differentiation process
in terms of morphology, immunophenotype and gene expression. This process is primed by BCL6 and requires the activation of BLIMP-1, which in turn represses c-MYC and BCL6
genes. Recent observations have indicated that BCL6 and
BLIMP-1 are targeted by different microRNAs (miRNAs), a
class of small non-coding RNAs acting as post-transcriptional
regulators of gene expression. Interestingly, recent data give
evidence that hsa-miR-127, which is the master regulator of
B cell fate through the interaction with BLIMP-1 and XBP1,
is strongly up-regulated only in EBV-positive BL, whereas
EBV-negative cases show levels of expression similar to GC
cells and reactive lymph nodes. It is not currently known how
EBV can regulate miRNAs expression but preliminary results
confirm the existence of an active interplay between EBV
gene product and cellular miRNAs.
However, epidemiological studies suggest that malaria and
EBV alone cannot account for the distribution of endemic BL
in high risk regions. Selenium, arboviruses and plant extracts
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
are additional environmental factors that appear to be important in the pathogenesis of eBL. It has been recently postulated
a possible role of Euphobia tirucalis, a plant commonly used
in the traditional medicine, as a cofactor in the development
of eBL by its modulation of the expression of the latency
genes of EBV, and the up-regulation of anti-apoptotic factor
as BCL-2.
The incidence of non-Hodgkin’s lymphoma (NHL) is greatly
increased in HIV-infected individuals, being the second most
common neoplasm (after Kaposi’s sarcoma). BL occurs in
less immunodeficient patients and develops when the CD4
count is relatively high, being the immunosuppression per se
not sufficient to explain the relatively high prevalence of BL
in this setting. Tat protein of HIV is a likely candidate to contribute to tumour pathogenesis in HIV-infected patients. Tat is
an early non-structural protein necessary for virus replication
which is secreted by infected cells and taken up by uninfected
cells. Deregulation of cellular genes and functions by Tat can
cause abnormalities that may contribute to AIDS pathogenesis
and to the development of AIDS-associated disorders. The
molecular mechanism underlying Tat’s pleiotropic activity
may include the generation of functional heterodimers of Tat
with cell cycle proteins, resulting in uncontrolled cell proliferation. Another mechanism, through which Tat may influence HIV-mediated transformation, is by hyper-activation
of transcription by interacting with chromatin remodelling
complexes. In addition, HIV and EBV through viral-encoding
miRNAs may interfere with the physiological regulation of
cell functions maintained by cellular miRNAs.
Morphology and molecular signature of BL. BL classically
shows a monomorphic, medium-sized cells diffusely infiltrating into the tissue, with a starry sky pattern. The stars representing tingible body histiocytes and the sky representing the
neoplastic lymphoid cells. The lymphoid cells show a regular
cytoplasmic border (non-cleaved), a non-vesicular nucleus
with two or four basophilic nucleoli. Mitoses are frequent, up
to 4%. The proliferation index as shown by Ki-67 is almost
always greater than 95%. The characteristic immunophenotype of BL is CD 19, CD 20, CD22 (B cell associated antigens) positive; CD10, BCL6, CD38, CD 77, CD 43 positive.
The cells are usually negative or weekly positive for BCL2
and Tdt-negative. Most tumours show MYC translocation at
band 8q24 to the Ig heavy chain region 14q32 or less commonly 22q11 or kappa, 2p12 light chain loci. Up to 10% of
cases may lack a demonstrable MYC translocation by FISH.
These tumors show a similar expression of MYC as the cases
carrying the typical translocation. There are several explanations for this, such as the fact that the break point can occur
in different regions that cannot be recognized by commercial
probes. Whatever the genetic mechanism may be, these cases
have a different molecular pattern due to miRNA deregulation
involved in MYC pathway. This supports the hypothesis that
is the over-expression of MYC independently by the genetic
mechanism that is important for the neoplastic transformation.
Furthermore, it should be considered that MYC translocation
in not specific for BL. Additional alterations may be then responsible for the typical BL signature. Other genetic and epigenetic aberrations, occurring in a subset of BL, can involve
p16, TP53, P73, BAX, P130/RB2, and BCL6.
Gene expression profile (GEP) studies have demonstrated a
consistent gene expression signature for BL, which is clearly
distinct from that of diffuse large B cell lymphoma (DLBCL),
but intermediate cases were also found. In addition, preliminary studies have shown also difference in GEP among
the clinical variants of sBL, eBL and ID-BL, having similar
GEP different from sBL. Of note, these differences regard
significant cellular pathways probably reflecting the different
pathogenetic mechanisms.
A look into the future. The different findings, point out at
the importance of a more discriminative and accurate GEP, as
that of miRNAs, to clarify differences between BL subtypes,
to better explain the pathogenetic mechanisms involved in
virus-associated diseases, and eventually to help designing
more tailored treatments.
Neoplastic pathologies of the bladder and prostate
Moderators: G. Mikuz (Innsbruck), R. Montironi (Ancona)
2009 International Society of Urological
Pathology consensus conference
on standardization of pathology reporting
of radical prostatectomy specimens
R. Montironi, A. Lopez-Beltran *, L. Cheng **
Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy;
* Department of Pathology, Reina Sofia University Hospital and Faculty of Medicine, Cordoba, Spain; ** Department of Pathology and
Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA
The 2009 International Society of Urological Pathology
consensus conference in Boston, made recommendations regarding the standardization of pathology reporting of radical
prostatectomy specimens.
Issues relating to the handling and processing of radical prostatectomy specimens were coordinated by working group 1.
Most uropathologists followed similar procedures for fixation
of radical and prostatectomy specimens, with 51% of respondents transporting tissue in formalin. There was also consensus
that the prostate weight without the seminal vesicles should be
recorded. There was consensus that the surface of the prostate
should be painted. It was agreed that both the prostate apex
and base should be examined by the core method with sagittal
sectioning of the tissue sample. There was consensus that the
gland should be fully fixed before sectioning Both partial or
complete embedding of prostates was considered to be acceptable as long as the method of partial embedding is stated. No
consensus was determined regarding the necessity of weighing and measuring the length of the seminal vesicles, the
preparation of whole mounts rather than standardized blocks
and the methodology for sampling of fresh tissue for research
purposes, and it was agreed that these should be left to the
discretion of the working pathologist.
Issues relating to the substaging of pT2 prostate cancers according to the TNM 2002/2009 system, reporting of tumor
size/volume and zonal location of prostate cancers were coor-
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Lectures
dinated by working group 2. A survey circulated prior to the
consensus conference demonstrated that 74% of the 157 participants considered pT2 substaging of prostate cancer to be of
clinical and/or academic relevance. The survey also revealed
a considerable variation in the frequency of reporting of pT2b
substage prostate cancer which was likely a consequence of
the variable methodologies used to distinguish pT2a from
pT2b tumors. Overview of the literature indicates that current
pT2 substaging criteria lack clinical relevance and the majority of conference attendees expressed dissatisfaction with the
current pT2 substaging system. Despite this, no consensus
was achieved relating to standardization of the method used
to distinguish the various pT2 substages. For these reasons it
was agreed that reporting of pT2 substages should, at present,
be optional. Several studies have shown that prostate cancer
volume is significantly correlated with other clinico-pathological features including Gleason score and extraprostatic
extension of tumor; however, most studies fail to demonstrate
this to have prognostic significance on multivariate analysis.
Consensus was reached with regard to the reporting of some
quantitative measure of the volume of tumor in a prostatectomy specimen, without prescribing a specific methodology.
Incorporation of the zonal and/or anterior location of the
dominant/index tumor in the pathology report was accepted
by most participants, but a formal definition of the identifying
features of the dominant/index tumor remained undecided.
Issues relating to extraprostatic extension (pT3a disease), bladder neck invasion, microvascular invasion, and the definition
of pT4 were coordinated by working group 3. It was agreed
that prostate cancer can be categorized as pT3a in the absence
of adipose tissue involvement when cancer bulges beyond the
contour of the gland or beyond the condensed smooth muscle
of the prostate at posterior and posterolateral sites. Extraprostatic extension can also be identified anteriorly. It was
agreed that the location of extraprostatic extension should be
reported. While there was consensus that the amount of extraprostatic extension should be quantitated, there was no agreement as to which method of quantitation should be employed.
There was overwhelming consensus that microscopic urinary
bladder neck involvement by carcinoma should be reported as
stage pT3a and that lymphovascular invasion by carcinoma
should be reported. It is recommended that these elements be
considered in the development of practice guidelines and in
the daily practice of urologic surgical pathology.
Issues relating to the infiltration of tumor into the seminal
vesicles and regional lymph nodes were coordinated by working group 4. There was a consensus that complete blocking of
the seminal vesicles was not necessary, though sampling of
the junction of the seminal vesicles and prostate was considered to be mandatory. Sampling of the vas deferens margins
was not considered as obligatory. There was consensus that
muscular wall invasion of the extraprostatic seminal vesicle
only should be considered as seminal vesicle invasion. Categorization into types of seminal vesicle spread was considered not to be necessary. For examination of lymph nodes,
special techniques such as frozen sectioning were considered
to be of use only in high risk cases. There was no consensus
on the optimal sampling method, though it was agreed that all
lymph nodes should be completely blocked as a minimum. It
was also agreed that a count of the number of lymph nodes
harvested should be attempted. It was agreed that in view of
recent evidence, the diameter of the largest lymph node metastasis should be measured.
Issues relating to surgical margin assessment were coordinated by working group 5. Pathologists agreed that tumor
extending close to the “capsular” margin, yet not to it, should
be reported as a negative margin, and that locations of positive
margins should be indicated as either posterior, posterolateral,
lateral, anterior at the prostatic apex, mid-prostate or base.
Other items of consensus included specifying the extent of
any positive margin as milimeters of involvement; tumor in
skeletal muscle at the apical perpendicular margin section, in
the absence of accompanying benign glands, to be considered
organ confined; and that proximal and distal margins be uniformly referred to as bladder neck and prostatic apex respectively. Grading of tumor at positive margins was to be left
to the discretion of the reporting pathologists. There was no
consensus as to how the surgical margin should be regarded
when tumor is present at the inked edge of the tissue, in the
absence of transected glands at the apical margin. Pathologists
also did not achieve agreement on the reporting approach to
benign prostatic glands at an inked surgical margin where no
carcinoma is present.
Variants and variations of bladder cancer
A. Lopez-Beltran
Cordoba University Medical School, Cordoba, Spain
Urothelial carcinoma has a propensity for divergent differentiation with the most common being squamous, followed
by glandular. Virtually the whole spectrum of bladder cancer
variants may be seen in variable proportions accompanying
otherwise typical urothelial carcinoma. The clinical outcome
of some of these variants differs from typical urothelial carcinoma; therefore, recognition of these variants is important
Urothelial carcinoma with mixed differentiation. About
20% of urothelial carcinomas contain areas of glandular or
squamous differentiation. Squamous differentiation, defined
by the presence of intercellular bridges or keratinization,
occurs in 21% of urothelial carcinomas of the bladder. Its
frequency increases with grade and stage. Detailed histologic
maps of urothelial carcinoma with squamous differentiation
have shown that the proportion of the squamous component
may vary considerably, with some cases having urothelial
carcinoma in situ as the only urothelial component. These
cases may have a less favorable response to therapy than
pure urothelial carcinoma. Of 91 patients with metastatic
carcinoma, 83% with mixed adenocarcinoma and 46% with
mixed squamous cell carcinoma experienced disease progression despite intense chemotherapy, whereas it progressed in
< 30% of patients with pure urothelial carcinoma. Low-grade
urothelial carcinoma with focal squamous differentiation has a
higher recurrence rate. Tumors with any identifiable urothelial
element are classified as urothelial carcinoma with squamous
differentiation, and an estimate of the percentage of squamous
component should be provided. Cytokeratin 14, L1 antigen
and Caveolin-1 have been reported as immunohistochemical
markers of squamous differentiation.
Glandular differentiation is less common than squamous
differentiation and may be present in about 6% of urothelial
carcinomas of the bladder. Glandular differentiation is defined
as the presence of true glandular spaces within the tumor.
These may be tubular or enteric glands with mucin secretion.
A colloid-mucinous pattern characterized by nests of cells
“floating” in extracellular mucin, occasionally with signet
ring cells, may be present. Cytoplasmic mucin-containing
cells are, present in 14-63% of typical urothelial carcinoma
and are not considered to represent glandular differentiation.
The diagnosis of adenocarcinoma is reserved for pure tumors.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
A tumor with mixed glandular and urothelial differentiation is classified as urothelial carcinoma with glandular differentiation, and an estimate of the percentage of glandular
component should be provided. The expression of MUC5ACapomucin may be useful as immunohistochemical marker of
glandular differentiation in urothelial tumors. When small
cell carcinoma is present in association with urothelial carcinoma, even focally, it portends a poor prognosis. Small cell
carcinoma is an important finding and usually dictates more
aggressive therapy (see following section).
Small cell carcinoma. Small cell carcinoma is a malignant
neuroendocrine neoplasm derived from the urothelium which
histologically mimics its pulmonary counterpart. Patients with
small cell carcinoma of the urinary bladder have a dismal prognosis. In a recent series of 64 cases of small cell carcinoma of
the urinary bladder, the mean age at diagnosis was 66 years
and the male to female ratio was 3.3:1; 88% presented with
hematuria. All the patients except one had muscle-invasive
disease at presentation. Thirty-eight patients (59%) underwent
cystectomy and 66% of patients had lymph node metastasis at
the time of cystectomy with regional lymph nodes, bone, liver
and lung being the most common locations. Twenty cases
(32%) were pure small cell carcinoma; 44 cases (68%) cases
consisted of small cell carcinoma with other histological types
(urothelial carcinoma, 35 cases; adenocarcinoma, 4 cases;
sarcomatoid urothelial carcinoma, 2 cases; and 3 cases with
both adenocarcinoma and urothelial carcinoma). Patients with
organ-confined cancers had marginally better survival than
those with non organ-confined cancer (p = 0.06). Overall, 1year, 18-month, 3-year, and 5-year cancer-specific survivals
were 56%, 41%, 23%, and 16%, respectively.
At histology, they consist of small, rather uniform cells, with
nuclear molding, scant cytoplasm and nuclei containing finely
stippled chromatin and inconspicuous nucleoli. Mitoses are
present and may be frequent. Necrosis is common and there
may be DNA encrustation of blood vessels walls (Azzopardi
phenomenon). Most cases have areas of urothelial carcinoma
sometimes in the form of flat urothelial carcinoma in situ and
exceptionally, squamous cell carcinoma, adenocarcinoma or
sarcomatoid carcinoma. This is important, because the presence of these differentiated areas does not contradict the diagnosis of small cell carcinoma. Neuroendocrine granules are
found with electron microscopy, but the immunohistochemical
profile reveals neuronal-specific enolase in 87% of cases, and
chromogranin A only in a third of cases. Some cases are also
reactive against synaptophysin, PGP 9.5, thyroid transcription
factor-1 (TTF-1), p53 (DO7), and Ki67 (MIB-1). The diagnosis of small cell carcinoma can be made on morphologic
grounds alone, even if neuroendocrine differentiation cannot
be demonstrated. Frequently small cell carcinoma expresses
cytokeratin which supports the hypothesis of urothelial origin.
The recent finding of c-kit and c-erbB2 expression by immunohistochemistry opens new possibilities for therapy in small
cell carcinoma of the bladder.
Nested variant. The nested variant of urothelial carcinoma
is an aggressive neoplasm with fewer than 50 reported cases.
There is a marked male predominance, and 70% of patients
died 4-40 months after diagnosis, in spite of therapy. This rare
pattern of urothelial carcinoma was first described as a tumor
with a “deceptively benign” appearance that closely resembles
Brunn’s nests infiltrating the lamina propria. Some nests have
small tubular lumens that eventually can predominate. Nuclei
generally show little or no atypia, but invariably the tumor
contains foci or unequivocal cancer with cells exhibiting enlarged nucleoli and coarse nuclear chromatin. This feature is
most apparent in the deeper aspects of the cancer. The differential diagnosis of the nested variant of urothelial carcinoma
includes prominent Brunn’s nests, cystitis cystica and glandularis, inverted papilloma, nephrogenic metaplasia, carcinoid
tumor, paraganglionic tissue, and paraganglioma.
Micropapillary carcinoma. Micropapillary carcinoma is a
distinct variant of urothelial carcinoma that resembles papillary serous carcinoma of the ovary, and approximately 60
cases were reported in the literature. There is a male predominance and the patients ages range from fifth to the ninth decade
with a mean age of 66 years. The most common presenting
symptom is hematuria. The first description of micropapillary
carcinoma consisted of 18 patients whose ages ranged from
47 to 81 years (mean 67) with a male-to-female ratio of 5:1.
Seven patients died of carcinoma. The micropapillary component is found in association with noninvasive papillary or
invasive urothelial carcinoma in 80% of reported cases, consisting of slender delicate filiform processes or small papillary
clusters of tumor cells; when present in invasive carcinoma,
it is composed of infiltrating tight clusters of micropapillary
aggregates that are often within lacunae that are negative for
endothelial markers. Twenty-five percent of cases show glandular differentiation, and some authors consider it as a variant of adenocarcinoma. 99Psammoma bodies are infrequent.
Vascular and lymphatic invasion is common, and most cases
show invasion of the muscularis propria or deeper, often with
metastases. Immunohistochemical studies in one large series
disclosed immunoreactivity of the micropapillary carcinoma
in 20 of 20 cases for MUC1 (EMA), Cytokeratin 7 and 20,
and Leu M-1. The presence of a surface micropapillary component in bladder biopsy specimens with cancer is an unfavorable prognostic feature, and deeper biopsies may be useful to
determine the level of muscle invasion. The main differential
consideration is serous micropapillary ovarian carcinoma in
women or mesothelioma in both genders.
Microcystic carcinoma. The microcystic variant of invasive
urothelial carcinoma is characterized by the formation of microcysts, macrocysts, or tubular structures with cysts ranging
from microscopic up to 1-2 cm in diameter. The cysts and
tubules may be empty or contain necrotic debris or mucin that
stains with periodic acid-Schiff stain with diastase predigestion. This variant of cancer may be confused with benign
proliferations such as florid polypoid cystitis cystica and
glandularis and nephrogenic metaplasia.
Lymphoepithelioma-like carcinoma. Carcinoma that histologically resembles lymphoepithelioma of the nasopharynx
has recently been described in the urinary bladder, with fewer
than 40 cases reported. Disease in the urinary bladder is more
common in men than in women (3:1 ratio) and occurs in
late adulthood (range: 52-81 years; mean: 69 years). Most
patients present with hematuria. The tumor is solitary and
usually involves the dome, posterior wall, or trigone, often
with a sessile growth pattern. Histologically, it may be pure or
mixed with typical urothelial carcinoma, the later being focal
and inconspicuous in some instances. Glandular and squamous differentiation may be seen. The tumor is composed of
nests, sheets, and cords of undifferentiated cells with large
pleomorphic nuclei and prominent nucleoli. The cytoplasmic
borders are poorly defined, imparting a syncytial appearance.
The background consists of a prominent lymphoid stroma
that includes T and B lymphocytes, plasma cells, histiocytes,
and occasional neutrophils or eosinophils. Epstein-Barr virus
infection has not been identified in lymphoepithelioma-like
carcinoma of the bladder. This tumor, thus far has been found
to be responsive to chemotherapy when it is encountered in
Lectures
its pure form. The epithelial cells of this tumor stain with several cytokeratin markers as follows: AE1/AE3, CK8, CK 7,
and they are rarely positive for CK20. The major differential
diagnostic considerations are poorly differentiated urothelial
carcinoma with lymphoid stroma, poorly differentiated squamous cell carcinoma, and lymphoma. Immunohistochemistry
reveals cytokeratin immunoreactivivity in the malignant cells,
confirming their epithelial nature. Most reported cases of the
urinary bladder had a relatively favorable prognosis when pure
or predominant, but when lymphoepithelioma-like carcinoma
is focally present in an otherwise typical urothelial carcinoma,
the disease behaves as it does in patients with conventional
urothelial carcinoma of the same grade and stage.
Plasmacytoid carcinoma. Zukerberg et al. described bladder carcinoma in two patients that diffusely permeated the
bladder wall and was composed of cells with a monotonous
appearance mimicking lymphoma. The tumor cells were medium-sized, with eosinophilic cytoplasm and eccentric nuclei
producing a plasmacytoid appearance. The epithelial nature
of the malignancy was confirmed by immunohistochemistry.
Differential diagnostic considerations include lymphoma
(plasmacytoid type) and multiple myeloma. Identification
of an epithelial component confirms the diagnosis. In a series report of 6 cases, the male-to female ratio was 2:1, and
the age range was 54-73 years. All cases stained positively
for cytokeratin cocktail, cytokeratin 20 and 7, and all were
negative for leukocyte common antigen. Five of six patients
died of disease (mean survival, 23 months). Some case may
express CD138.
Inverted papilloma-like carcinoma. The potential for misinterpretation of urothelial carcinoma with endophytic growth
as inverted papilloma is high. By definition, this variant of
urothelial carcinoma has significant nuclear pleomorphism,
mitotic figures, and architectural abnormalities consistent
with low- or high-grade urothelial carcinoma. In most cases,
the overlying epithelium has similar abnormalities and often
contains typical urothelial carcinoma. Inverted papilloma-type
carcinoma with minimal cytologic and architectural abnormalities have high mitotic activity. An exophytic papillary
or invasive component is often associated with the inverted
element. However, in cases of inverted papilloma fragmented
during transurethral resection, a pseudoexophytic pattern
may result. In some instances, both inverted papilloma and
inverted papilloma-type carcinoma are intimately mixed.
Large papillary tumors with prominent endophytic growth
“invade” the lamina propria with a pushing border. Unless this
pattern is accompanied by true destructive stromal invasion
the likelihood of metastasis is minimal, because the basement
membrane is not truly breached.
Urothelial carcinoma with syncytiotrophoblastic giant
Cells. Syncytiotrophoblastic giant cells are present in up to
12% of cases of urothelial carcinoma, producing substantial
amounts of immunoreactive beta-human chorionic gonadotropin (HCG) indicative of syncytiotrophoblastic differentiation.
The number of HCG-immunoreactive cells is inversely associated with cancer grade. Secretion of HCG into the serum
may be associated with a poor response to radiation therapy.
The most important differential diagnostic consideration is
choriocarcinoma; most but not all cases previously reported
as primary choriocarcinoma of the bladder represent urothelial
carcinoma with syncytiotrophoblasts.
Pleomorphic Giant cell carcinoma. High grade urothelial
carcinoma may contain epithelial tumor giant cells or the
tumor may appear undifferentiated, resembling giant cell
carcinoma of the lung. This variant is very infrequent. Malig-
191
nant giant cells in urothelial carcinoma, when present in great
numbers, portend a poor prognosis, similar to that associated
with giant cell carcinoma in the lung. The giant cells display
cytokeratin and vimentin immunoreactivity.
Clear cell (glycogen-rich) carcinoma. Up to two-thirds of
cases of urothelial carcinoma have foci of clear cell change resulting from abundant glycogen. The glycogen-rich clear cell
“variant” of urothelial carcinoma, recently described, appears
to represent the extreme end of the morphologic spectrum,
consisting predominantly or exclusively of cells with abundant clear cytoplasm that stains for cytokeratin 7.
Sarcomatoid carcinoma with/without heterologous elements (carcinosarcoma, metaplastic carcinoma). The term
sarcomatoid variant of urothelial carcinoma should be used
for all biphasic malignant neoplasms exhibiting morphologic
and/or immunohistochemical evidence of epithelial and mesenchymal differentiation (with the presence or absence of
heterologous elements acknowledged in the report). There
is considerable confusion and disagreement in the literature
regarding nomenclature and histogenesis of these tumors. In
some series, both carcinosarcoma and sarcomatoid carcinoma
are included as “sarcomatoid carcinoma”. In others they are
regarded as separate entities.
The gross appearance is characteristically “sarcoma-like,” dull
gray with infiltrative margins. The tumors are often polypoid
with large intraluminal masses. Microscopically, sarcomatoid
carcinoma is composed of urothelial, glandular or small cell
component showing variable degrees of differentiation. Carcinoma in situ is present in 30% of cases and occasionally
is the only apparent epithelial component. A small subset of
sarcomatoid carcinoma may have a prominent myxoid stroma.
The mesenchymal component most frequently observed is a
undifferentiated high grade spindle cell neoplasm. The most
common heterologous element is osteosarcoma followed
by chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma,
liposarcoma, angiosarcoma or multiple types of heterologous
differentiation may be present. By immunohistochemistry,
epithelial elements react with cytokeratins, whereas stromal
elements react with vimentin or specific markers corresponding to the mesenchymal differentiation. The sarcomatoid phenotype retains the epithelial nature of the cells by immunohistochemistry or electronmicroscopy. Recent molecular studies
strongly argue for a monoclonal origin of both components in
sarcomatoid carcinoma and carcinosarcoma. The mean age is
66 years (range, 50-77 years). Pathological stage is the best
predictor of survival in sarcomatoid carcinoma. The major
differential diagnostic consideration is urothelial carcinoma
with pseudo-sarcomatous stroma, a rare entity with reactive
stroma. In cases with exclusively spindle cells, the main
differential diagnostic consideration is sarcoma, particularly
leiomyosarcoma. Immunostaining with cytokeratin is helpful
in this setting. Sarcomatoid carcinoma with prominent myxoid and sclerosing stroma may be mistaken for inflammatory
pseudotumor.
Lipoid-cell variant. Lipoid cell variant, is a rare neoplasm
defined by the WHO (1999, 2004) as an urothelial carcinoma
which exhibits transition to a cell type resembling signet-ring
lipoblasts. It is currently considered to be an ill-defined tumor
variant, and whether it should be classified as carcinosarcoma
remains to be established. Clinicopathologic features and
the immunohistochemical findings in seven reported cases
showed gross hematuria as the initial symptom. All patients
were elderly men (mean age, 74 years; range, 63-94 years).
On microscopic examination, the extension of the lipid cell
pattern varied from 10%-30% of the tumor specimen, with
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
associated micropapillary (n = 1), plasmocytoid (n = 2), and
grade 3 conventional urothelial carcinomas (n = 4). The immunohistochemical results showed an epithelial phenotype
of the lipoid cell component characterized by diffuse staining
with cytokeratins AE1/AE3.
Undifferentiated carcinoma. This category contains tumors
that cannot be otherwise classified. To our knowledge, they
are extremely rare.
References
Lopez-Beltran A, Cheng L. Histologic variants of urothelial
carcinoma: differential diagnosis and clinical implications.
Hum Pathol 2006;37:1371-88.
Inverted bladder neoplasms: inverted papilloma
M. Colecchia, B. Paolini
Dipartimento di Patologia, Fondazione IRCCS IstitutoTumori di Milano, Italy
Inverted papilloma comprises less than 1% of urothelial
neoplasms and is generally considered a benign neoplasm.
The mean age at diagnosis is 64 years and a peak frequency
is in the 6th and 7 th decades. It is more common in men than
women. The etiology is uncertain 1. Recent molecular data
supports its benign nature based in the low amount of genetic
anomalies found in most cases 2. Most cases of inverted papilloma are located in the bladder trigone, but inverted papilloma can also be found in the uretere, renal pelvis, urethra 1.
Macroscopically it is characteristically sessile or pedunculated, smooth surfaced, small and single but large multifocal
lesions may occur 3. Microscopically inverted papillomas had
a relatively smooth surface covered by histologically and
cytologically normal urothelium and consists of intramucosal and submucosal anastomising islands and trabeculae of
urothelium. There are two main patterns: the trabecular and
glandular patterns 4. In both patterns of inverted papilloma,
the epithelial elements are surrounded by an intact membrane
and delicate fibrovascular stroma. Unusual growth patterns
of inverted papilloma include basaloid, hyperplastic, spindle
cell and neuroendocrine patterns. Mild cytologic atypia could
be observed in inverted papilloma, and the precise demarcation with carcinoma is unresolved; in rare cases nuclear
atypia may be prominent but these atypical nuclear features
are considered degenerative in nature. Immunohistochemical
expression of a number of biomarkers, including Ki 67, p
53 and CK 20has been shown to be of diagnostic value. In
a study by Jones 0/15 inverted papillomas stains positively
for Ki67 and CK 20 and only 1/15 stained positively for
p 53. Urovysion FISH produced normal results for all cases
of inverted papilloma 5. Mitotic figures are rare or absent in
inverted papilloma, unlike carcinoma and generally are less
than 1/10 hpf, while in inverted carcinoma mean number
was 8/10 HPF in a large series study 5 6. The number of cases
with coexistent urothelial carcinoma in situ or carcinoma has
increased recently. Inverted papilloma are usually diploid 7.
Sung and collagues examined a series of 39 inverted papillomas using LOH analysis and showed a very low incidence
of LOH at genetic loci that are frequently lost in both urothelial carcinomas and papillary urothelial neoplasms of low
malignant potential 2. Recurrent lesions have been observed in
< 1% of the reported cases 8.
References
1
Sauter G. Inverted papilloma. In: Eble JN, Sauter G, Epstein JI, et al.
(eds). Pathology and genetics of Tumors of the Urinary System and
Male Genital Organs. Lyon: Iarcc Press 2004.
2
Sung MT, Eble JN, Wang M, et al. Inverted papilloma of the urinary
bladder: a molecular genetic appraisal. Mod Pathol 2006;19(10):128994.
3
Rozanski TA. Inverted papilloma: an unusual recurrent, multiple and
multifocal lesion. J Urol 1996155(4):1391.
4
Kunze E, Schauer A, Schmitt M. Histology and histogenesis of two different types of inverted urothelial papillomas. Cancer 1983;51(2):34858.
5
Jones TD, Zhang S, Lopez-Beltran A, et al. Urothelial carcinoma with
an inverted growth pattern can be distinguished from inverted papilloma by fluorescence in situ hybridization, immunohistochemistry, and
morphologic analysis. Am J Surg Pathol 2007;31(12):1861-7.
6
Amin MB, Gómez JA, Young RH. Urothelial transitional cell carcinoma with endophytic growth patterns: a discussion of patterns of
invasion and problems associated with assessment of invasion in 18
cases. Am J Surg Pathol 1997;21(9):1057-68.
7
Cheville JC, Wu K, Sebo TJ, et al. Inverted urothelial papilloma: is
ploidy, MIB-1 proliferative activity, or p53protein accumulation predictive of urothelial carcinoma? Cancer 2000;88(3):632-6.
8
Cheng CW, Chan LW, Chan CK, et al. Is surveillance necessary for
inverted papilloma in the urinary bladder and urethra? NZ J Surg
2005;75(4):213-7.
Clinical governance
Moderators: G. Coggi (Milano), G. Barresi (Messina)
Clinical pathways: is there a role for
pathologists?
E. Bonoldi, A. Parafioriti *, G. Coggi
Unit of Pathology, IRCCS Ospedale Maggiore Policlinico, Milan,
Italy; * Unit of Pathology Gaetano Pini Institute, Milan, Italy
A critical or clinical pathway (CP) is the coordinate, accurately planned sequence of interventions by health care professionals for a single patient or a group of patients requiring
treatment for a particular diagnosis or problem. CPs are tools
used as references for Evidence Based health-care. They have
been implemented internationally since the 1980s. by different health care systems all over the world.
CPs are characterized, according to the European Pathways
Association by five parameters: 1) multidisciplinary approach to plan care 2) translation of guide lines or evidence
into local structures 3) detailed description of the steps
in a course of diagnosis and treatment, with regard to the
algorithms, guide-lines, protocols and procedures adopted
4) evaluation in progress of the timeframes of intervention
5) standardization of care for a specific clinical problem in a
specific population.
CPs are developed through collaborative efforts of clinicians,
case managers, nurses and other allied health care professionals with the aim of improving the quality of patient care and
minimizing costs.
Indeed CP aims to improve, in particular, the continuity and
coordination of care, across different medical specialties.
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Lectures
Although the choice of a standardized CP is mainly a clinician’s task, still an important role is left to other health care
professional in the field of Laboratory Medicine, Radiology,
Radiotherapy etc.
With regard to Laboratory Medicine, a significant role is
played by Pathologists.
The latter, in fact, can deeply contribute to the development of
CPs, for both neoplastic and non-neoplastic diseases, in prevention, diagnosis, control of response to therapy and follow-up.
The histopathological diagnosis itself is in fact of paramount
relevance in addressing a patient’s health process towards the
most appropriate CP or shifting it, from one CP to another.
For example, in case of superficial lymphoadenopathy, the
choice of CP will be considerably different with a diagnosis of
metastatic disease, rather than of lymphoproliferative disease,
or of a reactive process. Even more different will be the CP
should a fine needle biopsy approach or a surgical excision be
chosen, on the basis of specific diagnostic clinical questions.
The CP followed by patients affected by breast cancer, for
example, is going to become more and more tailored on the
basis of pathological and biological features of the single
case, just in virtue of the increasingly appropriate pathological
diagnosis, which must fulfill the criteria of Evidence Based
Pathology, reporting prognostic and predictive factors.
The multidisciplinary team of health professional involved
in planning and construction of a CP will refer to guide lines
(systematically developed statements to assist practitioners
for making decision in diagnostic process and/or health
care), protocols and procedures (diagnostic and/or treatment
recommendations based on guide lines), in order to decrease
individual variability. Development of optimal CP is gained
through the following steps:
– Select topic: topic selection generally concentrates on high
frequency, high cost diagnoses and procedures.
– Select a team: crucial to success is to develop a sound
multidisciplinary team, in order to guarantee coverage of
the different fields and professionals involved in patient
care. Lack of active commitment and participation plays a
dramatic role in failure of a CP.
– Evaluate the current process of care in order to understand
current variation.
– Evaluate medical evidence and gold-standard practice.
– Determine the format of CP: a simple check list could be
an optimal method for both attending and implementing the
pathway.
– Document and analyze variance using performance indicators corresponding to key features in process improvement
– Pathway implementation by educational projects addressed
to the staff and accurate selection of individual roles.
Among the benefits to health care organization in introducing
CP, special interest gains the reduction of unnecessary variation in patient care, of delay in discharge and the improvement
of cost-effectiveness of clinical services.
Notwithstanding the potential barriers to the introduction of
CPs, integrated processes running the whole course from prevention to diagnosis, treatment and rehabilitation can really
help to provide explicit and well-defined standards of care,
while supporting clinical effectiveness, risk management and
clinical audit.
References
Coffey RJ, et al. Qual Manag Health Care 1992;1(1):45-54.
Campbell SS, et al. British Medical Journal 1998;316(7125):133-7.
Coffey RJ, et al. Qual Manag Health Care 2005;14(1):46-55.
Talmor D, et al. Crit Care Med 2006;34(11):2738-47.
Patkar V, Fox J. Stud. Health Technol Inform 2008;139:233-42.
The control process in pathology
G. Angeli
Pathology Unit, Vercelli Hospital, Italy
The control process consists of sequential actions taken by
management to establish performance standards, measure
and evaluate performance and take corrective actions where
indicated.
The control process is practiced by all areas and levels of
an organization. The basic process remains the same: 1) setting performance standards; 2) measuring the performance;
3) evaluating the performance; 4) making effective use of
feedback and taking corrective actions when necessary.
Feedback information can be used either to confirm or to correct organizational performance. Effective use of feedback is
a powerful tool for the control of work performance. Using
feedback is a crucial point, because if the actual performance
does not meet the performance standard, management will
take corrective actions. This presupposes that the standards
are fair and can be met. This is particularly true when we are
dealing with the objectives assigned to the Pathology Unit,
which have to be by definitions realistic and related to the
given resources.
Control is a dynamic and ongoing process. Such a process assumes as given the aims and strategies of the organization and
takes place in the context of the strategic planning.
The first step is the definition of performance standards, that
are organizational goals stated in concrete and measurable
performance terms. If the standards are unrealistically high,
the organization will not obtain the desired results and will be
judged a failure. If the performance standards are set too low,
an organization may easily get the desired standards and be
considered a success when a much more productive use of the
assigned resources was actually possible. The aim is to create
fair and equitable standards. This can be done by examining
past performance as well as the performance of other institutions with similar characteristics in a benchmarking manner.
In the context of control process it is necessary to decide
what performance to measure, when to measure, and how to
measure. Most important point is to define suitable and easily
measurable indicators of the different phases of the process.
Such indicators may be of quantitative or qualitative types, as
for example those of the balance score card. The indicators so
identified become part of reporting documents.
Once the performance standard has been defined and the measures taken, the next step of the control process is to evaluate
the performance. This is the process in which the measured
performance is compared with the performance standard. The
information derived from the performance evaluation is in
each case used in a constructive manner, either confirmative
or corrective, on the job itself. This is the feedback mechanism.
The control process can operate at three different levels: input,
process and output. When the control process operates before
the actual activity is called input control. It allows the organization to correct defective performance before making the
final commitment of resources. Example of input control is
budget. Process control takes place as the work is performed,
so it can assures that the actual performance meets the desired standards. Examples of this kind of control are quality
controls. The output control operates at the end of the process
and involves the final product of the process itself. It includes
quality controls of the final product and audits.
No organization has unlimited resources, so controls are
necessary. Among financial controls budget has a primary
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
importance, because it can be used as financial performance
standard. Since budgets are created for organizations with
limited resources, they give the financial framework and
define the boundaries within which the allocation of those
resources must take place. Budgets aid management in the
performance of the basic managerial functions: planning,
organizing and control.
Budget is a plan expressed in quantitative, usually monetary,
terms referred to a given period of time, generally one year,
with a cost center or a responsibility center approach. So responsibility centers commit themselves to reach the objectives
with assigned resource, then superior levels assure a positive
evaluation of this behavior.
There are two different kinds of approach to budgeting process: top-down, when the budget is prepared by top management; bottom-up, when the budget is prepared by lower levels
managers and then submitted to top management for approval.
The second approach has the advantage that the budgeting
process is carry out by the same that have created it.
The budgeting process needs regular reports which allow to
compare expected inputs and outputs with the actual ones and
to start confirmative or corrective feedbacks. Such a process
should be flexible and subject to periodic review. Flexible
or probabilistic budgets take into account potential environmental changes, and may be changed in front of a modified
context.
Audits are another kind of process control, either external or
internal to the organization. Audits are formal evaluations
of the performance of an organization in term of financial
situation or quality of the final product (clinical audits, for
example).
Most important function of control process is the behavioral
control, in term of evaluation and motivation of the people,
who represent the most valuable non material resource of an
organization.
References
Anthony RN, Young DW. Management control in nonprofit organization. The McGraw – Hill Co. 1999.
Montana PJ, Charnow BH. Management. Barron’s Educational Series
2008.
Evidence based pathology
A. Parafioriti, E. Bonoldi *, E. Armiraglio *, G. Coggi
Unit of Pathology Gaetano Pini Institute, Milan, Italy; * Unit of Pathology, IRCCS Ospedale Maggiore Policlinico, Milan, Italy
Evidence Based Pathology (EBP) is an integral part and an
off-shoot of Evidence Based Medicine (EBM), specialized
in diagnosis and prognosis of human diseases. Pathologists
play a vital role in clinical diagnosis: decisions based on the
pathological contribution to diagnosis are made on a daily
basis and have far-reaching consequences for patients and the
health service. The same diagnostic role also plays a key part
in screening, in classifying disease and as a reference standard
for clinical disease. EBM has mostly focused on therapy and
has provided a solid basis for some treatment, emphasizing the
importance of the randomized controlled trial to determine,
through perfectly designed experiments, the best treatments.
Diagnosis is more complex than therapy and less attention
has been paid to developing a rigorous, systematic approach
to this field of medicine. Well-tried methods of EBM can be
used for diagnosis. The process starts with identifying a real
clinical problem and then, following rules of EBM, translating this into an answerable question. Possible solutions are
obtained through a search for evidence and critically evaluated. When solutions are appropriate they can be applied in
practice, otherwise we need for further research.
In anatomic pathology, the pathway of diagnostic inference
is usually leaded by clinical instinct, supported by experience
according the so-called “skill full eye”.
Such a trajectory could look like an “artistic process” rather
than a scientific procedure.
Incorporation of EBP in everyday practice can really help to
plan a diagnostic method informed by objective evidences
derived from well-designed studies, rather than based on
problem-solving experience.
In placing a lesion in a defined category, pathologists should
always link the observation of morphological elements to the
information obtained by ancillary tests, clinical data and to the
knowledge derived from the literature. This requires to rely
upon a rigorous algorithm defining the hierarchy of morphological and biotechnological features, so that, the final report
can be reproducible.
Actually, many pathology practices are neither objective, nor
precise because guidelines for standardization are lacking.
EBP helps to unify the current methodology primarily based
on “pattern recognition” to newer types of tissue-based testing, including but not limited, to analyses of scores, cut-off,
genomic or proteomic features.
A final diagnosis, EBP-directed, allows a real customized
therapy, in observance of the unique biology of an individual
patient’s disease and more accurately can predict outcome and
effectiveness of treatment.
Moreover, we must not forget that everything that is done in
healthcare system has an “opportunity cost”. Reproducibility
of a diagnosis, obtained according to EBP criteria, can help to
minimize both error possibility and potential costs, including
medico-legal consequences.
In conclusion, we believe that EBP is concerned with ensuring that all the aspects of generating a test result (sampling,
morphological assessment and final report) are based both on
judgment ability stemming from experience and on reproducible and clinically relevant criteria.
Evidence based diagnosis needs more systematic reviews and
appropriate tools, with special regards to electronic databases
and meta-analysis (Pub-Med, Cochrane Library, Clinical Evidence), and more high quality research information in order to
translate the data obtained to real decision making.
The challenge to clinicians, pathologists, educators, researchers, funders, journal editors, and publishers is to work together
to make this happen.
References
Annual Meeting of the Association of directors of Anatomical and Surgical Pathology; March 24, 2007; San Diego, CA.
Booth A. Mapping the evidence base of pathology. J Pathol 1999;188:34450.
Crawford J.M. Original research in pathology: judgment, or evidencebased medicine. Lab. Invest 2007;87:104-14.
Marchevsky AM, Wick MR. Evidence-Based Medicine, Medical Decision Analysis, and Pathology. Hum Pathol 2004;35:1179-88.
Wick MR, Marchevsky AM, Foucar E. Evidence-Based Medicine. Semin
Diagn Pathol 2005;22(2):105-77.
195
Lectures
Procedures and guidelines
Moderator: R. Giardini (Cremona)
Procedures and guidelines
R. Giardini, E. Tavani, D. Ientile
Istituti Ospitalieri, Cremona, Italy, Rho Hospital, Rho, Italy, Bucheri
La Ferla Fatebenefratelli Hospital, Palermo Italy
Procedures manuals and guidelines are born as tools with
the aims to permit to the professionals to make “informed
choices” based on the analysis of scientific tests and on the
evaluation of risk and benefit of every action. Moreover, procedure manuals and guidelines proved to be a tool of updating
for professionals, of education and information for patients
and an external reference to verify what the pathologist is able
to produce.
Given the high level of information interchange among pathologists of the same hospital and among different structures
(counselling activity, data centralization, regional quality
controls, screening campaigns), the operative practice in a
Division of Anatomical Phatology, is usually and, to say,
physiologically, based on standard protocols, well defined
and accepted among professionals, even if not very diffused
trough divisions and known only in very specialized (by organs or pathology) divisions.
Nevertheless there is often an implied information interchange, not formalized and not standardized by methodologically rigorous procedures clearly declared. On the other hand
there is an implicit need of validated references proved by
those almost always high level procedures, optimized over the
time, by study groups of the National Scientific Society or into
regional branches of the same society.
Therefore it seems to us appropriate to start again a “new-old”
discussion in our National Scientific Society about the proper
and practical use of work tools such us the procedures manual
and the diagnostic guidelines. We think that such tools are
more and more relevant in the diagnostic activity and express
a specific value both for crediting and certification of the Anatomical Pathology divisions and also for the role that their use
assume in the risk management related to our work.
A relevant remark concerning an appropriate consideration
about the real meaning of the terms “Procedures manual” and
“Guidelines”, often not properly utilized as synonyms.
In the Anglo-Saxon literature, due to the prevalence of health
insurance systems imposing more rigorous rules, a better defined meaning of those words is used, whereas in our language
those rules are vaguer.
If from one side we can agree about the value of “procedures”
in order to standardize as much as possible the “technicalmethodological” course in different extent of a pathology
field, on the other side we have to ask ourselves if there is a
meaning in talking about our “guidelines”, out of an indispensable multidisciplinary context, where we are working in
the every day life.
As suggested by the “manual of direction” published by the
Italian Superior Institute of Health, perhaps it would better not
to talk about anatomopathological guidelines but about “diagnostic algorithms”, intended as supports to the diagnostic
iter, based on the literature data (EBM) and/or on necessaries
consensus conferences.
In summary our terms “procedures” and our “interpretative
algorithms” should find their natural context in real guidelines, established by the contribution of multi-specialist teams.
In this sense, the FONCAM manual looks as a very good
example to us.
Based on this not only semantic, but also substantial, argueing, stay the medicolegal aspect of the use of procedures and
guidelines, more and more relevant in our professional field.
There is a very open question about that and at this point an
indispensable discussion among expert pathologist should be
open.
The aim of this session is to make possible a workgroup that
can guarantee, with the collaboration of corporate specialists
and single interested pathologists, the necessary homogeneity
and coverage of as much diagnostic fields as possible, constituting a valid support to our professional activity, in every
context could be expressed.
Slide seminar: Lymphoma surgical pathology
Moderators: V. Franco (Palermo), S. Pileri (Bologna)
Primary large B cell lymphoma presenting as
soft tissues mass: a case report and review of
the literature
U. Gianelli, E. Bonoldi, E. Armiraglio *, A. Di Bernardo, A.
Moro **, A. Parafioriti *
Dipartimento Interospedaliero di Anatomia Patologica: U. O. C.
Anatomia Patologica, Università di Milano, Fondazione IRCCS “Ca’
Granda”, Ospedale Maggiore Policlinico, Milano, Italia; * U.O. Anatomia Patologica A.O. Istituto Ortopedico Gaetano Pini, Milano, Italia; ** U.O. Anatomia Patologica A.O. San Paolo, Milano, Italia
We describe a case of a 73 years old man, who was admitted
at the Istituto Ortopedico “G. Pini” of Milan, because of a
continuous pain in the pelvis, for 7 months.
The patient had a previous history of a car accident occurring four years before, from which he had reported multiple
fractures of the ribs and of the left pelvis, together with lung
contusions.
At the time of the first observation the patient suffered of
cruralgia and the physical examination revealed a swelling of
the left buttock.
Peripheral blood examination showed the following parameters: WBC: 11,1 × 109/l, RBC: 4,72 × 1012/l, Hb: 12,3 g/dl,
PTL: 422 × 109/l (LF: N = 81, Eo = 0, Ba = 0,3; l = 11,
Mo = 6,7), ESR 100 mm.
The CT scan revealed a mass 4 cm. thick, covering the foramen ovale and extending to the pelvis, located posterior to the
iliac muscle and anterior to the muscle of the left buttock.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Bone scintigraphy was negative and didn’t show any pathological uptake.
Ecothomograpy displayed a grossly hypoecogenic mass growing between the iliac muscle and the bone.
In the suspect of soft tissue malignancy an open biopsy of the
mass was performed.
Histological examination revealed a solid proliferation of
large cells with abundant clear to granular and eosinophilic
cytoplasm, with a nested and diffuse growth pattern, focally
alveolar, infiltrating the skeletal muscle and the adipose tissues. The nuclei of tumoral cells were irregular with prominent single or multiple nucleoli. Differential diagnosis included: clear cells sarcoma, amelanotic melanoma, metastatic
large cells carcinoma or diffuse large cells lymphoma.
Neoplastic cells presented the following immunophenotype:
CD45 (+), CD79a (+), CD20 (+), CD10 (-), bcl-6 (+), MUM1 (+), NKIC3 (+/-), CD3 (-), CD5 (-), CD30 (-), EMA (-),
S-100 (-), HMB45 (-), Melan-A (-), CD68R (-), Actin (-),
Desmin (-), CK (MNF116, CAM 5.2, AE1-AE3) (-). The
proliferation index was ki-67 (MIB1): 60-70%.
The morphology and the immunophenotype supported a diagnosis of diffuse large B-cell lymphomas (DLBC), NOS, of
non-germinal centre-like immunophenotype. Because of all
other staging procedures did not reveal any localization of
the neoplasm, a final diagnosis of primary DLBC of the soft
tissues was performed.
Primary DLBC of the soft tissue are extremely rare and the
two most important series of these types of NHLs were described about 20 years ago.
The Armed Forces Institute of Pathology collected 75 cases of
primary lymphomas of the soft tissues in a period spanning 20
years. The Mayo Clinic was able to find 8 of these cases out
of 7000 NHLs collected in period of 10 years. Other sporadic
cases have been described in the literature as case report.
All histological types of NHL have been found in soft tissues,
including low- and high-grade tumours. Most of the tumours
occur in the soft tissues of the thigh, followed by abdominal
wall, arm and leg. Intermuscolar tissue rather than the muscle
itself appears to be the tissue most frequently involved.
This case emphasizes the importance to include Non Hodgkin
large B cell lymphoma in the differential diagnosis of poorly
differentiated neoplasms arising in soft tissues and skeletal
muscle.
Detection of primary lymphoma, by means of immunohistochemical investigation may spare the patient invasive surgical
treatment and provide properly clinical management.
A case of cd5 negative, diffuse Large b-cell
lymphoma with unusual expression of cyclin D1
M. Lucioni1, R. Riboni1, F. Novara2, G. Fiandrino1, S. Kindl3,
O. Zuffardi2, M. Paulli1
1
Anatomic Pathology, Foundation IRCCS Policlinico San Matteo,
University of Pavia, Pavia Italy; 2 Human Genetics, University of
Pavia, Italy; 3 Anatomic Pathology, Ospedale Guglielmo da Saliceto,
Piacenza, Italy
Background. The nuclear protein cyclin D1 plays a major
role in cell cycle control since it promotes transition from G1
to S-phase and, therefore, cell proliferation. Based on its well
defined molecular function, cyclin D1 deregulation may contribute to the pathogenesis of certain lymphoma subtypes 1.
Overexpression of cyclin D1 is, with few exceptions, restricted to three lymphoma subtypes: mantle mantle cell lymphoma
(MCL), plasma cell myeloma and hairy cell leukemia 2.
Cyclin D1 is aberrantly expressed in 90% of MCL as a result
of t(11;14)(q13;q32), between the immunoglobulin heavy
chain (IGH) and cyclin D1 (CCDN1) genes. Expression of
CD5 is also highly characteristic, being present in at least 90%
of mantle cell lymphomas; that notwithstanding, existence of
CD5- cases is well recognized. Plasma cell myelomas express
cyclin D1 in about 40% of cases, some of which show t(11;14).
In hairy cell leukemia, deregulated expression of cyclin D1 is
very common, apparently in the absence of the t(11;14) 2.
In these settings, immunostain for cyclin D1 may be diagnostically useful, also providing prognostic information in the
case of multiple myeloma. Cyclin D1 detection is crucial to
identificate the pleomorphic and blastoid variants of MCL,
two aggressive forms of MCL characterized by large cells
(different from those of classical MCL), high label index and
complex kariotypes. Cyclin D1 expression is of paramount
importance in the differential diagnosis between these MCL
variants and diffuse large B-cell lymphoma (DLBCL), a heterogeneous group of aggressive lymphomas with respect to
morphology, phenotype, genetic features and clinical behaviour, that is usually not associated with t(11;14). Herein we
present an unusual case of DLBCL expressing cyclin D1 in
the absence of CCND1 translocation, addressing the dilemma
of the differential diagnosis for B-cell lymphomas consisting
of large cells and its possible biological significance.
Case history. A 69-year-old male patient presented with
superficial laterocervical and submandibular lymphadenopathies, since 2 months. He was well and reported no systemic
symptoms. Laboratory investigations showed normal white
cell count and a raised lactate dehydrogenase level. Because
of clinical suspicion of a lymphoproliferative disorder, the
patient underwent surgical excision of a right laterocervical
lymph node.
Histological examination revealed complete nodal architecture
effacement by a lymphoid proliferation consisting of medium
to large atypical cells, with round, elongated or pleomorphic
vescicular nuclei, one or more prominent nucleoli and relatively abundant cytoplasm. Numerous mitoses were observed.
The lymphoid proliferation showed a predominant diffuse
growth pattern, with residual vaguely nodular appearance still
detectable at low magnification. Accompanying interstitial
collagenous fibrosis was found, sometimes in broad bands.
By immunohistochemistry, the neoplastic cells strongly expressed CD20, CD79a and PAX5 but were negative for CD3
and CD5. Immunostains for cyclin D1, using both mouse
monoclonal antibody P2D11F11 and rabbit monoclonal antibody SP4, showed moderate nuclear expression in about 60%
of tumor cells. Additional staining for T-cell leukaemia 1
(TCL1) oncogene and the nuclear transcription factor SOX11
were negative. Histogenetic profile (CD10-, bcl-6+, MUM1+)
was consistent with a post-germinal center (GC) derivation.
Lymphoma cells were also strongly positive for bcl-2 and
p53, whereas CD23, IgD and IgM immunostains were negative. Variable expression of CD30 was also found in 30-40%
of lymphoma cells. Mib1/Ki-67 label index was about 40%.
Molecular biology analysis documented monoclonal IGH
gene rearrangement, with use of IGHV 1-69 gene segment and
high load of somatic mutations (8,59%) at direct sequencing.
Search for BCL-1/CCND1 a BCL-2 gene rearrangements by
PCR was negative.
Interphase DNA fluorescence in situ hybridization (FISH)
with IGH/cyclin D1 fusion probes (Vysis®) and cyclin D1
split probes (DakoCytomation®), failed to detect any translocation involving CCND1 gene. Further FISH studies revealed
no translocations involving BCL-2 or c-MYC genes. We also
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performed whole genome array-CGH analysis using the 60K
platform (41.5 KB overall median probe spacing) according
to the manufacturer’s protocol (Agilent Technologies®). Array
CGH experiment revealed complex kariotype, characterized
by the following copy number alterations (CNAs): losses of
1p36.33-p12 (120 Mb), 1p35.3-p35.2 (1,6 Mb), 3, 10, 15,
16q12.12-q12.2 (2,9 Mb), 18, Y; gains of 1q21.1-q44 (102
Mb), Xp22.11-q11.1, Xq11.1-q28. The observed log2ratios of
all CNAs suggested that the rearrangements were in mosaic.
Based on the integration of morphologic findings, tomor cell
phenotype, molecular and genetic data, our final diagnosis
was of DLBCL, centroblastic with aberrant cyclin D1 expression. Following this diagnosis the patient began immunochemotherapy with rituximab, cyclophosphamide, doxorubicin,
vincristine and prednisolone (R-CHOP).
Discussion. The present case is interesting because of unusual
expression of cyclin D1 in a DLBCL centroblastic subtype.
Immunohistochemical detection of cyclin D1 in a B cell lymphoma consisting of medium to large cells usually suggests a
diagnosis of pleomorphic or blastoid MCL; however in our
case several features argued against this hypothesis. Actually,
t(11;14)(q13;q32) or variants, that are found in virtually all
cases of cyclin D1+ MCLs, were lacking. Aberrant phenotypes have been described in MCL and also in blastoid and
pleomorphic variants; rare cases seem to express bcl-6 (1,6%
in a series) 3 CD10, or MUM1, others lack CD5 or IgM/IgD 4.
In spite of this, our case showed a CD5-, bcl-6+, MUM1+
phenotype, which is far more in keeping with DLBCL. In
addition to bcl-6 and MUM1 co-expression, the presence of a
high degree of somatic hypermutation in IGHV genes would
be exceptional in MCL and suggests post-GC histogenesis 5.
Array CGH revealed a highly complex kariotype, but the most
frequent chromosomal aberrations accompanying CCND1
translocation in MCL were lacking 2.
TCL1 and SOX11 are two immunohistochemical markers recently employed in the characterization of B-cell lymphomas,
being positive in the vast majority of MCLs. In particular,
strong TCL1 expression has been detected in the majority of
lymphomas of pre-GC derivation, including MCL, whereas
lymphomas deriving from GC and post-GC B-cells are usually negative, with the exception of Burkitt lymphoma 6.
SOX11 is a neural transcription factor that is expressed in
lymphoblastic lymphoma (almost always), MCLs (up to
93% of cases), and in a subset of Burkitt lymphoma (33%).7
Negativity for both TCL1 and SOX11 in the present case also
seems to exclude MCL, thus confirming the usefulness and
specificity of these novel markers in the differential diagnosis
of mature B-cell lymphomas.
Cyclin D1 positivity is exceptional in DLBCL and mainly
restricted to single case reports 8 9. Few studies have systematically examined the immunohistochemical expression of cyclin D1 by DLBCL. Most authors found no or only occasional
cyclin D1 expression in DLBCL, but we can not exclude that
the recent introduction of more sensitive and reliable antibodies (such as SP4), may result in the detection of an increased
number of cyclin D1+ cases 10. Indeed, a more recent study by
Ehringer et al reported the immunohistochemical expression
of cyclin D1 in 10 (4,3%) of 231 DLBCL, some of which
showing structural aberrations at CCND1 locus, but only one
carrying t(11;14) 11.
These and our findings confirm the existence of CD5-, cyclin
D1+ DLBCL, in the absence of t(11;14), even if these cases
are very rare. Therefore, in current haematopathology practice cyclin D1 immunopositivity alone may be not sufficient
in distinguishing pleomorphic/blastoid MCL from DLBCL.
FISH detection of a t(11;14)(q13;q32) appears preferable for a
definitive diagnosis of MCL, at least in equivocal cases.
The mechanism of aberrant expression of Cyclin D1 in the
present case is unclear. The normal pattern of FISH analysis
using CCND1 probes and the absence of gains at the 11q13
locus suggest that the overexpression of cyclin D1 is related
to posttranslational mechanisms. DLBCL represents a biologically and genetically heterogeneous group of aggressive
lymphomas. Cyclin D1 is one of the key regulators of the
cell cycle and elevated levels of cyclin D1 expression may
accelerate G1/S-phase transition and therefore tumor cell proliferation. Our and other findings seem to suggest that cyclin
D1 overexpression may play a role in the pathogenesis of a
subset of DLBCL.
References
1
Baldin V, Lukas J, Marcote MJ, et al. Cyclin D1 is a nuclear protein
required for cell cycle progression in G1. Genes Dev 1993;7:812821.
2
WHO Classification of Tumours of Haematopoietic and Lymphoid
Tissues. Eds Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA,
Stein H, Thiele J, Vardiman JW. Lyon: IARC Press 2008.
3
Camacho FI, Garcia JF, Cigudosa JC, et al. Aberrant Bcl6 protein
expression in mantle cell lymphoma. Am J Surg Pathol 2004;28:10516.
4
Gualco G, Weiss LM, Harrington WJ Jr, et al. BCL-6, MUM1 and
CD10 expression in mantle cell lymphoma. Appl Immunohistochem
Mol Morphol 2010;18:103-8.
5
Kienle D, Krober A, Katzemberger T, et al. VH mutation status and
VDJ rearrangement structure in mantle cell lymphoma: correlation
with genomic aberrations, clinical characteristics, and outcome.
Blood 2003;102:3003-9.
6
Herling M, Patel KA, His ED, et al. TCL1 in B-cell tumors retains its
normal B-cell pattern of regulation and is a marker of differentiation
stage. Am J Surg Pathol 2007;31:1123-9.
7
Mozos A, Royo C, Hartmann E, et al. SOX11 expression is highly specific for mantle cell lymphoma and identifies the cyclin D1-negative
subtype. Haematologica 2009;94:1555-62.
8
Rodriguez-Justo M, Huang Y, Ye H, et al Cyclin D1-positive diffuse
large B-cell lymphoma. Histopathology 2008;52:889-904.
9
Teruya-Feldstein J, Gopalan A, Moskowitz CH. CD5 negative, Cyclin
D1-positive diffuse large B-cell lymphoma (DLBCL) presenting as
ruptured spleen. Appl Immunohistochem Mol Morphol 2009;17:2558.
10
Cheuk W, Wong KOY, Wong CSC, et al. Consistent immunostaining
for cyclin D1 can be achieved on a routine basis using a newly available rabbit monoclonal antibody. Am J Surg Pathol 2004;28:801-7.
11
Ehringer M, Linderoth J, Christensson B, et al. A subset of CD5- diffuse large B-cell lymphomas expresses nuclear cyclin D1 with aberrations at the CCND1 locus. Am J Clin Pathol 2008;129:630-8.
Diagnosing splenic marginal zone lymphoma in
the bone marrow
C. Tripodo, E. Iannitto *
Dipartimento di Patologia Umana, Università di Palermo; * Unità di
Ematologia con Trapianto di Midollo Osseo, Università di Palermo
Splenic marginal zone lymphoma (SMZL) is an uncommon
B-cell neoplasm listed as a distinct pathological entity in
the WHO classification of tumours of haematopoietic and
lymphoid tissues. SMZL is characterized by a commonly
asymptomatic presentation, indolent clinical course, and
overall survival usually exceeding ten years. SMZL diagnosis
has been classically based on spleen histology, following the
evidence that the spleen harbours most of the disease burden.
Nevertheless, in most cases, the diagnosis of SMZL can be
achieved by the combination of clinical and laboratory data,
peripheral blood examination, and bone marrow histopathology, thus avoiding splenectomy.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Here we discuss the approach to SMZL diagnosis on BM histopathology, by describing the prototypical case of a 63 years
old female patient. The patient presented to the Haematology
Unit of our University Hospital following the occasional finding of splenomegaly during a routine clinical examination.
The spleen was palpable 3cm below costal margin and displayed a maximum diameter of 15cm on ultrasound imaging.
No superficial or deep-sited lymphadenopaty was identified
by physical examination and CT scan, respectively.
Peripheral blood counts were the following: Hb 11 g/dl,
HCT 37, WBC 5.8 × 109/l, Neut. 1.5 × 109/l, Lym. 3.8 × 109/l,
Mono 0.4 × 109/l, PLT 270 × 109/l. LDH was within normal
range and β2-microglobulin was 3.3 mg/ml. Total serum protein levels were normal. However, electrophoresis revealed
an inconspicuous monoclonal band in the gamma region. On
immuno-electrophoresis, the monoclonal component proved
to be of the IgM-kappa type.
Peripheral blood smear analysis revealed the presence of a
fraction of circulating lymphocytes (about 8%) with characteristic “villous” morphology (i.e. the presence of large polar
villi). On flow cytometry, circulating lymphocytes showed the
following phenotype: CD19+, CD20+, CD3-, CD5-, CD4-,
CD8-, CD10-, CD23-, CD25-, CD43-, kappa-light-chain
restricted.
Bone marrow histopathology highlighted a slightly hypercellular marrow (55% overall cellularity) with preserved haematopoiesis and showing the presence of a mixed nodular,
interstitial, and intra-sinusoidal infiltration by medium-sized
lymphocytes, accounting for nearly 40% of the haematopoietic parenchyma. Immunohistochemistry confirmed the
CD20+CD79a+CD5-CD2-CD23-CD10-IgM+ phenotype of
the neoplastic lymphoid cells.
On the bases of these data, a diagnosis of SMZL was performed and the patient was followed-up adopting a watchful
waiting policy.
Bone marrow examination is a crucial step in the diagnosis of
splenic lymphomas. The presence of an intrasinusoidal pattern
of infiltration (either alone or in combination with other patterns) can be frequently observed in B- and T-cell lymphomas
with preferential splenic localization other than SMZL, such
as hairy cell leukemia (HCL), HCL-variant, hepatosplenic Tcell lymphoma, all sharing a tropism for sinusoidal vascular
niches.
A pediatric natural killer lymphoma/leukemia
with indolent course
M. Ungari
I Servizio di Anatomia Patologica, Spedali Civili di Brescia, Italia
Case report. A 3 years old girl showed several erythematous
skin lesions as well as scabby nasal lesion characterized by
spontaneous regression. During the following 5 years, these
lesions occurred every two months. At the age of 8, lesions
recurred weekly. Physical examination showed enlarged right
cervical and inguinal lymph nodes, hepatomegaly and normal
spleen. A computed tomography (CT) scan revealed multiple
adenopaties in the laterocervical, submandibular, subclavian
and axillary regions.
Haematological findings showed a slight anemia (10g/dl) and
leucocyte counts (4,800 µl) with lymphocytosis (70%). The
flow cytometric analysis of peripheral blood showed a natural
killer lymphocyte population (CD2+, CD3-, CD7+, CD16+,
CD56+). Morphological and immunophenotypical analysis of
the bone marrow were normal.
Antibodies vs EBV were tested and the IgG titer was1/128,
whilst IgM titer was negative. The CT scan and a lumbar
puncture were negative.
A skin and a cervical lymph node biopsies were performed.
The skin showed extensive necrosis, infiltration of atypical lymphocytes and histiocytes with angioaggressive and
angiodestructive behavior. The lymph node parenchyma
revealed an effaced architecture, with a diffuse infiltration
of small-to-medium atypical lymphoid cells, that frequently
enchroached upon the wall of large vessels; subcapsular areas
of necrosis were also evident. On imprints, numerous cells
with azurophilic granules were identified. Immunophenotypical examination performed on frozen and paraffin sections revealed a dominant infiltration of cells displaying a NK
phenotype, with expression of CD2, CD56, TIA1, Perforin,
and CD94, and negativity for CD3, CD4, CD5, CD8, CD30,
CD57, the B-cell associated antigens CD19 and CD20, and
those associated with dendritic plasmacytoid cells CD123 and
BDCa2. Finally on both skin and lymph node sections high
number of cells were positive for EBV, detected with EBER
in situ hybridation technique. TCR-gamma rearrangement
study did not show clonal bands.
A chemotherapy according NHL protocol for anaplastic
lymphoma was administered. A good clinical response in
both skin and lymph nodes was abtained since the first cycle
of treatment; flow cytometric analysis of PB cells showed
a decrease (< 20%) of the peripheral NK-lymphoma cells.
Following the second cycle of chemotherapy, full remission
was obtained. Forty months after interruption of treatment,
complete recovery was still enduring.
Discussion. Cytotoxic lymphomas are tumors derived from
T or NK lymphocytes with a cytotoxic phenotype. Neoplastic
cells typically express at least one cytotoxic protein such as
T-cell intracellular antigen (TIA)-1, granzyme B, or perforin 1 2. The World Health Organization (WHO) 3 lists them
as distinct (extranodal NK/T-cell lymphoma nasal type and
cutaneous γ/δ-cell lymphoma) or provisional entities (primary
cuteneous aggressive epidermotropic CD8+ cytotoxic T-cell
lymphoma). A closely related entity seen mainly in children
is hydroa vacciniforme-like lymphoma. This disease shows
overlap with NK/T-cell lymphoma, nasal type, of which it can
be considered a variant 4. However the expression of cytotoxic
proteins is not restricted to a specific group of lymphomas as
they can be observed in micosis fungoides, cutaneous CD30+
lymphoproliferative disorders and subcutaneous “panniculitis-like” T-cell lymphoma. Cytotoxic proteins do not have
any diagnostic or prognostic value per se, and their expression
should be evaluated in the context of the clinico-pathologic
and molecular features of the lesions 4.
Negativity for T-cell markers and germline rearrangement of
T lymphocytes, together with positivity for EBV in neoplastic
cells, should be interpreted as a strong hint towards a diagnosis
of extranodal NK/T cell lymphoma, nasal type 5. This lymphoma is commonly located in the nasal cavity, but involvement
of the skin can be observed. The prognosis in adults is usually
unsuccessfull. Fifteen pediatric cases showed a better outcome
than adults 6. Complete remission was obtained and remained
steady in two-thirds of the patients with localized disease.
Among the 6 cases in stage IV, two had a successful outcome
after treatment with high dose chemotherapy and hematopoietic stem cell transplantation. Moreover, it has been suggested
that NK/T-cell lymphomas that express CD94, a lectin that
inhibits NK function, may have a better prognosis 7.
This case showed unexpected long interval between the first
signs of the disease and the diagnosis. At the age of 3, the
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first cutaneous lesions were evident and characterized by
spontaneous regression. Unfortunately, no skin biopsy was
performed at the age of 3.
References
1
Massone C, Chott A, Metze D, et al. Subcutaneous, blastic natural
killer (NK) NK/T-cell and other cytotoxic lymphomas of the skin: a
morphologic, immunophenotypic and molecular study of 50 patients.
Am J Surg Pathol 2004;28:719-35.
2
Kluin PM, Feller A, Gaulard P, et al. Peripheral T/NK-cell lymphoma:
a report of the IXth Workshop of the European association for Haematopathology. Histopathology 2001;38:250-70.
3
4
5
6
7
Swerdlow SH, Campo E, Harris NL, et al. (eds). WHO Classification
of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC
Press 2008.
Nava VE, Jaffe ES, The Pathology of NK-Cell Lymphomas and Leukemias. Adv Anat Pathol 2005;12:27-34.
Cerroni L, Gatter K, Kerl H. Skin Lymphoma – The Illustrated Guide;
3rd Edition, 2009.
Shaw PH, Cohn SL, Morgan ER, et al. Natural killer cell lymphoma:
report of two pediatric cases, therapeutic options, and review of the
literature. Cancer 2001;91:642-6.
Lin CW, Chen YH, Chuang YC, et al. CD94 transcripts imply a better prognosis in nasal-type extranodal NK/T-cell lymphoma. Blood.
2003;102:2623-31.
Network of biobanks of archived tissues in Italy
Moderators: V. Eusebi (Bologna), G. Stanta (Trieste)
Bioethics in Research on Human Tissue
C. Faralli
Bologna
Research on human tissue, and in particular the collection and
storage of biological samples to that end, raises bioethical
problems relative to the tissue donor’s consent and privacy as
well as to the commerciability of the human body.
Consent. Informed consent is one of the keystones on which
bioethics is built, because through informed consent the basic
human liberties are exercised.
Indeed, the principle of consent had been codified, and the
occasion for it was the aberrant human experimentation carried out in the first half of the twentieth century: this led to
the Nuremberg Code. And the principle has also been made
part of other international documents, such as the Declaration
of Helsinki and the Oviedo Convention, as well as in all European recommendations and directives directly or indirectly
concerned with health.
Under the principle of informed consent, such as it has
evolved through the aforementioned documents and through
the opinion of the Supreme Corte di Cassazione in Italy, no
consent is valid unless accompanied by an adequate notice
providing the following information, especially where the
collection of human tissue is concerned: 1) the purpose for
drawing and storing samples of tissue; 2) the techniques
used to this end; 3) the location of the facilities that will be
analyzing and storing the samples; 4) the lenght of storing
samples; 5) the means used to guarantee the donor’s privacy;
and, not least; 6) a requirement expressly stated by the Italian
Data Protection Commissioner for genetic data, the methods
enabling donors to withdraw their consent and destroy their
samples if they so choose.
Since biobanks cannot be created without processing biological samples, the issue that has emerged in this connection is
that of the “right not to know:” this right, stating that a human
being should be able to decide not to know his or her health or
life prospects, was codified into law in Italy in 2007 through
an authorization by the country’s Data Protection Commissioner.
The hypothesis of an open consent or a trust consent has
emerged in Northern European countries.
A fundamental twofold requisite needs to be met in compliance with ethical, scientific, and legal standards: on the one
hand, someone must be appointed who will be responsible for
the tissue samples; on the other hand, dedicated ethical committees must be set up to which to turn in seeking an opinion
on the use of a biobank and the single biological samples in
it.
Privacy and Discrimination. Clearly, as is the case with
DNA sequences, biological samples can be used in ways that
seriously infringe a person’s privacy, the risk being that of
genetic discrimination, all the more so that the advance of
knowledge is making for greater and greater possibilities,
correspondingly increasing the potential for research to be
conducted with such aims as bring greater harm.
Indeed, the use of databanks containing tissue samples constitutes “processing of sensitive data” and can prove enticing to insurers, employers, and, not least, drug companies,
among others. Under European law – and under Italian law
in particular (especially the rules stated in the aforementioned
authorization by the country’s Data Protection Commissioner
for genetic data, an authorization applying as well to the
processing of biological samples)—much attention is paid to
the risk of unauthorized access to tissue samples, and for this
reason strong security measures are provided for to restrict access to data as far as possible and to subject the tissue samples
themselves to the most exacting formal, physical, and technological controls, while also limiting as far as possiple the
maximum period over which such samples may be stored.
Commercialization. Biobanks containing human tissues are
additionally exposed to the risk of being improperly or illegally used for commerical purposes. The Oviedo Convention
on the human genome and biomedicine accordingly expressly
provides that the human body and its parts may not be used for
profit, a provision based in part on an altruistic and solidarity
principle.
Network di biobanche europeo
G Stanta
A.C.A.D.E.M. Department, University of Trieste, Italy.
Translational research on prognostic and therapy predictive
biomarkers is strictly connected to the availability of normal
and pathological tissues. In the last ten years the traditional
biobanking system has been collecting many thousands of
frozen tissues, but these repositories are being rapidly used.
In the meantime the need for tissues has been increasing
and their unavailability could slow down research. For these
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
reasons a European project “Archive Tissues: Improving Molecular Medicine Research and Clinical Practice (IMPACTS)”
(www.impactsnetwork.eu) was implemented in the past years
to validate molecular methods in formalin-fixed and paraffinembedded tissues, which are usually preserved for a very long
time in any pathology department of any hospital (Archive
Tissues – AT). The project was also devoted to start biobanking procedures on this kind of tissues and on bioethical implications 1. Recently the usefulness of these tissues has been
evaluated by the Biobanking and Biomolecular Resources
Research Infrastructure (BBMRI) 2.
The IMPACTS group, together with the European Society
of Pathology (ESP) and BBMRI is trying to develop a panEuropean network of archive tissue biobanking that could
be a very important instrument for accelerating the clinical
application of molecular medicine. The possibility to carry
out multicentric projects on a voluntary and collaborative
basis with the collection of a very high number of human
pathological tissues correlated with clinical information or
the possibility to collect a sufficient number of rare lesions
is an important issue that must be pursued. The reason for
this is that AT are those tissues available for any patient in
any European hospital and in most of the cases the only tissues available. That’s why any clinical procedure must take
AT into consideration, and pathologists must take in charge
molecular analysis in this kind of tissues. The role of pathologists starts from their ability to identify a huge number
of pathological entities and to recognize the heterogenity of
pathological tissues. In this way effective microdissections
can be performed before any molecular examination. The
unrecognized tissue variabilities could be the basis of many
mistaken results reported in literature. Such mistakes could
lead not only to wrong, but also to confusing research with
retardation of a correct development.
After performing multicentric studies to give validated methods for this kind of analysis, the IMPACTS group prepared
the final version of “Guidelines for molecular analysis in archive tissues”. Pre-analytical conditions and molecular procedures were carefully evaluated to try to standardize the results
of research. The network itself can represent a reference point
for this kind of molecular procedures.
The pan-European network can be organized as a virtual network that can be activated on a voluntary and collaborative
basis. The existing biorepositories in the pathology departments are the basis for the development of the network. When
a pathology department decides to participate in a multicentric
project it can very easily modify its function of biorepository
to the function of biobank just through the anonymization
of tissue samples. The reason for this is that pathologists are
already privacy guarantors, since they are involved in the diagnostic process and therefore subject to professional secrecy.
They can explore the already existing computerized clinical
files to connect tissues with clinical data and follow-up information. They are also in charge of choosing the cases and
defining microdissection criteria. The best choice of cases,
tissues and the validity of methods can guarantee the results
of the studies in the best way ever.
References
1
Stanta G, Cescato A, Bonin S, et al. Bioethics considerations for
medical research in human archive tissues: the point of view of the
researcher” Virchows Arch 2008;453:117-9.
2
Hainaut P., Bevilacqua G, Bosman F, et al. The role of the pathologist in tissue banking: European Consensus Expert Group Report.
Virchows Arch. 2010 Feb 16. [Epub ahead of print] PubMed PMID:
20157825.
Quality control in biobanking
G. Bevilacqua
Division of Surgical, Molecular and Ultrastructural Pathology, University of Pisa and Pisa University Hospital, Italy
Introduction. Over the past 20 years, biobanking of human
specimens has become a central activity underpinning all
aspects of biomedical research as well as the development
of personalized medicine. Biobanking encompasses a wide
range of specimen types and sample collection designs, ranging from population-based biobanking of specimens from
healthy subjects in large, epidemiological cohorts to specific
biobanking of diseased tissues obtained in the course of clinical interventions. Human tissue biobanking is of particular
importance for implementation of novel biomarkers into
clinical trials, as well as for the application of a wide range
of new technologies (-“omics”) to the discovery and validation of new, molecular patterns of disease. Heterogeneity and
variability of pre-analytical practices is a major source of error
in analyzing biobanked specimens. In recent years, large international efforts have converged towards the harmonization
of standard operating procedures for biobanking, providing a
basis for improving reproducibility and comparability of molecular data as well as for designing large, multicentric studies
involving specimen exchanges among different centers. The
most critical steps in the workflow of biospecimen acquisition and annotation for biobanking involve hospital pathologists. Pathology is the cornerstone of tissue biobanking. The
most basic minimal standard for any biobanking operation
is to identify and define the nature and origin of the tissues
to be kept in the biobank. This requires specialized pathology expertise. Furthermore, pathologists also make decisions
on what should be biobanked, making sure that the timing
of all operations is consistent with both the requirements of
clinical diagnosis and the optimal preservation of biological
products. Pathologists also play a central role in the design
of studies involving banked biospecimens and in the dialogue
between clinicians and researchers. The rapid development
of biobanking as an essential process in translational research
and personalized medicine places strong demands on the work
of the pathologist. This document summarizes the conclusions of a Pathology Expert Group Meeting that took place
in Munich in December 2008 within the European Biological
and Biomolecular Research Infrastructure (BBMRI) Program.
The experts have considered all aspects of the involvement of
the pathologist in the biobanking process. They also discussed
the impact of biobanking on pathology practice. The recommendations developed in the document are aimed at providing
guidance for pathologists as well as for institutions hosting
biobanks on how to better integrate and support pathological
activities within the framework of biobanks that fulfill international standards.
Scope and definition. 1. The focus of the working group is
the banking for research of human tissues in a clinical context.
This activity is hereby defined as “tissue banking”. It includes,
but is not limited to, the banking of residual specimens obtained in the course of clinical procedures as well as of “postmortem material.” 2. Tissue banking is a chain of operations
that includes informing patients and obtaining the proper
consent (depending on local requirements), data acquisition,
tissue procurement, annotation, preservation, storage, quality control, cataloguing, managing of access, processing and
distribution. Pathology expertise is required at several steps.
Tissue banking also requires expertise in cryobiology, quality
management, legal/ ethical aspects, project management, staff
Lectures
management, administration and networking. 3. “Pathology
archives” represent a special type of tissue repository that may
support tissue banking, provided that they fulfill required standards with respect to a. documentation of variations; b. cataloguing; c. rules of access; d. fulfillment of legal requirements
for use as research resource. The primary role of these archives
is to document diagnosis and to support later/metachronous
diagnostic analyses but they should be developed in a way that
allows them to fulfill roles in research as well.
Tissue banking: critical role in articulating translational
research and personalized medicine. 1. Tissue banking in
a clinical context is essential for the procurement of high
quality samples for translational research aimed at biomarker
discovery and validation as well as identification of new
targets for therapy. It is therefore a strategic activity for research and innovation in biomedicine. 2. Tissue banking is
critical for implementing and applying biomarkers in clinical practice. It lays the foundations for the discovery of new
targets for therapy and for drug discovery. It sets conditions
and procedures allowing patients to benefit from new developments in biomarkers as well as personalized medicine and
is therefore beneficial for future diagnosis and treatment and
for public health. In this vision, each patient contributes to
the care that will be provided to the future patients. 3. Translational research on biomarkers encompasses three overlapping phases: discovery, validation, and implementation. Each
phase has different requirements in terms of tissue banking.
4. Discovery phase is aimed at identifying biomarkers and
molecular targets for therapy, establishing their prevalence
and formulating hypotheses on their biological and medical
significance in ex vivo analyses. This requires access to well
annotated and pathologically reviewed case series, either
based on specimens collected and processed in the course
of clinical diagnostic activities or in specific tissue collection protocols. 5. Validation phase is aimed at demonstrating
the effect and significance of a potential biomarker. This
requires applying ex vivo analyses within study designs with
adequate epidemiological and statistical power. Such designs
may be comparable to those of clinical trials except that they
do not necessarily imply de novo specimen collection using
invasive procedures. In a number of cases, these studies can
be constructed using retrospective or prospective collections.
6. Implementation phase is aimed at translating biomarkers
into clinical practice in affordable, cost-effective conditions
and at integrating new biomarkers into diagnostic practice.
This requires applying biomarkers to a large series of specimens collected using standard operating clinical protocols.
Role of the pathologist. 1. The pathologist has an essential
role in tissue banking. His medical and scientific expertise is
required at two distinct phases in the process of tissue banking: a. in making diagnostic decisions, providing specific annotations and overseeing specimen procurement and preservation, and b. in reviewing specimens and providing information
prior to specimen processing and distribution to research laboratories. 2. Through his role in tissue banking, the pathologist
is a key actor in the continuity between research and medical
care. 3. The pathologist adds value and expertise to the definition of the banked tissue and is a critical scientific contributor
to research carried out on the specimen. 4. The pathologist
validates the appropriateness of the banked tissue specimen
and its use for a particular research purpose, excluding conflicts with diagnostic purposes. 5. The pathologist has a key
role as custodian of the banked specimens. Tissue collections
are best developed in the context of a pathology department
or pathology service.
201
Role of institutions. 1. Tissue banking is not the exclusive responsibility of pathology departments. It should be run in the
context of institutions (mainly hospitals or universities) that
are responsible for providing the whole chain of expertise and
the organizational frame required for tissue banking. 2. Institutions are responsible for the maintenance, sustainability, and
accessibility of tissue banks, adequate level of training of the
staff and the protection of patient rights. Full cost calculation
is an essential step in guaranteeing the sustainability of the
tissue bank.
Tissue banking in clinical trials. 1. Clinical trials offer a
wide range of designs with added value for the discovery,
validation and implementation of potential new biomarkers.
2. Using biomarkers is critical for the interpretation of many
therapeutic trials in particular for defining the characteristics
of responders vs. non-responders. 3. In future medical care,
biomarkers will become mandatory for allocating patients
to appropriate therapeutic protocols. 4. The participation of
a biobank into a clinical trial should obey to the same strict
technical, legal, and ethical standards independently of the
type of promoter, academic, or industrial.
Improving standards for tissue banking within clinical
practice. 1. There are technical differences in current standards for tissue processing in pathology practice and in tissue
banking. 2. Many protocols used in tissue banking, e.g., for
duration of fixation, optimal time for preservation and duration of storage, are mainly based on experience rather than
evidence. 3. There is a need for more adequate markers of
quality for the tissue-banking process for the qualification of
banked tissue specimens for specific research applications.
4. Discovery, validation, and implementation of biomarkers
and therapeutic targets in the clinics require a very large series
of specimens with inter-laboratory comparison. Such studies
need strong networking between dedicated platforms using
harmonized, comparable protocols.
Incentives for increasing the participation of pathologists.
1. Tissue banking is an important mechanism by which pathologists participate in generating and increasing knowledge
in biomedicine. 2. In many instances, the involvement of the
pathologist adds scientific value to the banked specimens beyond the requirements of routine diagnosis. This added value
corresponds to an intellectual property. 3. Tissue-banking
activities entail considerable costs and demands on pathology
staff time.
Conclusions and perspectives: a strategic vision for tissue
banking in Europe. Today, tissue banks have a key role in
the process of biomarker and drug target discovery through the
procurement of annotated specimens to innovative research
programs. In addition to this research role, the use of cellular
and molecular biomarkers is rapidly becoming a standard part
of hospital pathology practice and of therapeutic decision
schemes. Tissue banking is the key mechanism for pathologists to get involved in translating newly discovered biomarkers into clinical practice. Furthermore, tissue banking will rapidly become an intrinsic part of pathology requirements in the
context of standard clinical care. Given its strong linkage with
clinical activities, tissue banking is best performed at the local
level, and its sustainability requires investment in infrastructure at the local and/or regional and national levels, to avoid
duplication of effort and achieve critical mass necessary to address major academic research programs, as well as to secure
a strong position in addressing the needs of industry. Therefore, tissue banks must be organized in operational networks.
Implementation of biomarkers will require large networks interconnecting tissue banks, analysis and distribution platforms
202
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
and several other data resources such as databases of clinical
information and population-based disease registries. Biobank
networks should have fully documented standard operating
procedures, share tissue bank catalogues, and clear rules for
access. They should also be able to run research projects based
on collections developed in several tissue banks. Such projects
may be retrospective (using previously banked specimens) or
prospective. Running the same, hypothesis-driven collection
protocol through a large network of tissue banks that adhere
to the same standards will allow assembling large case series
addressing a wide range of clinical conditions. In developing
such protocols, the diversity of European populations and
ecological contexts is an asset for the design of sophisticated
case–case comparison studies. To achieve this vision, it is essential to perform innovative research on improving all aspects
of specimen processing, including the development of quality
controls applicable to retrospective collections. This requires a
dedicated effort from funding agencies and from the scientific
and medical publication community. Training of highly qualified tissue-banking professionals will increase the standards
of tissue banking as well as the recognition of tissue banking
as an integral part of biomedicine. This will also facilitate the
development and dissemination of a corpus of harmonized,
evidence-based tissue-banking procedures.
Past President SIAPEC-IAP; Unit of Anatomic Pathology, “Antonio
Cardarelli” Hospital, Naples, Italy
are daily managed by pathologists, these latter looked to be
excluded from any kind of initiatives concerning the topic,
being confined to a marginal role.
The first goal of the Group has been that to involve “the pathologist” and possibly put him at the center of any discussion,
guidelines extension or tissue banking management project,
on the basis of the well recognized competence in selecting,
preserving, storing and studyng human cells and tissues.
Following these ideas, the Group was invited to contribute to
write the “Guidelines for the Institution and certification of
Biobanks”, a document commissioned by Italian Government
in 2006 1.
In 2008 the Group published a thematic issue of Pathologica 2
where several papers on national and international experiences, legal and ethic aspects, sample conservation and quality control, informatics,cost analysis and other related topics
have been presented by most of the Group’s members. All the
published papers have been presented in Italian and English
languages.
Moreover, Congress Sessions, Specific educational Courses,
National and International meetings have been organized
under the patronage of the SIAPEC-IAP and his Biobanking
Group.
Next step is the constitution of a permanent “SIAPEC-IAP
Working Group on Biobanking” connected also to European
Society of Pathology, that should continue to produce documents and organize educational events, having the perspective
of building a progressive Italian biobanking network within a
wider European one, that can involve the majority, if not all,
of national anatomic pathology services.
In 2005, the Executive Committee of Italian Society of Pathology and Cytopathology, IAP Italian Division (SIAPECIAP) decided to institute a “Project tissue biobanking task
force Group”. At that time, the Biobanking was becoming
an emerging issue with several problems and much confusion. The main problem was that,,although cells and tissues
References
1
Linee guida per l’istutuzione e l’accreditamento delle biobanche.
Presidenza del Consiglio dei Ministri. Comitato Nazionale per la biosicurezza e le biotecnologie. Rapporto del Gruppo di lavoro. 19 Aprile
2006.
2
AA.VV. Tissue banking: a tool in Anatomic Pathology. Pathologica
2008;100:43-148.
Biobanking and SIAPEC-IAP
O. Nappi
The cancer crisis in Africa: diagnostic anatomo-pathology
using a multidisciplinary approach
Moderators: F. Bonetti (Verona), C. Clemente (Milano)
Ultrasound and fine needle aspiration:
a low-costs multidiciplinary approach
S. Guzzetti
Department of Histopathology, Ospedale Evangelico Valdese – ASL
TO1, Turin, Italy; Member of “Patologi Oltre Frontiera”, NGO
Recently, the role of pathology in developing countries has
grown and diversified: the increasing demand for projects
not only dedicated to the improvement of the diagnostic level
in low-resource settings but also the mandatory involvement
of pathology in establishing specific programs of preventive
medicine is making our specialization even more essential in
these contexts.
Regardless the project type, the major problems are due to the
shortage of skilled personnel and to the rational use of available economical resources.
Since 1999, the Association “Patologi Oltre Frontiera, NGO”
(APOF) has developed projects dedicated to the improvement
of pathology in developing countries in cooperation either
with local institutions or with other Italian specialists in order
to provide sustainable and multidisciplinary diagnostic tools.
In some of these projects, the combined use of ultrasound with
fine needle aspiration (FNA) made possible a quick, safety and
cheap diagnose for a large group of detectable pathologies.
In Mwanza, Tanzania, APOF restructured and reorganized
the Department of Pathology of the local referral hospital, the
Bugando Medical Center, also improving the cytology and
applying it first for a specifically set outpatient clinic for FNA
on palpable masses, then for ultrasound-guided FNA even for
inpatients.
Similarly, at the Mtendere Mission Hospital in Chirundu,
Zambia, APOF not only provides for the building of a new
Pathology Department, but also organized several missions of
pathologist and radiologists in order to introduce ultrasound
and cytology, together with a program of remote diagnostics
through a system of telepathology.
203
Lectures
The use of ultrasound has proven essential in another project
in Bethlehem, Palestine, where, together with the local Ministry of Health and the Italian Cooperation, APOF is developing
a pilot program for breast cancer screening.
In conclusion, multidisciplinarity and low-cost technologies
can play a key role in the improving of diagnostics in developing countries.
An Italian-Palestinian cooperation project.
The role of pathology dept. in the prevention
and treatment of cancer: the experience of Beit
Jala Government Hospital
R. Shriam, S. Guzzetti *, D. Fenocchio **, P. Giovenali **
Dept. Of Pathology, Beit Jala Government Hospital, Palestine (West
Bank); * Associazione Patologi oltre Frontiera, Serv. Anatomia Patologica, Ospedale Evangelico Valdese, Torino; ** Associazione Patologi oltre Frontiera, Serv. Citologia e Istologia Diagnostica, Ospedale
“S. Maria della Misericordia”, Perugia
Introduction. The aims of “Associazione Patologi oltre Frontiera” (APOF) a nonprofit organization (NGO), established
since 1999 are to implement and improve diagnostic oncology, prevention and treatment of cancer in developing and
emerging countries.
Background. Under agreement between Bethlehem Municipality, the Provincial Authority of Venice, United Nation Developing Program (UNDP), Italian Cooperation: Unità Tecnica Locale (UTL) Of Jerusalem and Palestinian Ministry of
Health (MOH), pathologists of APOF assessed the feasibility
of a project to advance pathology service for the West Bank
area in Beit Jala Government Hospital (BJGH) and submitted
a proposal with a 3-years plan to strengthen and stabilize cytological and histopathological diagnostics, which are essential
for the development of oncology services.
The proposal was accepted by the Ministry of Health and by
UNDP in February 2006.
The project started in collaboration with UTL, including the
construction of the pathology laboratory on 4th floor (160 m2)
of the Beit Jala Hospital and the technical assistance in the
program for prevention and early detection of breast cancer
in West Bank.
At the starting of the project, despite major efforts by UNDP
to recruit at least one physician specialized in pathology from
the Palestinian Territory, the position for pathologist to run
and manage the pathology service at BJGH was vacant, and
the laboratory was not yet able to prepare adequate specimens
for histopathology; for this reason, a pathologist from Nablus
Rafidia Hospital was recruited on a part-time contract, as
consultant, for three years and a Palestinian physician, Dr
Riad Shriam, was identified to complete his studies in surgical
pathology in Italy (University of Pisa, Scuola di Specializzazione in Anatomia Patologica) and since 2009 he is the head
of Pathology Dept. of BJGH
The following staff was recruited and trained during the years
2006-2009:
• Two technicians; one of them was trained in Italy for a 3months stage in immunohistochemistry and tumor markers.
• One medical secretary.
• Three specialist pathologists.
Another physician has nearly completed the specialty fellowship in pathology in Jordan.
Nowadays the lab is fully equipped with all of the instruments
needed.
The Palestinian Ministry of Health, the main partner in this
project, whose input has been essential for the successful
maintenance of this project, will provide reagents and disposables needed for the pathology lab at BJGH.
APOF, in collaboration with UTL and MOH, organized in
April 2009 a training course in Bethlehem with the following
aims:
• staff training;
• development of guidelines ad protocols and procedures of
breast cancer management.
The main aim was to raise the capacity of the staff in identifying the cytological patterns of breast cancer and precursor
lesions and to gain knowledge of pathological classification,
grading, staging and prognostic markers of breast cancer.
The course consisted in training on:
• pre-operative diagnosis by means of FNAC and micro-biopsy (needle core biopsy);
• breast surgical pathology;
• evaluation of prognostic/therapeutic markers and
• developing local guidelines and protocols on breast cancer
management, according to European Guidelines.
In the year 2009, MOH, in collaboration with Italian Cooperation, started a national Palestinian program for early detection
of breast cancer. Bethlehem Governorate was identified as
pilot area and breast screening by mammography started in
BJGH with fully involvement of Pathology Dept.
Results. The reported data are relative to admitted patients,
outpatients and specimens referred to Pathology Lab of BJGH
from other 6 hospital in South West Bank, between January
2006 and May 2010
1) Dept of Pathology
Histo-cytological cases in BJGH (2006-2010)
n. of cases
Breast
Gastro-intestinal
Urinary tract + Prostate
Lymph nodes
Thyroid
Lung
Gynecopathology
Other
Total
747
419
249
205
356
129
2860
9,414
14,379
malignancy
n
191
144
70
49
29
18
75
261
837
%
25.6
34.4
28.1
23.9
8.1
14.0
2.6
2.8
5.8
204
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
FNAC’S in BJGH (2006-2010).
n. of cases
Breast
Thyroid
Other
Total
malignancy
n
45
5
34
84
%
19.4
4.9
50.0
20.9
malignancy
45
9
137
191
controlled
53
5
=
232
102
68
402
Breast pathology cases in BJGH (2006-2010).
n. of cases
FNAC
232
23
Core biopsies
492
Surgical specimens
747
Total
2) Screening
The Breast Unit of BJGH opened in January 2009: since then
and until April 2010, were performed 1.919 mammograms
and/or ultrasounds, 103 out of those were diagnosed as suspicious or positive (5.4%) and FNA was suggested. FNA was
actually performed in 57 women; 20 were positive or highly
suspicious (1.0%), 6 were diagnosed as benign but with uncertain malignant potential (0.3%), 23 were negative and 8
unsatisfactory.
Conclusion. The APOF cooperation project, in collaboration
with Palestinian MOH, leaded to the establishment and organization of a modern and effective Pathology Dept. A specific
false positive
1
0
=
false negative
5
1
=
program was developed to train and bring up-to-date physicians and technicians of the pathology laboratory at BJGH.
The main beneficiaries of the project are the Palestinians,
resident in the West Bank who can benefit from the improved
health services, the medical staff who received training to better
fulfill their specialist tasks, and the Ministry of Health who has
improved medical facilities to serve the Palestinian people
The general objective to improve the level of advanced medical services in the West Bank, has obtained a good support by
the establishment of this new pathology service, needful in a
general hospital with surgical and oncological depts. and for
screening programs.
Men and women in pathology, the strength of the foundation
Moderators: C. Bondi (Parma), S. Uccini (Roma)
Camillo Golgi: a genius of observation
P. Mazzarello
Museo per la Storia dell’Università di Pavia, Dipartimento di Medicina Sperimentale, Sezione di Patologia Generale, Università di Pavia,
Pavia Italy
Camillo Golgi was born at Corteno (today Corteno Golgi),
a small mountain village in the North of Italy on the 7th July
1843. He studied medicine at the University of Pavia where
he graduated in 1865. After his graduation, Golgi started his
clinical activity at the San Matteo Hospital in various medical, surgical and dermatological wards. However he soon
became assistant at the Psychiatric Clinic headed by Cesare
Lombroso who sparked his vocation to study the brain. Following the tenets of the positivist scientific philosophy, advocated by Lombroso, anatomical and anthropological data
became, at that time, the tools by which biology could explore neuropsychiatric diseases. Thus Golgi, in collaboration
with Lombroso, began to investigate the etiology of mental
and neurological illness from an experimental and antimetaphysical point of view. Meanwhile in the free time that
his hospital duties allowed, Golgi attended the Institute of
General Pathology under the direction of Giulio Bizzozero,
the rising exponent of the new experimental medicine which
had as its emblem the microscope. From Bizzozero, Golgi
acquired a passion for histological investigation, the direct
means of penetrating the formidable unknown of the archi-
tecture of the nervous system. Even if three years younger
than Golgi, Bizzozero thus became his master, patron and
the “catalyst” of his mind. Under his direction, Golgi began
to publish works between 1870 and 1872, the most important
of which were dedicated to the study of the neuroglia and
which were flatteringly quoted in international literature. By
1872 Golgi had acquired a solid reputation as a clinician and
histologist but this was not considered enough to earn him a
satisfactory position at the University. On 1872 pressured by
his father, Golgi took part in and won the competition for the
post of Chief Physician at the Pio Luogo degli Incurabili, a
hospital for chronic diseases, at Abbiategrasso near Milan.
Everything suggested that, with Golgi’s arrival in a small
town hospital, his research activity were about to end for
good. However after some initial difficulties, Golgi set up a
rudimentary laboratory consisting of a microscope and a few
instruments in the kitchen of Golgi’s small quarters. On 16
February 1873, Golgi in a rush wrote the following words
to his friend Nicolò Manfredi: “I spend long hours at the
microscope. I am delighted that I have found a new reaction
to demonstrate, even to the blind, the structure of the interstitial stroma of the cerebral cortex. I let the silver nitrate
react with pieces of brain hardened in potassium dichromate. I have already obtained magnificent results and hope
to do even better”. This is the first record of the invention
of the black reaction known nowadays as “Golgi staining”
or “Golgi impregnation” that was a breakthrough for brain
Lectures
structure research. The black reaction consists of a first phase
of hardening the tissue in potassium dichromate followed by
the impregnation of the nervous elements by silver nitrate.
The final result is a preparation in which the silhouette of the
nerve cell appear in all its morphological complexity with all
its ramifications, which could be followed and analysed even
at a great distance from the cell body. The great advantage
of this technique is that, for reasons that are still unknown,
a precipitate of silver chromate randomly stains only a few
cells in black (usually from 1 to 5%), and completely spares
other surrounding cells, allowing the individual elements to
emerge from the nervous puzzle. The discovery of the black
reaction provided the spark to a truly scientific revolution
which allowed the morphology and the basic architecture of
the cerebral tissue to be displayed in all its complexity, thus
contributing to the foundations of modern neurosciences.
Golgi remained in Abbiategrasso until January 1876; there he
discovered the constant presence of the axon in nerve cells,
the branching of the axon, the presence of striatal and cortical
lesions in a case of chorea and performed studies on the structure of the cerebellum (describing the so-called Golgi cells
of the cerebellar cortex), and of olfactory bulbs. Meanwhile
he began to elaborate on a general theory of brain organization, the so-called “diffuse nervous net” according to which
the axons are connected (through direct fusion or intimate
contact) in a diffuse network along which the nervous impulse is propagated. Although this concept was in polemical
opposition to the “neuron theory”, ironically the indefatigable
paladin of this theory, the Spaniard Santiago Ramón y Cajal,
became such by using the Golgi stain.
After the discovery of the black reaction Golgi became Professor of Histology at the University of Pavia in 1876 and
from 1879 onward, he also became Professor of General
Pathology and honorary chief with direct clinical responsibilities of a medical ward at the San Matteo Hospital. In 1878
he described two kinds of tendinous sensory corpuscles: the
Golgi tendon organ (proprioceptors) and the Golgi-Mazzoni
corpuscles (transductor of pressure stimuli). Then he invented
the staining method with potassium dichromate and mercuric
chloride (1878-79), discovered the myelin annular apparatus
(horny funnel of Golgi-Rezzonico, 1879) and analysed several
regions of the nervous system in detail providing beautiful
illustrations of them (Golgi, 1885). Between 1885 and 1892
he concentrated on studying human malaria. He was soon
able not only to determine the entire intraerythrocytic cycle of
development of the malaria parasites for tertian and quartan
(Golgi cycle), but he also discovered the temporal relation
between the recurrent febrile bout and the segmentation of
the parasite (Golgi law). Meanwhile he concentrated on the
study of kidney histology, histopathology and histogenesis
(1884-1889) and discovered the important relationship between the vascular pole of the Malpighian glomerulus and the
distal tubule, which plays an important role in the regulation
of blood pressure.
A skilled physician who always refused private activity, he
also published important clinical studies on peritoneal blood
transfusion, intestinal worm infection, regeneration and pathological changes of the kidney. He also observed independently
from the Swedish histologist Erik Müller, the canaliculi of the
parietal cells of the gastric glands, often called Müller-Golgi
205
tubules. At the end of 1893 he was elected, for the first time,
Rector of the University of Pavia, and held the position until
1896. Thereafter Golgi returned to the study of the nervous
system and using a variant of his black reaction he was able
to observe, in 1897, a “reticulum” in the cytoplasm of cells of
spinal ganglia, the so-call internal reticular apparatus, subsequently christened the Golgi apparatus or Golgi complex.
Meanwhile Golgi observed the perineuronal net which constitutes a reticular structure enveloping many neurons.
On the twentieth century, Golgi’s scientific creativity faded.
His time was divided between new responsibilities in the
direction of Pavia University (of which he was again made
Rector from 1901 to 1909) and the Senate of the Italian Kingdom, of which he was elected member from 1900. In 1906
he reached the pinnacle of his international fame, when he
received the Nobel prize for Physiology or Medicine, which,
ironically, was also won by his eternal scientific rival Ramón
y Cajal.
During the First World War, Golgi directed the Military
Hospital Collegio Borromeo of Pavia, and promoted the rehabilitatory treatment for the war-wounded. In 1918 he retired
from the University of Pavia at the age of 75, but continued
to teach Histology as Professor Emeritus until the beginning
of the 1920’s.
During his life he was elected honorary doctor of the Universities of Cambridge, Geneva, Kristiania (Oslo), Athens and
Paris (Université de la Sorbonne). He had been Dean of the
Medical Faculty of the University of Pavia and member of a
number of international academies and scientific societies.
He died in Pavia on 21 January 1926.
In Golgi’s laboratory Carlo Martinotti identified the cell
named after him in the cerebral cortex, Aldo Perroncito described the phases of regeneration in the peripheral nerves,
Emilio Veratti observed the T system linked to the sarcoplasmic reticulum and Adelchi Negri discovered the intraneuronal
inclusions (the Negri bodies) in animals and humans infected
with the rabies virus. Many other scientists spent periods
of study and specialization in Golgi’s laboratory such as
Giovanni Battista Grassi, the discoverer of the Anopheles
which transmit human malaria, Antonio Carini who discovered the Pneumocystis carinii (recently renamed P. Jiroveci)
and Fritjof Nansen a Norwegian zoologist and a Nobel Prize
winner for Peace in 1922.
References
Golgi G. Sulla struttura della sostanza grigia del cervello. Gazzetta
Medica Italiana – Lombardia 1873;33:244-6.
Golgi G. I recenti studi sull’istologia del sistema nervoso centrale.
Rivista critica. Rivista Sperimentale di Freniatria e Medicina Legale,
1875;1:121-30 (first part), 260-74 (second part).
Golgi C. Sulla fina anatomia degli organi centrali del sistema nervoso.
Reggio Emilia: Tipografia di Stefano Calderini e Figlio 1885.
Golgi C. Opera Omnia. Vol. I-III. Fusari R, Marenghi G, Sala L (eds).
Milano: Hoepli Editore 1903.
Golgi C. Opera Omnia. Vol. IV. Sala L, Veratti E, Sala G. (eds). Milano:
Hoepli Editore 1929.
Mazzarello P, Garbarino C, Calligaro A. How Camillo Golgi became “the
Golgi”. Febs Letters 2009;583:3732-7.
Mazzarello P. Golgi. A biography of the founder of modern neuroscience. Transl. by and A. Badiani and H. Buchtel. New York: Oxford
University Press 2010.
Mazzarello P. The rise and fall of Golgi’s school. Brain Research Reviews (in press).
206
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Giuseppina Cattani
Woman and scientist
In the reconstruction and internationalization
process in post-unification Italy
S. Tugnoli Pàttaro
Department of Philosophy, University of Bologna, Italy
Background. “We regret to announce the death of Dr. Giuseppina Cattani [1859-1914], lecturer on general pathology,
first in the university of Turin, later in that of Bologna. Her
name is associated with that of Tizzoni in the investigation
of tetanus. She was also the author of several memories embodying the results of independent research. The state of her
health made it impossible for her to continue her labours as a
university teacher, but she continued to direct the laboratories
of the civil hospitals and the observation asylum of her native
town, Imola” 1.
After Cattani died, her name seemed to have been consigned
to oblivion, as evidenced by the fact that it does not appear in
any general historico-scientific bibliographical dictionary, not
only abroad but also in Italy.
Today, however, it is much easier to talk about Giuseppina
Cattani than it was even only a few years ago.
Indeed, in gender studies – an area of investigation which
originated in 1968-70 in parallel with the developing feminist
movement, first in the United States and then in Europe, and
which has been intensifying since the 1980s – a broad and
intense historiographical effort has been undertaken leading to
unprecedented historiographical findings of great interest, no
doubt heralding further developments down the line.
These findings have really lead to the “rediscovery” of documents bearing witness to the role that female scientists have
played in the history of scientific thought. In this process,
involving an effort to recover forgotten documentary sources,
Giuseppina Cattani has herself begun to regain in the history of science the sort of visibility she certainly deserves.
For which reason she now receives special mention even in
foreign dictionaries of female scientists. Suffice it to mention
in this regard that in a listing of female scientists, she figured
among the “leading contributors,” along with the FrenchAmerican neurologist Augusta Dejerine Klurapke; more to the
point, the vast research Cattani has done on tetanus has earned
for Italy a position as a “leading Western country for pre-1901
bacteriology research by women” 2.
Methods. The method to be used will be as follows. We will
first provide an outline pointing out key moments in the life
and training of Giuseppina Cattani as a woman and as a scientist, to this end relying on recently discovered documents
to which we will refer the reader for further study. We will
then proceed to an interpretation of the primary and secondary
sources collected, offering a new, contextualized reading of
Cattani at the historical moment in which she lived.
Results. We will have two main objectives.
1) The first of these is to illustrate three closely interconnected
perspectives from which Cattani as a historical figure can be
rendered. One such perspective will be that of her scientific
contribution, recognizing a primary role for her discovery of
a tetanus vaccine with Guido Tizzoni, a discovery that immediately gave her prominence in the international effort in
search for the best cure against tetanus, and owing to which
Guido Tizzoni was awarded a Nobel Prize in 1895; the second
perspective is that of her civil-political commitment, militating in the ranks of internationalist and socialist movements
that university students and scholars across all areas of study
(from literature to medicine) took part in at that time; lastly,
we will discuss her role in the women’s liberation movement,
where she fought for equal rights with men – political rights,
and especially the right to education and the right to practice a
profession (the medical profession where she was concerned,
including research and medical teaching at university) – in a
society that denied women both types of rights.
2) Our second objective will be to illustrate the interest that
Cattani has for us today once we take the three aforementioned aspects of her activity and view them in the context of
the social, political, economic, and scientific ferment that was
stirring in her time in Europe and especially in Italy. She was
born on the eve of Italian Unification (1861) and died five
months after the outbreak of World War I (1914). She thus
lived at a time when the construction of the Italian state was
in full swing, a construction that even in the face of enormous
difficulties, and sometimes of contradictions, was pursued
according to a specific design, on a political level as well as
on a cultural and a scientific level, in which last respect the
effort was to lift the country from its provincialism and make
it an international player, singling out strategic points around
which to develop academic research and teaching. Cattani
partook in full of the élan and civil and scientific commitment
that distinguished many of her contemporaries, from humanists to scientists—but with a huge additional hurdle, that of
being a woman in a “world without women,” in the words of
David F. Noble (1992).
References
1
Obituary. BMJ 1915;1:577-8.
2
Creese MRS, Creese TM. Ladies in the Laboratory II: West European
Women in Science, 1800-1900; A Survey of Their Contributions to
Research. Lanham, MD: Scarecrow Press 2004, p. 287.
207
Lectures
Trophoblast pathologies
Moderators: E. Fulcheri (Genova), A. Salerno (Bologna)
Management of Gestational Trophoblastic Disease
M.J. Seckl
Gestational Trophoblastic Disease Centre, Charing Cross Hospital
campus of Imperial College London, UK
Introduction. Gestational trophoblastic disease (GTD) is a
spectrum of pregnancy related disorders comprising the premalignant conditions of complete (CHM) and partial (PHM)
hydatidiform moles (HM) through to the malignant conditions
of invasive mole, choriocarcinoma (CC) and the rare placental
site trophoblastic tumour (PSTT). The latter three conditions are also collectively known as gestational trophoblastic
tumours or neoplasia (GTN). Sixty years ago, most women
with this group of diseases could expect to die. However, with
modern management and careful follow-up protocols, overall
cure rates can exceed 98% with retention of fertility. This
success can be explained by 3 factors: 1) the development of
effective therapies, 2) the use of human chorionic gonadotrophin (hCG) as a biomarker and 3) centralization of care.
What is Hydatidiform Mole (HM) and who gets it? HM
affect 1-3 per 1000 pregnancies. About 10% of hydatidiform
moles subsequently transform into one of the malignant forms
of GTD. HM are abnormal conceptions resulting in excessive
placental, and little or no fetal, development. HM can affect
women throughout the reproductive-age range but are more
common at the extremes of childbearing age. Thus, women
< 16 years old have a six-fold higher risk of developing the
disease compared to women aged 16-40, whilst those conceiving aged > 50 have a 1 in 3 chance of having a molar pregnancy. Interestingly, the previously documented higher incidence
in women of far-eastern origin, although still greater than for
Caucasions, is now falling to more closely match the rates
seen in the UK and other western countries. The reasons for
this are not clear but might reflect dietary changes. HM can
also rarely develop (1:100,000 pregnancies) as part of twin or
multiple gestations.
How does GTD present clinically? In the United Kingdom,
most women with HM present with vaginal bleeding and/or
suspected miscarriage in early pregnancy, prompting a pelvic
ultrasound examination, although in one observational study
of 41 women with confirmed CHM, 40% were entirely asymptomatic, being detected following routine early pregnancy sonographic examinations. The remaining majority presented with
vaginal bleeding; only 2% reported symptoms of hyperemesis
and none had any other systemic manifestations. Vaginal bleeding in early pregnancy is of course common and is often innocent, but such symptoms should precipitate an early ultrasound
examination. The presence of material in the uterus in the absence of a viable pregnancy will lead to uterine evacuation with
examination of products for identification of pathology.
How is a diagnosis of GTD made? The grape-like or hydropic change most commonly found with CHM occurs mainly in
the second-trimester and an ultrasound performed at this stage
shows a classical snow-storm like appearance. However, most
women develop vaginal bleeding in the first trimester and now
undergo uterine evacuation around 8-9 weeks of gestation in
the UK. At this time, there is minimal hydropic change which
makes early sonographic diagnosis of hydatidiform moles less
reliable. Two large, recent retrospective studies from centres
in London have reported that correct pre-evacuation identification of molar pregnancy by ultrasound in the first and early
second trimester is achieved in around only 40-60% of cases
in routine clinical practice. In the largest study, of > 1,000
patients referred to a regional trophoblastic disease centre,
only 40% had a pre-evacuation ultrasound diagnosis suggesting molar pregnancy, including 80% of complete and 30% of
partial moles. The sonographic diagnosis in the majority of
cases was simple miscarriage, with the diagnosis of HM being
dependent on subsequent routine histological examination of
the products of conception. The implications of not sending
evacuated uterine contents for histology are clear, since if the
diagnosis is not made, subsequent monitoring for malignant
change is not instituted and such women have a significantly
increased risk of life threatening complications such as uterine
perforation and severe haemorrhage; in a retrospective study
of 51 women with HM following pregnancy termination,
women without a known histological diagnosis were significantly more likely to have subsequent life-threatening complications, or require surgical intervention and chemotherapy
compared to those in whom a histological diagnosis of HM
made been made.
Pathology of GTD. It follows from the above that pathological diagnosis of HM is essential. All GTD is derived from
components of the normal human placenta, hydatidiform
moles (HM) plus CC, and PSTT, representing villous and
interstitial trophoblast, respectively. Most CHM and PHM
have distinctive morphological characteristics, although these
features have changed in recent years with earlier gestational
ages at evacuation (median 8-9 weeks in the UK). First-trimester CHM show characteristic abnormal ‘budding’ villous architecture with trophoblast hyperplasia, stromal karyorrhectic
debris and collapsed villous blood vessels. In contrast, early
PHM show patchy villous hydrops with scattered abnormally
shaped irregular villi with trophoblastic pseudoinclusions and
patchy trophoblast hyperplasia. The morphological distinction
between non-molar miscarriage (NMM) and PHM can sometimes be difficult, since villous dysmorphism may be present
but NMM do not show trophoblast hyperplasia characteristic
of PHM. Ancillary techniques may rarely be required including immunostaining with p57KIP2, the product of cyclin
dependent kinase inhibitor CDKN1C. This is expressed from
the maternal allele as nuclear staining of cytotrophoblast and
villous mesenchyme in placenta of all gestations other than
androgenetic CHM. In addition, ploidy analysis by in-situ
hybridisation or flow cytometry can distinguish diploid from
triploid conceptions, so facilitating the diagnosis of PHM but
not distinguishing CHM vs diploid non-molar, or molar vs
non-molar triploidy, which require molecular investigations.
CC are malignant hCG-producing epithelial tumours demonstrating central necrosis and characteristic biphasic architecture recapitulating cytotrophoblast-like cells and multinucleate, pleomorphic syncytiotrophoblast-like areas; mononuclear
cells may predominate in some cases, especially post-chemotherapy. Intraplacental CC may occur and probably represent
the source of metastatic CC following term pregnancies.
Neonatal choriocarcinoma is well-described, with most cases
now thought to represent metastatic spread from an intraplacental choriocarcinoma. PSTT is the malignant equivalent of
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
extravillous interstitial implantation site-like trophoblast and
forms uterine lesions with less haemorrhage and necrosis,
and lower hCG levels, than CC. A specific variant of PSST
with distinctive hyalinization has been reported, Epithelioid
Trophoblastic Tumour (ETT) which is clinically thought to
behave like PSTT, but data are still relatively sparse.
How are HM initially managed? Since in many cases the diagnosis of HM is unsuspected until histological examination,
it is important that all products of conception from non-viable
pregnancies and those suspected of molar disease are submitted for routine pathological evaluation. In clinically suspected
cases, initial management is suction uterine evacuation, (sharp
curettage should be avoided to minimise the risk of uterine
perforation). Usually, initial evacuation is sufficient to remove
most molar material and any residual tissue subsequently involutes. A second uterine evacuation when there is evidence
of persisting disease with further vaginal bleeding, regrowth
of molar material and a plateaued or rising hCG is not usually
recommended because 70% of patients undergoing a second
evacuation will still need chemotherapy which is safe and curative, and each procedure carries a risk of uterine perforation,
infection and massive haemorrhage.
What’s the risk of malignant disease and should women be
screened? In benign disease, patient hCG levels spontaneously return to normal, but in those who develop GTN, the hCG
concentration plateaus or rises. The risk of malignant sequelae
following a complete or partial HM is 15% and 0.5% respectively. This is detected in almost all cases by regular hCG
monitoring using an hCG assay capable of detecting all the
different forms of the hormone that can be produced in cancer,
with sensitivity and specificity of almost 100%. In general, in
cancer medicine one would perform a tissue biopsy to prove
malignancy. However, malignant GTD is highly vascular and
re-biopsy is contra-indicated, since it may be associated with
life-threatening haemorrhage. To ensure reliable monitoring
of hCG levels after a molar pregnancy, all patients in the UK
are registered with one of three centres: Ninewells Hospital
(Dundee), Weston-Park Hospital (Sheffield) and Charing
Cross Hospital (London). Although most other countries do
not have a centralized screening program, the majority will
have designated regional centres to manage GTN.
Any patient with a diagnosis of HM, either clinically or following routine histological diagnosis, should be registered
for surveillance with a specialist centre. In the UK, this is
usually done by the Gynaecologist either using paper or webbased registration. The patient and managing doctors are then
posted an information pack and the referring hospital asked
to provide histological material for central pathology review
where available. Patients then submit regular samples for hCG
monitoring. The protocols for monitoring hCG differ slightly
between centres regarding the frequency and type of samples
required but the principle is to monitor the patient at least until
hCG levels have returned to normal. At Charing Cross Hospital hCG is measured in serum and urine for several months of
normal values and following this protocol the risk of missing
treatable disease is about 1:1400. Intriguingly, reactivation
of molar disease may occasionally occur after a subsequent
pregnancy, even several years later, therefore repeat hCG
monitoring at 6 and 10 weeks after any further pregnancy is
recommended. Following a molar pregnancy, the risk of a
subsequent mole rises to 1:80, but most women have normal
pregnancies after their first hydatidiform mole. Other centres
in the world have advocated using abbreviated hCG followup protocols, particularly for partial mole, where the risk of
malignant progression is lower. However, this increases the
risk of undetected malignant disease and since hCG testing is
cheap and prevents life-threatening complications, we do not
advocate such shortened follow-up.
Factors that increase the risk of malignant progression of
HM. Logically, one might expect that hydatidiform moles that
progress to later gestations before evacuation should acquire
more genetic changes with increased malignant transformation. However, two studies, one of twin pregnancies comprising a mole and healthy co-twin and the other in singleton
molar pregnancies, indicate that gestational timing of molar
evacuation does not affect the risk of developing malignant
disease. Conversely, the method of evacuation does appear
important since procedures that induce uterine contractions
could theoretically increase the risk of persistent disease and
systemic spread. Finally, evidence based on retrospective series of patients from the UK suggest that the hormones in the
combined oral contraceptive pill (OCP) may increase the risk
of malignant sequelae in a subset of women in whom hCG
levels remains raised, therefore UK centres currently recommend avoidance of the OCP until hCG levels have returned to
normal. This area remains controversial, however, with data
from other countries suggesting that OCP use may be safe.
Who requires chemotherapy after HM? Most patients will
exhibit plateaued or rising hCG levels indicative of GTN
(usually invasive mole or choriocarcinoma) with or without
vaginal bleeding. If bleeding is severe this is, in itself, an indication for chemotherapy to reduce haemorrhage even if the
hCG level is falling. Women with hCG levels > 20,000 IU/l
one month after molar evacuation are at risk of uterine
perforation and chemotherapy is required to help preserve
fertility, and histological diagnosis of choriocarcinoma or placental site trophoblastic tumour, or the presence of metastases
should prompt urgent referral for treatment. The commonest
metastatic disease site is lung, which may be associated with
dyspnoea, cough, haemoptysis, and/or chest pain, but any site
can become involved. Consequently, any woman of childbearing age presenting with possible metastatic disease should
have GTN included in the differential diagnosis. A positive
serum or urine hCG test will suggest the diagnosis and should
prompt referral to a GTD centre.
What happens to patients referred for specialist treatment? In the UK treatment is provided at two specialist centres (Sheffield and London), with similar treatments offered
in many countries. In order to determine the chemotherapy
regimen required, women are assessed to estimate their risk
of having disease which might become resistant to single
drug therapy with methotrexate. Risk scoring will usually be
determined based on history, examination, serum hCG concentration, Doppler pelvic ultrasound and chest radiograph.
About 2/3 of women with low risk (score 0-6) disease will
be cured with methotrexate alone, whilst women at high risk
(score > 7) require combination drug chemotherapy, usually
involving etoposide, methotrexate and Dactinomycin alternating weekly with cyclophosphamide and oncovine (EMA/CO).
Analysis of UK data reveals that the vast majority of women
following molar pregnancy have low-risk disease, since they
are on hCG surveillance and the onset of malignant disease
is detected early. They receive methotrexate injections intramuscularly alternating daily, with folinic acid tablets, for
one week, repeated every two weeks. Therapy is continued
until the hCG has been normal (< 5 IU/l on the Charing Cross
hCG assay) for 6 weeks. This regimen is well tolerated, with
only 2% suffering troublesome side-effects such as mouth
ulcers and sore eyes, which are managed with mouthwashes
and hypromellose eyedrops respectively, and sometimes by
Lectures
increasing the folinic acid dose. Patients are admitted for their
first cycle of methotrexate due to the potential risk of bleeding. Additional treatment courses are usually administered by
a practice nurse, GP or local hospital.
Response to therapy is assessed by a falling hCG serum concentration monitored twice weekly. In one third of women,
treatment is changed either because of drug resistance or, very
occasionally, severe toxicity (mouth ulcers or methotrexateinduced serositis). Those developing methotrexate resistance
at relatively low hCG concentrations (hCG < 100 IU/l) are
usually cured with Dactinomycin, which is slightly more
toxic, causing hair loss, myelosuppression, mouth ulcers and
nausea. The remaining resistant patients, and the occasional
patients not cured by Dactinomycin, are effectively salvaged
with EMA/CO chemotherapy. This requires an overnight hospital stay every two weeks, and is more toxic, inducing alopecia, myelosupression, lethargy, nausea and other short-term
problems. Moreover, in contrast to methotrexate which has
no long-term toxicity, EMA/CO hastens the menopause by
about 3 years, and increases the risk of a second malignancy
by about 1.5 fold compared to the general population. None
of the therapies affects fertility and the overall outlook is excellent with an almost 100% cure rate for women developing
GTN after an HM based on a study of 485 patients with GTN
following HM.
Presentation and Management of High Risk GTN. Most
high risk GTN patients present with multiple metastases
months or years after the causative pregnancy which could
have been of any type. Symptoms and signs will vary depending on the location of disease. Patients with brain metastasis
may present with seizures, headaches or hemiparesis whilst
those with lung metastasis or disease in the pulmonary vasculature might have haemoptysis, shortness of breath and/or
pleuritic chest pains. Menstrual irregularity may be present but
is not universal, so unless clinicians consider GTN in the differential of metastatic disease and measure the serum or urine
hCG the diagnosis can be overlooked. If the hCG is raised, the
patient should be immediately discussed with the nearest GTD
centre regarding further management. Imaging investigations
should include CT body, MRI brain, Doppler ultrasound and
MRI pelvis. If the brain scan is normal then a lumbar puncture
to assess the CSF:serum hCG ratio (normal less than 1:60) can
help to exclude occult CNS disease. Biopsy of these highly
vascular tumours should be avoided to prevent life threatening
haemorrhage. However, where a lesion is easily accessible and
bleeding can be controlled then excision biopsy may be helpful. This is particularly important if a PSTT or non-gestational
tumour might be present, since their management differs from
gestational choriocarcinoma. Fortunately, PSTT has a distinct
histological appearance and comparison of microsatellite polymorphisms in the tumour with DNA from the patient and her
partner can determine whether the tumour is gestational. The
phenotypic appearance of the tumour is not always reliable
and rarely non-gestational carcinomas may appear morphologically very similar to gestational choriocarcinomas, and
conversely, the latter can occasionally mimic other epithelial
tumours. Chemotherapy is effective at curing the gestational
tumours whilst the chance of survival from a non-gestational
tumour reflects the primary site of origin.
The patients scoring over 7 are at high risk of developing drug
resistance and so are very unlikely to be cured with single
agent chemotherapy. Consequently, several different multiagent therapies have been developed. At Charing Cross, after
many years of progressive experience, a regimen was developed consisting of etoposide, methotrexate and actinomycin
209
D (EMA) alternating weekly with cyclophosphamide and
vincristine (CO). This has been widely adopted worldwide
because it appears to be effective with predictable and easily
managed short-term toxicity. Indeed, a retrospective comparison from the Korean GTD centre’s experience of MFA, MAC,
CHAMOCA with EMA-CO demonstrated a remission rate
of 63.3% (31/49), 67.5% (27/40), 76.2% (32/45) and 90.6%
(87/96), respectively. The EMA/CO regimen requires one
overnight stay every 2 weeks and causes reversible alopecia.
It is myelosuppressive but G-CSF support helps to maintain
neutrophil count, treatment intensity and avoid neutropaenic
febrile episodes.
The cumulative 5-year survival of patients treated with this
schedule varies between 75-90%, and of 272 cases at Charing
Cross was 86.2% (95% CI 81.9% to 90.5%). While these results were good, the presence of liver or brain metastases correlated with only 27% or 70% long-term survival, respectively
and was just 10% with both liver and brain metastases. The
reasons why these patients have adverse outcomes is unclear
but most did not have a prior HM, were not registered for
follow-up and consequently presented with extensive disease.
Furthermore, many deaths occurred soon after admission from
haemorrhage or metabolic complications of overwhelming
disease. Indeed, if deaths within 4 weeks (before adequate
chemotherapy can be given) are excluded, survival of patients
with brain metastasis is similar to other patients. The situation
with liver metastasis may be similar; of 37 patients with liver
metastasis treated between 1977-2005 at Charing Cross, overall survival had increased to approximately 50% at 5 years
but if early deaths were excluded, survival was nearly 70%
(ISSTD Conference 2009). In addition to disease extent, other
factors associated with poor outcome include the type of, and
duration from, the antecedent pregnancy and the prior use of
chemotherapy.
To reduce early deaths in patients with very advanced disease,
we have found that commencing chemotherapy gently with
low dose etoposide and cisplatin (100 mg/m2 and 20 mg/m2,
respectively for two days) combined with dexamethasone
24 mgs in 24 hours to diminish tumour oedema has been
helpful. Further details on the management and modifications
of treatment required for these and other challenging clinical situations such as brain metastasis and pulmonary failure
are beyond the scope of the present review but are contained
within the following references.
Similar to low-risk disease, therapy is continued for 6 weeks
of normal hCG values or 8 weeks if poor prognostic features
such as liver or brain metastases are present (19). Patients are
then re-imaged to document the post-treatment appearance for
future comparison. Removal of residual masses is unnecessary as it does not reduce the risk of recurrence which is less
than about 3%.
Follow-up post-chemotherapy. Post-treatment, patients are
followed-up with hCG measurements weekly for 6 weeks,
two-weekly for 3 months and then with diminishing frequency
until just 6-monthly urine samples are requested according to
the Charing Cross protocol In the UK, the follow-up continues
indefinitely since insufficient data is available to determine a
safe time to stop, but is variable in other countries. Of 1708
GTN patients at Charing Cross Hospital, including women
presenting after non-molar pregnancies, the overall relapse
rate was 3.5%, most of which occurred within the first year
post-treatment. Therefore, women are advised not to become
pregnant for 12 months since this may mask early detection
of relapsed disease. Fertility is unaffected by either low risk
methotrexate or high risk combination agent chemotherapy
210
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
with EMA/CO. However, the latter brings forwards the date
of the menopause by about 3 years. Second cancer are also increased by combination agent chemotherapy by about 1.5 fold
compared to the general population but single agent therapy
has no measurable effect.
Survey on the incidence of gestational
trophoblastic disease in histopathology:
rare or underdiagnosed?
A. Salerno
Bologna
Background. The epidemiology of gestational trophoblastic
disease (GTD) is not well understood. Despite extensive epidemiological data spanning more than 50 years, the extent to
which genetic and environmental factors including race, age
and geographic location influence the variably in reported
differences in incidence rates for GTD throughout the world
is uncertain. Three obstacles limit the interpretation of most
published studies: case definition, case detection and identification of the population at risk.
Case definition: many reports lack a precise and reproducible
case definition of the disease entities encompassed by the
term “gestational trophoblastic disease”. Until recently there
was no universally accepted classification for GTD. Several
systems continue to be used for staging, including the World
Health Organization(WHO) Scoring Index, the FIGO system
and others. WHO currently divides GTD variants into hydatiform mole, choriocarcinoma, placental site thophoblastic
tumour, miscellaneous trophoblastic tumour (exaggerated
placental site, placental site nodule, or plaque), and unclassified trophoblastic lesions. Gestational trophoblastic neoplasia includes invasive mole, choriocarcinoma, and placental
site trophoblastic tumor. Low risk patients according to the
FIGO system will follow the same terapies regardless of the
histologic features. Likewise, treatment for a trophoblastic
pulmunary nodule would be the same regardless the initial
histological feature of the uterine contents. An accurate nosologic definition of GTD is important to understanding its
epidemiology, but histologic classification schemes may have
little clinical usefulness.
Case detection: published reports are subject to errors in ascertaining cases of GTD disease. Over-reporting of pregnancies involving GTD relative to other pregnancies can occur in
hospital-based studies, especially in less developed countries
because patients with problem pregnancies or cancer are more
likely to receive hospital care than are those with uncomplicated deliveries, which may occur routinely at home. Underreporting of cases may also be common: hydatiform moles
may be spontaneously expelled in many women who never
receive medical attention and choriocarcinoma may not be diagnosed in women who died without medical care or without
pathological diagnosis.
Identification of the population at risk: reported rates of GTD
are difficult to interpret because different denominators are
used in published studies since only pregnant women are at
risk of GTD, traditional census statistics, which do not take
fertility levels into account, are inappropriate for use in calculating incidence rates. The preferred denominator for women
at risk of GTD disease is all women who have conceived.
This number is not known for populations and several approximations have been used as denominators. The number
of pregnancies usually includes live births, stillbirths, and
abortions (and ectopic pregnancies) and this represents the
closest approximation to the population at risk. The number of
deliveries is not so good as a denominator because it excludes
spontaneous abortions and induced abortions. The number of
live births is the poorest approximation of the population at
risk because it excludes still more conceptions at risk, but it
can be the only or the most complete information available.
Since different denominators have been used in calculating
the incidence rates, comparison of rates may be misleading.
To the extent that denominator underestimate the size of the
population at risk, estimates of the incidence of GTD will
be too high. Use of births or live births in hospitals as the
denominator, especially in regions where childbirth at home
is customary, may account in part for high rates of gestational
trophoblastic disease reported from less-developed countries.
Populations-based studies relying on centralized pathology
institute or comprehensive hospital surveillance should provide the most accurate estimates of the incidence of GTD.
Several epidemiological studies using cases recorded by regional and national registries have been published. They are
clearly superior to hospital-based studies, but in some cases
only malignant variants were recorded and other cases without
histologic confirmation were excluded. Few have identified
all GTD cases by histological sub-type, and all potential risk
factors including dietary, environmental gravidity, parity, age
at diagnosis and ethnicity have not always been recorded.
Nevertheless, because population-based registries permit the
calculation of incidence rates using women residents, live
births and pregnancies within a well-defined geographic region, comparison can be made across registries through the
world. The reported incidence of gestational trophoblastic
disease varies dramatically among different regions of the
world. The published incidences, wich express the rates per
1000 live births, range from 0.7 in Australia to 4.6 in Hawaii.
Some of the highest rates were reported from hospital-based
studies in Indonesia, the Philippines and Mexico. There is
little epidemiological data from the Indian sub-continent, with
the exception of a reported rate of choriocarcinoma of 19.2 per
1000 pregnancies. In North America and in Europe the rates
of hydatiform mole and GTN are approximately 0.5-1/1000
pregnancies and 0.2-0.7/1000 pregnancies, respectively. Italian studies reported rates of 0,8 hydatiform mole/1000 deliveries and 1GTD/1500 pregnancies.
The aim of this study is to recognize the difficulties in collecting data from different type of Hospital in different part
of Italy and to compare the rates obtained with reported data,
using different denominators as pregnancies, deliveries and
live births.
Methods. Pathologists (mostly belonging to APEFA-Gruppo
Italiano di Anatomia Patologica dell’Embrione, del Feto e dei
loro Annessi) from hospitals located in many regions of Italy
were asked to retrieve from their hospital records the number
of histopathological diagnosis of Complete Hydatiform Mole,
Partial Hydatiform Mole, Gestational Choriocarcinoma and
Placental site Trophoblastic Tumour, excluding referral cases.
They were asked also to provide the number of total pregnancies (live births, still births, spontaneous abortions, induced
abortions) registered in their hospital. Public official data
from Regions or National statistic registries were also used
when available.
Pathologists and Hospitals. Giovanni Botta, Ospedale
Sant’Anna, Torino; Francesca Garbini Ospedale Careggi,
Firenze; Giovanni Angeli Ospedale S. Andrea, Vercelli;
Mario Abrate Ospedale di Savigliano (CN); Gaetano Pietro
Bulfamante Università di Milano, Polo S. Paolo Milano; Valeria Lucchini Ospedale San Gerardo Monza; Gertrud Fichtel
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Lectures
Azienda sanitaria dell’Alto Adige, Bolzano; Yuri Musizzano
AOU S. Martino, Università di Genova; Luigi Caliendo ASL
2 savonese Ospedale S. Paolo, Savona; Cristina Vignale
Ospedale Città di Imperia, Imperia; Massimo Palladino EO
Spedali Galliera Genova; Francesca Saro ASL 4 Chiavarese
Ospedale di Sestri Levante (GE); Eugenio Merlo Ospedale Civile Padre Antero Miconi, Genova; Filippo Licausi Ospedale
S. Corona Pietra Ligure (SV); Gianfranco Carfagna Ospedali
Civili di Sanremo; Marina Gualco Ist. Nazionale Ricerca sul
Cancro-IST, Genova; Maria Paola Bonasoni IRCCS Istituo
Gianna Gaslini, Genova; Tommaso Ragusa A.O. Villa Scassi,
Genova; Paolo Dessanti Ospedale S. Andrea, La Spezia;
Daniela Danieli Ospedale ASL n. 6, Vicenza; Tiziana Salviato Ospedale S. Maria Degli Angeli, Pordenone; Adriano
Zangrandi Ospedale Civile, Piacenza; Giovanna Giordano,
Università-Azienda Ospedaliera, Parma; Maria Carolina Gelli
Arcispedale S. Maria Nuova, Reggio Emilia; Francesco
Rivasi Università di Modena; Angela Salerno Ospedale Maggiore AUSL Bologna; Luigi Serra Ospedale di Forlì; Evandro
Nigrisoli Ospedale Bufalini, Cesena; Silvia Zago Ospedale
Civile S. M. delle Croci, Ravenna; Monica Ricci Ospedale Infermi Rimini; Vincenzo Nardini Azienda Ospedale-Università
di Pisa; Fiovo Marziani Ospedale di Terni; Evelina Silvestri
Ospedale Camillo Forlanini, Roma; Gianfranco Zannoni Università Cattolica, Roma; Maria Teresa Ramieri Ospedale di
Frosinone; Ugo Buonocore AORN Cardarelli, Napoli; Maria
D’Armiento Università di Napoli Federico II; Leonardo Resta
Anatomia Patologica Policlinico Universitario, Bari; Gabriella
Ottoveggio Presidio Ospedaliero G. F. Ingrassia, Palermo
Results. The data retrieval has resulted in various difficulties. The most frequent causes of incompleteness or lack of
data on the numbers at numerator were: partial computerization; the computer system has changed over time and the old
archives are not readily available; encoding specific disease
has changed over time and may be ambiguous or incorrect;
diagnoses (especially partial mole) not confirmed clinically or
by other means can not be counted or could have been coded
differently.
The most frequent causes of incompleteness or lack of denominator data were: data (most often those of recent years) have
not yet been developed or are not available; the aggregated
data are available but in a different way from hospital to hospital (live births and stillbirths combined) and not comparable;
the data from different hospitals referring to the same institute
of pathology are not comparable.
In preliminary data the rates of complete hydatiform mole
and choriocarcinoma are approximately 0.6/1000 pregnancies
(0.9 /1000 deliveries) and 0.02/1000 pregnancies (0.04/1000
deliveries) respectively.
References
Fasoli M., Ratti E, Franceschi S, et al. Management of gestational trophoblastic disease: results of a Cooperative study. Obstet Gynecol
1982;60:205.
Golfier F, Raudrant D, Frappart L, et al. First epidemiological data from
the French Trophoblastic Disease Reference Center. Am J Obstet
Gynecol 2007;196:172e1-e5,.
Grimes DA. Epidemiology of gestational trophoblastic diseases. Am J
Obstet Gynecol 1984;150:309-18.
Ngan S, Seckl MJ. Gestational trophoblastic neoplasia management: an
update. Curr Opin Oncol 2007;19:486-91.
Smith HO. Gestational trophoblastic disease epidemiology and trends.
Clin Obstet Gynecol 2003;46:541.
Steigrad SJ. Epidemiology of gestational trophoblastic diseases. Best
Pract Res Clin Obstet Gynaecol 2003;17(6):837-47.
Thama BWL, Everardb JE, Tidyc JA, et al. Gestational trophoblastic
disease in the Asian population of Northern England and North Wales.
BJOG 2003;110(6):555-9.
Diagnostics of Early Spontaneous Abortion
E. Fulcheri, Y. Musizzano
Anatomic Pathology, DISC, University of Genoa
Classically, early spontaneous abortion (ESA) can be defined
as occasional, repeated, or recurrent; we discussed the diagnostic problems of these three groups several years ago, in a
leading article considering every aspect of embryo, fetal and
neonatal pathology 1. The role of the pathologist has acquired
more and more importance in the diagnostics of miscarriages
and in the interpretation of the related infertility. Actually,
a different socio-anthropological attitude has changed the
features of female population over the last decades and is still
inducing significant mutations in the composition of rural
and urban ethnic groups. The most striking aspects of this
phenomenon can be resumed in: 1) an increasing number of
pregnancies in advanced maternal age; 2) the wish of only
one pregnancy, well planned and programmed with a definite timing; 3) the characteristics of immigrant populations,
whose needs about maternity and fecundity are very different
from those of the past and present aboriginal population. It is
evident that, nowadays, a generic diagnosis formulated on the
abortion specimen cannot be considered satisfactory. At the
same time, it is clear that accurate and adherent diagnoses can
be made only in optimal conditions, and when clinical data
and cytogenetics are available. Anyway, given the abovementioned conditions and the fact that occasional abortion
does not permit accurate collection of clinical informations,
the problem should be considered from different viewpoints;
in brief, four different types of specimen exist, representing
the following situations. 1) First occasional ESA in a woman
with negative clinical history; only the date of last menses is
known, and a few more data can be referred. 2) First ESA in
a patient treated for infertility or other disease; in this case,
clinical data should be very exhaustive, with particular regard
to the patient’s history. 3) Repeated or recurrent ESA; even in
this case thorough clinical data should be provided. 4) Review
of chorio-decidual specimens from previous ESAs in a recurrent aborter.
These specimens can be available in the same laboratory or
elsewhere; unfortunately, sometimes they are not available
owing to the lack of previous histological examination. This
item will be discussed later, in the forthcoming debate.
While facing any of these situations, we need to define the
level of diagnostic accuracy needed and the appropriate type
of histological report. Undoubtedly, the perspective of an
indisputable diagnosis is unrealistic and this occurrence is feasible in a very few situations. Hence, in some cases we shall
formulate our diagnosis in terms of highest likelihood; due to
the above-discussed problems, even this approach is possible
only in selected cases. Thus, in the majority of cases we’ll
make orientating diagnoses that, particularly in the setting of
infertility and repeated ESAs, take on great importance. Nevertheless, some situations remain in which it is only possible
to rule out a given condition; even this approach, can play
an important role in the evaluation of repeated or recurrent
ESAs.
In order to follow this scale based on diagnostic complexity,
the ability of the pathologist to explain chorionic and decidual
findings is essential. First, some fundamental differences in
the cause-and-effect mechanism underlying the generic definition of detachment of gestational sac should be defined:
actually, in some cases the detachment following any cause
of abortion can be itself the cause of the pregnancy loss (e.g.
abruptio placentae), while in other instances it represents the
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
common evolution of other noxae From this point of view,
a diagnosis such as “abortion owing to detachment of gestational sac” appears inconclusive.
Before outlining the diagnostic procedure, it seems necessary
to focus on the sampling methods. As a rule, the whole specimen should be submitted to histology, requiring 4 to 6 biocassettes. Preliminarily, the fragments should be examined on a
large Petri dish. The latter allows to recognize the presence
of a gestational sac and its macroscopic features (complete
and sealed, complete and open, presence of parts of embryo
and adnexa, according to Fujikura classification) The embryo
examination should be discussed as a distinct issue. Microautopsy of the embryo, as we defined it in a previous meeting
in Pisa 2, requires the use of a dissecting steromicroscope, and
inclusion in epoxy resins is unavoidable; in our institution,
this method is routinely used since 1994.
Diagnostic flow chart. When approaching an abortion specimen (Fig. 1), adequacy should be first considered; the latter
relates not only to the amount, but also to the representativeness of the received fragments. A specimen should be
regarded as adequate when it includes parts of basal decidua
(marked by the presence of Nitabuch’s stria), parietal decidua
and chorionic villi. Other structures can be seldom identifed,
such as the cord, amniotic sac, or yolk sac 3.
The identification of the basal decidua represents the first step
in the evaluation of the specimen, since it allows to recognize
superficial maternal vessels and to evaluate the modifications
induced by extravillous (intermediate) trophoblast. Conversely, it is virtually impossible to investigate deep decidual
vessels, which are usually not included in the specimen. Investigation of the parietal decidua, apart from pregnancy
Fig.1. Checklist for the microscopic examination of abortion
specimens according to UNI EN ISO 9001-2000 norm, no. 9122.
AO10 (modified from: Musizzano et al. 6).
stromal modifications and Arias-Stella reaction, permits the
evaluation of decidual vessels that were not modified by extravillous trophoblast and so very similar to deep vessels in
the implantation site; the features of intima and vascular wall
should be described.
The most important feature of the chorionic plate is the
branching of chorionic villi. Trophoblast layer is bilaminar
all over the first trimester, featuring a cap of cytotrophoblasts
only in terminal villi. These normal features should be accurately researched and documented in the histopathological
report. All modifications, in terms of uneven branching, abnormal trophoblast proliferation and degeneration, as well as
every modification of the villous outline (invagination, inclusion, angular or slender shape) are unmistakable findings that
suggest karyotype abnormalities.
Other important features of the villi are represented by the
type and distribution of capillaries and by the presence of
red blood cells and erythroblasts in the lumen, according to
well established proportions. Similarly, every abnormality
in vascular distribution, as well as the presence of incomplete vascularization in the villi, shall suggest karyotypic
abnormality.
Finally, stromal degeneration and fibrinoid deposition, though
common findings in many conditions and hence not peculiar
to a given etiology, are useful to correlate and sharpen the
observations in the setting of the above discussed protocol.
While approaching ESA pathology, it is useful the knowledge
of the features, histopathological modifications and diagnostic
findings peculiar to some large categories.
The first condition is represented by abnormal karyotype, that
is responsible for a huge amount of cases, at present estimated
around 70%.
Morphological and structural findings in the villi peculiar to
karyotype abnormalities: abnormal branching; avascular villi;
Irregular outline of the villi; trophoblast invagination; trophoblast inclusion; stromal hydrops (edema).
Morphological and structural findings in the villi suggestive
of karyotype abnormalities: irregular branching; uneven,
sometimes lacking blood vessels; Uneven, discontinuous
trophoblast double layer (cyto- and syncytiotrophoblast);
vacuolated syncytiotrophoblast; hystiocyte-like stromal cells;
fibrinoid degeneration of villous stroma.
Secondly, acute or chronic infections should be considered,
the term chronic indicating long standing, eventually remittent or steady and latent, conditions. Nowadays, the majority
of infections and subsequent inflammatory states can be reliably identified. Unfortunately, the isolation of microorganisms (viruses, bacteria) is today more difficult despite the
large amount of antibodies available, hence in many cases the
villitis or chorioamniositis remains “aspecific”.
Morphological and structural findings in the villi peculiar of
acute infections: stromal edema of the villi; dilated villous
vessels; granulocytic infiltrate (villitis/intervillitis)
Morphological and structural findings in the villi suggestive
of chronic infections: mild stromal fibrosis of the villi; collapsed vessels; mineralization of trophoblast basement membrane; syncytiotrophoblast hyperplasia (sprouts).
Another diagnostic category is represented by maternal vasculopathies and related conditions. The latter include, foremost,
maternal autoimmunity (even subclinical), hypertension, and
decidual vasculopathy as defined by the AFIP Atlas of Placental Pathology 4. Frequently, these pictures partially overlap
thus further complicating diagnostics. In the last years, our efforts were aimed at the definition of an examination protocol
mainly based on vascular decidual findings both in routine
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Lectures
H&E slides and after some cost effective and easy-to-do histochemical and immunohistochemical stainings.
Although in many cases only orientating diagnoses are feasible, some histopathological findings, eventually confirmed
by histochemistry and immunohistochemistry, tend to recur
and sometimes to cluster, so that at least three situations can
be defined 5: unconverted decidual vessels strongly suggest
maternal luteal defect; diminished or disrupted smooth muscle
cells, intimal thickening, and inflammation are more probably
related to autoimmune maternal diseases; finally, fibrinoid
necrosis (staining blue with Weigert-fibrin) and/or hypertrophy of vascular walls (confirmed by the increase of smooth
muscle actin-positive cells) are the usual findings in classical
decidual vasculopathy according to the AFIP.
References
1
Fulcheri En et al. Pathologica. 2006;98(1):1-36.
2
Fulcheri E. Pathologica 1997;89(6):624.
3
Fulcheri E, Mariuzzi GM. Patologia della gravidanza. In: Mariuzzi
GM (ed). Anatomia Patologica e correlazioni anatomo-cliniche. Padova: Piccin Nuova Libraria 2007, pp. 1946-8.
4
Kraus FT, et al. AFIP Atlas of nontumor pathology. N. 3. Placental
pathology 2004.
5
Musizzano Y, Fulcheri E. Virchows Arch 2010;456:543-60.
6 Musizzano Y, Fulcheri E. Decidual vascular patterns in first-trimester
abortions. Virchows Arch 2010;456:543-60.
SINOMED-NAP
Moderators: F. Crivelli (Gallarate), C. Francescutti (Udine)
A review on pathology report coding practices
V. Della Mea
Medical Informatics, Telemedicine & Health Lab; Dept. of Mathematics and Computer Science, University of Udine Italy
Background. Text included in clinical documents, including anatomic pathology reports is often complemented by a
concise version of the content, obtained by coding them using
terms (and corresponding alphanumeric codes) coming from
one or more terminologies or classifications.
Coding may be aimed at different applications, where the
most traditional are administration and finance aspects (e.g.
healthcare intervention reimbursements) and epidemiology (e.g., disease statistics provided by national statistics
institutes and WHO). The practical advantage provided by
coding to the coder (e.g., the reporting pathologist), when
applied into some computerized information system, is the
possibility to retrospectively retrieve reports according to
coded content. Coding also provides the opportunity of
comparing and collecting anatomic pathology data independently from document language, and also automated
decision support.
In the anatomic pathology report, coding may involve various
aspects, not always and not all present and coded, including:
– site of sampling, for which a terminology describing human
anatomy is needed;
– macroscopic and microscopic description of the sample, for
which a terminology is needed to describe morphological
aspects;
– the diagnosis, or which a terminology of diseases is needed
that provides the adequate amount of detail to describe anatomic pathology diagnoses;
– and finally, procedures applied to the sample (e.g., stainings), for which a terminology collecting all possible procedures is needed.
SNOMED is historically a very large terminology that provides all above mentioned components, plus others aimed at
describing other content of a general clinical record including
other reports.
In the world, SNOMED is likely the most used terminology
for anatomic pathology report coding, but is not the only one
available, nor is used in just one, i.e., the last available, version. In fact, in some countries national terminologies have
been developed, like ADICAP in France and READ codes in
UK, while in other countries WHO’s diagnostic classification
have been used (ICD9-CM, ICD10, ICD-O).
SNOMED was initially developed by the College of American Pathologists, but since few years is maintained by the
International Health Terminology Standards Development
Organisation (IHTSDO), based in Denmark and currently involving 15 countries 1. While for long time it was used only in
Pathology, application is broadening towards other areas 2.
The present paper reviews the current practices of pathology
report countries in various countries.
Methods. A questionnaire was developed to survey pathology
report coding practices in the different countries, starting from
those participating into the COST action IC0604 “Telepathology Network in Europe: EURO-TELEPATH” 3 4. Survey was
implemented using GoogleApps in order to provide an online
fillable version, but was also collected by interviewing participants to the European Congress of Telepathology and Virtual
Microscopy, held in Vilnius in 2010.
Questions in the survey were means at collecting information
on the presence and application of a national policy for pathology report coding, on who established the policy, on which
codings are used for the various sections of the report, on
IHSTDO involvement and on SNOMED translation plans.
Results. Representatives of 12 countries answered the questionnaire, of which 10 from EU countries. All countries present at least some coding practice, but is applied by the totality
of pathology institutes in 25% of countries. In two cases, this
is made without a national policy, although when present (8
countries) it is provided by professional associations in all
but one case. This also means that usually coding decisions
are left at the Pathology Institute level, so that heterogeneous
behaviour can be found in a country for both the coding and
the terminology used.
SNOMED-CT is formally adopted in two countries, although
this does not mean that is also practically used by a majority
of their Pathology institutes. Older versions of SNOMED,
often nationally or even locally adapted to purpose, are most
often used, together with national terminologies. When coding is done, site of disease and diagnosis are always coded.
Procedures are much less subject to coding, and description
almost never, although sometimes morphological aspects are
enclosed in the diagnostic coding.
Conclusions. Pathology report coding seems a common practice in most countries, although the terminologies used for
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
that are actually heterogeneous. This makes sharing of data
more difficult, although trans-coding (i.e., converting one
coding into another) is already used for other terminologies
and classifications.
In a previous assessment of SNOMED implementations, Giannangelo and Fenton 5 found out that a considerable amount
of software vendors need a business case for why SNOMED
CT should be deployed in their systems. In addition to that,
some vendors indicated that if institutions were to require
it, then they would proceed with including SNOMED CT in
their products. Unfortunately, report coding policies are not
always available or mandatory in countries involved in the
present survey.
Translation in national languages has been started and done by
few countries 6, but it can be a problem due to the large size
of the whole SNOMED-CT terminology. Pathologists use just
a part of the whole vocabulary, which could be more easily
translated than the whole SNOMED-CT.
Although respondents were among people involved in telepathology and digital pathology, awareness about policies and
plans about coding, IHSTDO and SNOMED was not much
diffused, perhaps because perceived as a secondary aspect of
digitalization.
Uniform implementation of a single coding systems seems
still more easily rechaed where a common information system
is adopted throughout the country, like in Netherlands.
However, pathologists produce a considerable amount of
information that would be better exploited if made available
also in a coded, sharable form.
Acknowledgements. The present review has been carried
out inside the activities of the COST Action IC0604 “Telepathology Network in Europe: EURO-TELEPATH”, Working
Group 2 “Informatics Standards in Pathology”.
References
1
International Health Terminology Standards Development Organisation (IHTSDO): http://www.ihtsdo.org/
2
Cornet R, de Keizer N. Forty years of SNOMED: a literature review.
BMC Med Inform Decis Mak. 2008;8(Suppl 1):S2.
3
COST Action IC0604 “Telepathology Network in Europe: EUROTELEPATH”: http://www.conganat.org/eurotelepath/
4
Garcia Rojo M, Punys V, Slodkowska J, et al. Digital pathology in
Europe: coordinating patient care and research efforts. Stud Health
Technol Inform 2009;150:997-1001.
5
Giannangelo K, Fenton SH. SNOMED CT survey: an assessment of
implementation in EMR/EHR applications. Perspect Health Inf Manag
2008;5:7.
6
Klein GO, Chen R. Translation of SNOMED CT - strategies
and description of a pilot project. Stud Health Technol Inform
2009;146:673-7.
Personal data protection in italian pathology
services
G. Negrini
Ospedali Area Ovest AUSL Bologna, Italy
Background. In Italy, since 2003 there is a regulatory body of
personal data, so called Privacy Code (Dlgs 196/2003) *.
According to this legal framework, special rules were established in order to protect all sensitive data, especially those
relating to health.
Moreover, within these ones, genetic data have a far greater
protection.
Pathologist’s activities usually come across a lot of privacy
matters, for instance:
– informed consent to personal data treatment, related to patient care;
– use of personal data for clinical studies, epidemiology, research;
– biological samples collections;
– electronic documentation.
Discussion. Firstly, we have to answer the question if the
patient consent must be related only to clinical needs or even
further occurrences.
Nowadays most physicians deem data as essential for purposes other than each patient’s treatment.
Nevertheless, it is not enough to give generic information to
patients about foreseeable purposes of study or research, consequently we have to clear for which study or research data
could be used.
All that may seem too restrictive.
When we ask for the patient consent we couldn’t know some
needs that arise only later.
Our legislation ** relieves us from the patient consent only if
a research or study was provided by the National Research
Program, 45 days after the notice to Privacy Authority.
Otherwise, when seeking consent is too expensive, because of
a great deal of patients or actual difficulties, we have to acquire a favorable opinion of a local ethic committee, followed
by the approval of the Privacy Authority.
About genetic data, some Authors speculated about what they
are really and confuted genetic exceptionalism 1, but our legal
system requires a separate, written consent ***.
About that one, a previous information should explain: detailed list of all specific purposes to be achieved, possible
findings, the right to object to data processing, whether the
data subject is allowed to limit the scope of their communication and the transfer of biological samples, retention period of
genetic data and biological samples.
When the consent to search is withdrawn, the related biological samples must be destroyed, if they are still identifiable.
Many expedients should be arranged to prevent the risks of
undue accesses, for instance: every room where genetic data
are stored needs special controls.
People, who enter it, after the closing time, must be identified
and recorded.
Storage, use and transport of biological samples must be
carried out to ensure their quality, integrity, availability and
traceability.
Genetic data should be transmitted electronically by certified
electronic mail after encrypting and digital signature.
If genetic data or biological samples are acquired for clinical
purposes, a different use is allowed only for a purpose related
to the former, unless a new consent or the anonymization of
data or samples.
If seeking new consent is too expensive, they can get positive
evaluation of local ethic committee, followed by approval of
Privacy Authority.
However, we consider that, except for rare hereditary cancers,
genetic characteristics of tumor tissues can not be qualified as
genetic data, because they are limited to some body parts and
don’t affect germ cells.
With regard to biological samples, we should ask ourselves:
‘Who owns? Who can decide what to do with them? Is there
a real property right of biological materials 2-5?
The answers determine the resolution of several issues: their
use for additional purposes, responsibility of their retention,
storage time, right of access (for example: could patients get
their samples back?).
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Lectures
The Convention on Human Rights and Biomedicine ****
– Oviedo, 1997 – article 22 provides:
“Disposal of a removed part of the human body.
When in the course of an intervention any part of a human
body is removed, it may be stored and used for a purpose
other than that for which it was removed, only if this is done
in conformity with appropriate information and consent procedures.”
Well, what happens if the patient dies?
May the relatives – the heirs – succeed to the rights of the
deceased?
All these issues have been discussed for a long time in many
countries, but we are still waiting for a definite answer.
Recently, some Authors have suggested an interesting innovation with regard the patients’ consent and their rights 6.
They argued the need of rethinking the relationship between
patients - or donors not patients- and researchers and proposed
to replace the current informed consent with trusted consent,
at least with reference to research biobanks.
New themes and questions are now related to the development
of electronic health records.
In our country, regional governments are building health information systems, designed to create files with the medical
history of each person, potentially from birth to death 7 8.
Our Privacy Authority has recently launched guidelines on
reports on line as well as on electronic record and electronic
file *****.
Patients have the right to give or withhold their consent to the
implementation of such a file.
It should be possible excluding some items of medical information, so that patients can decide, after being duly informed,
whether or not they want to disclose certain events (blanking).
However, blanking can become a threat to clinical decisions
when these are based on partial information.
Despite of the easy electronic consultation, we must ensure
only professionals who provide care to the patients are entitled
to consult their records.
Another issue is the patients right of access to their data: may
the documents containing severe diagnosis be directly known
by the patient, without previous discussion with a doctor, as
it is now possible (e.g.: on line reports, access to electronic
health record)?
To avoid such an impact, some health organizations block
critical reports until an interview with a doctor.
What’s above is a limited summary: how can we extricate
ourselves from all these entanglements?
Pending regulatory precise answers, we should make an effort
to go beyond the rule of thumb, to seek balanced solutions,
in compliance with existing legal rules and prevalent ethical
principles.
References
1
Rothstein MA. Genetic Exceptionalism and Legislative Pragmatism.
Hastings Center Report 2005;35:27-33.
2
Negrini G. Materiali biologici donati: incertezze di inquadramento
giuridico. Rischio Sanità 2009;34:16-21.
3
Negrini G. Raccolte di materiali biologici: interrogativi. De Qualitate
2008;2:8-25.
4
Furness PN, Nicholson ML. Obtaining explicit consent for the use of
archival tissue samples: practical issues. J Med Ethics 2004;30:5614.
5
Negrini G, La Pietra L. Campioni biologici conservati nelle strutture sanitarie: interrogativi e problemi aperti. Professione. Cultura e
pratica del medico d’oggi 2005;1:34-41.
6
Boniolo G, Di Fiore P, Pece S. Trusted Consent and Research Biobanks. Towards a new alliance between researchers and donors.
7
8
Bioethics 2010 JUN doi:10.1111/j.1467-8519.2010.01823.x
Negrini G, la Pietra L. Opportunità e criticità del fascicolo sanitario
elettronico. Professione & Clinical Governance 2009;7:30-7.
Moruzzi M. Health e Fascicolo Sanitario Elettronico. Il Sole 24 Ore.
Milano 2009
Notes
*
www.garanteprivacy.it
**
Art. 110 Dlgs 196/2003
***
General Authorisation for the processing of genetic data, 27/2/2007
http://www.garanteprivacy.it/garante/doc.jsp?ID=1389918
****
http://www.coe.int/t/dg3/healthbioethic/Activities/01_
Oviedo%20Convention/
*****
http://www.garanteprivacy.it/garante/doc.jsp?ID=1681147
http://www.garanteprivacy.it/garante/doc.jsp?ID=1634116
NAP Italia – The new nomenclature for the
anatomic pathology
P. Crucitti, A. Bondi
U.O. Anatomia Patologica, Maggiore Hospital - AUSL Bologna,
Italy
Background. Classification is a recognised method to organize in a systematic way all scientific informations. In medical
field, the broad diagnosis terminology has produced the internationally utilized SNOMED code, with the subset “Microglossary for Pathology”, translated in Italian. SNOMED has
an international copyright and is registered by the College of
American Pathologists (CAP): use of the code is under payment of the rights for workstation.
Adverse events during SNOMED distribution in Italy caused
a consequent autonomous development of “self-made” codes,
often not in line with international version and that not allow
exchanges of data between different Anatomic Pathology.
All this situation became an obstacle for data extraction from
different Institutions and, as a consequence, for Tumour Registries end official epidemiological archives, raising the need
of one national language. From these assumptions, role of
SIAPEC has been to join different codes from many Anatomic
Pathology in Italy and re-direct Italian Pathology to the international scenario, favouring and coordinating cultural and
scientific collaboration between varies Associations involved
in medical informatics, diagnostic coding, epidemiology and
tumour registry. Actors of this project are Scientific Corporations of the field (SIAPEC-IAP and AIRTum), the Italian
Contributor Centre of OMS for Sanitary Codes (CC-OMS)
and few Regional Sanitary Agencies (Friuli-Venezia Giulia,
Liguria and the cooperation of Emilia-Romagna and Lombardia). Aim of the Nomenclature for Anatomic Pathology
(NAP) is to become the national reference, for improvements
and updating, shared from the Pathologist community.
Methods. NAP development is on-going, but we can briefly
schematize the general proceeding:
1.Identification of a work group, constituted by Pathologies
from different Italian Anatomic Pathology, an operator editing different tables (see below), sometimes in collaboration
with other centre; a group coordinator.
2.Census of main code versions utilised between Italian
pathologists. Requirement to software houses (SH) of the
area, to get different nomenclature distributed. Eventually
other sources can be Institutions, that have enriched autonomously the Nomenclature.
3.Definition of coding rules and preparation of NAP, mainly
for section related to non-tumours definitions: extra-tumour
morphologies, topography, procedures, other sections.
4.ICD-O 3 acquisition to create the core of NAP.
216
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
5.Realization of other axes, starting from Microglossary from
Pathology of 1995, lately integrated with varies versions
collected from produced from the work group promoted
from the executive in 2005.
6.Definition of a first NAP edition and official SIAPEC approval.
7.Creation of transcoding tables for new terms (point 2) and
distribution from SH, in application to each administration
management.
8.Realization of a transmission protocol among archives,
eventually according to HL7.
Through the construction of intermediate “tables of comparison” we found “alignment rules”, containing all indications
for records to be modified, deleted or added to uniform any
table according to these rules.
Results. At present NAP sections already completed are:
a table containing morphology of tumours (M-8 and M-9),
based on ICD-O 3 perfectly in line with SNOMED CT 2002,
a table containing topography codes and - to be released - a
table containing all morphology (extra-tumour). All tables are
organised according to a record track, including alphanumeric
code, Italian and English description, an index code to trace
source of each record, a link to SNOMED / ICD-O topography and finally a hierarchy code to structure each table (see
below). The NAP code for tumours is completely compatible
with SNOMED International and it’s composed by: Axis definition (1 capital letter) / dash / tumour definition (4 Digits) /
behaviour (1 digit) / Differentiation (1 digit) / 1 Extra digit /
Synonyms (1 letter).
Id link allows to trace each code/definition for correction,
modification.
The reference table is also modified and enriched with new
terms deriving from different Anatomic Pathology: new
tables and codes will be available through the Italian Portal of
Classifications, tool of the Italian Collaborating Centre, for a
continuous upgrade of NAP.
NAP code is also related to the creation of a digital Atlas
for rare cases promoted from Emilia-Romagna region and
presented in current SIAPEC congress (see presentation from
S. LEGA et al)
Record track:
Field name
cod
Length
10
Data type
Text
txt
txt_en
id
120
120
8
Text
Text
Number
icd
15
Text
ref
80
Text
Super
10
Text
Function
NAP code - ICD-O
/SNOMED
Italian description
English description
Unique identification
of the term
Reference to ICD-O
topography
Link to SNOMED
topography
Hierarchy code
Germ cells in tumoural pathology
Moderator: C.A. Beltrami (Udine)
Stem cells and genetic skeletal diseases – Role
in pathogenesis and therapy
M. Riminucci
Department of Experimental Medicine, University La Sapienza,
Rome, Italy
Fibrous dysplasia (FD) (polyostotic fibrous dysplasia, McCune/Albright syndrome, OMIM#174800), is a non inherited
skeletal dysplasia caused by mutations of GNAS, the gene
encoding the alpha subunit of the stimulatory G protein (Gs).
Mono and polyostotic forms of the disease are recognized,
the latter frequently associated with extraskeletal disorders
in complex clinical settings such as the McCune-Albright
syndrome (MAS, polyostotic FD, café-au-lait spots and endocrine lesions) and the Mazabraud’s syndrome (MS, FD and
muscular myxomas).
FD lesions develop during the post-natal growth and replace
normal skeletal tissues (bone, adipose and hematopoietic marrow) with woven bone and fibrotic marrow. Affected skeletal
segments often become mechanically insufficient and in most
patients, severe invalidity ensues from recurrent fractures and
deformities.
Long thought of as an undefined fibro-osseous disease, FD has
become over the last few years a unique model of human disease affecting both embryonic and post-natal stem cells. The
somatic nature of the mutation and the distribution of mutated
cells in derivatives of all germ layers, point to a pluripotent
embryonic cell as the initial (although silent) target in which
the molecular lesion occurs. The profound derangement of the
bone/bone marrow microenvironment observed at affected
sites and reproduced upon ectopic transplantation of FD osteprogenitors, indicate post-natal skeletal stem cells (originating from the mutated embryonic clone) as the late effectors in
which the mutational event displays its adverse effects.
Reinterpretation of FD as a stem cell disease has opened new
avenues to investigation of its pathogenetic mechanisms, generation of suitable experimental models and development of
specific therapeutic approaches.
Many complex changes occurring at affected skeletal sites,
such as the inappropriate bone resorption and bone matrix
hypomineralization, which cannot be explained based on the
activity of mutated osteoblasts solely, have been reinterpreted
in light of the abnormal expansion and function of mutated
osteoprogenitors.
Lentivector-mediated trasduction of normal ES and post-natal
skeletal stem cells with the disease gene has provided appropriate experimental models to investigate the molecular
responses induced by the GNAS mutation at different developmental stages and to seek new potential pharmacological
targets.
Finally, the recognition of FD as a stem cell disease has emphasized the need for innovative, stem cells based therapeutical approaches as the only possibility to cure the disease radically. However, the develoment of new strategies based on
either the replacement or the genetic manipulation of mutated
FD skeletal stem cells requires the availability of suitable in
vivo models. To this aim, we have recently generated the first
transgenic murine models of the diseases.
217
Lectures
Saturday, September 25th, 2010
Slide seminar: Histopathology
Moderators: V. Eusebi (Bologna), G. Pelosi (Milano)
Case n. 1
Aggressive psammomatoid cemento-ossifying
fibroma of the sinonasal region
L. Roncati, A. Maiorana
Department of Laboratory Services, Pathologic Anatomy and Forensic Medicine, Section of Pathologic Anatomy, University of Modena
and Reggio Emilia, Modena, Italy
Background. An 18 years-old woman with a clinical history of chronic sinusitis and headache was evaluated for
nasal airway obstruction associated with recent, painful and
widespread left hemifacial swelling. No history of trauma
was elicited. Computed tomography of the maxillo-facial area
showed an expansive-erosive neoformation, predominantly
isodense to surrounding soft tissues, that occupied the left
maxillary sinus, eroding the left hemipalate and the left jawbone. The left orbital floor was thinned and slightly raised.
The right maxillary sinus, together with both sphenoidal and
frontal sinuses and ethmoid cells were regularly pneumatized.
A biopsy of the left maxillary sinus was performed. Following
the diagnosis, the patient underwent a first surgical intervention under the intraoperative direction of the pathologist.
A left hemimaxillectomy with reconstruction of the orbital
floor, postero-lateral wall of the maxillary sinus (maxillary
alveolar process) and zygomatic process using revascularized
fibula was performed. Nine months later a recurrence in the
left orbital floor required surgical revision of the orbital area.
After a recurrence-free follow-up period of two years, the
patient underwent another surgical intervention for removal
of fixation devices in the orbital floor and remodelling of the
homologous bone graft with alloplastic tissue.
Methods. All specimens were routinely processed for histopathological examination (fixation in 4% formaldehyde,
paraffin embedding, staining of sections with hematoxylineosin). Immunohistochemistry for CD34 (Ventana), EMA
(Ventana) and MIB-1 (Dako) was performed.
Results. The bioptic sample was a small fragment measuring
cm 1 × 0.5 × 0.5. The surgical material consisted of numerous
white, hard tissue fragments that, when put together, measured
approximately cm 3.8 × 3.5 × 3.2. All fragments showed a
fibro-osseous lesion composed of a fibrous stromal component
of monomorphic spindle or polygonal cells that embedded bony
trabeculae of varying shapes, mostly curvilinear, and numerous
round-to-oval calcific elements similar to psammoma bodies
(so-called “psammomatoid bodies” or “ossicles”). Some bony
trabeculae were rimmed by osteoblasts accompanied by isolated osteoclasts. A cystic component with hypocellular fibrous
septa, reminiscent of aneurysmal bone cyst, was also present.
Nuclear atypias and mitotic activity were not detected. Immunohistochemical reactions for CD34 and EMA were negative.
The histological findings led to the diagnosis of “aggressive
psammomatoid cemento-ossifying fibroma” of the sinonasal
region, also known as “extragnathic cemento-ossifying fibroma”.
Discussion. The definition of “aggressive (or juvenile) psammomatoid cemento-ossifying fibroma” identifies a complex
extragnathic fibro-osseous mesenchymal proliferation that
may arise in every site of the sinonasal tract (nasal cavity,
paranasal sinuses, turbinates, nasolacrimal duct). The tumor
is similar to cemento-ossifying fibromas that occur in the
gnathic region and was postulated to arise from mesenchymal
elements of the periodontal ligament 1. In the common definition, the use of descriptive adjectives such as “aggressive”,
“active” or “juvenile” refers to the tumor capability to exhibit,
especially in young patients (first and second decades) of
both sexes, a locally aggressive behaviour, mainly characterized by invasion and destruction of surrounding anatomic
structures (cranial cavity, orbit, palate, nasopharynx), with
tendency to recur after surgical resection, in particular in cases
of simultaneous involvement of multiple sites. Some authors
have suggested the adjective “juvenile” to be dropped, since
the lesion is not limited to the young age and can even affect
people in the fifth or sixth decades of life. Although isolated
cases were able to cause death of the patient, due to local involvement of vital intracranial areas, the aggressive behaviour
does not imply an evolution into metastatic disease. The most
common symptoms are sinusitis, headache, nasal obstruction,
facial swelling, visual disturbances and progressive blindness,
exophthalmos and proptosis. Epileptic seizures, smell disturbances and facial deformities are rarely observed. A case of
juvenile aggressive ossifying fibroma presenting as mucocele
of the ethmoid sinus has also been reported 2. Clinicopathological integration, with detailed analysis of the radiographic
projections, is essential to reach the correct diagnosis. At radiological imaging, the lesion is round-shaped and initially appears hyperdense suggesting the presence of a calcified matrix.
In advanced stages, it can either show a multiloculated internal
appearance with variable density, usually surrounded by a
sclerotic bone rim, or can exhibit lytic growth in the adjacent
bones or soft tissues. Aggressive growth is evidenced by invasion of anatomic compartments and bulging or displacement
of surrounding bone structures. Grossly, the tumor can assume
a compact tight aspect or a multicystic appearance with accumulations of coagulated blood material, resulting in a color
change from greysh to brownish. Histologically, it shows a
cellular stroma stuffed by numerous, spherical, concentric
mineralized elements (psammomatoid ossicles) with variable
foci of stromal myxoid degeneration and occasional multinucleated giant cells, in particular around vascular spaces. The
differential diagnosis 3 4 of sinonasal psammomatoid cementoossifying fibroma includes a group of pseudoneoplastic proliferations (fibrous dysplasia, aneurysmal bone cyst, giant cell
reparative granuloma) and benign or malignant neoplastic conditions such as osteoma, ossifying fibroma, giant cell tumor,
myxoma/fibromyxoma, chondromyxoid fibroma, psammomatous meningioma 5, osteoblastoma and osteosarcoma. The support of radiological findings associated with the detection of a
large number of pathognomonic psammomatoid ossicles in the
histological section does usually allow the correct diagnosis to
218
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
be made. Surgical treatment may be conservative (endoscopic
tumor resection) or highly demolitive (craniofacial resection,
enucleation), with cosmetic deformities and increased risks for
secondary blindness, meningitis and brain abscesses. Adjuvant
therapeutic strategies (radiation therapy) have proved ineffective and can, at times, promote tumor dedifferentiation. The sinonasal psammomatoid cemento-ossifying fibroma is a prime
example of how a radiosurgical multidisciplinary approach,
directed by the intra-operative diagnosis of the pathologist, is
imperative in order to define the extent of the disease and allow its total excision.
References
1
Wenig BM, Pilch BZ. Tumors of the upper respiratory tract. In:
Fletcher CDM (ed.) Diagnostic histopathology of tumors, II ed, vol. 1
Churchill-Livingstone 2007.
2
Vaidya AM, Chow JM, et al. Juvenile aggressive ossifying fibroma
presenting as an ethmoid sinus mucocele. Otolaryngol Head Neck
Surg 1998;119:665-8.
3
Wenig BM, Vinh TN, et al. Aggressive psammomatoid ossifying fibroma of the sinonasal region. A clinicopathologic study of a distinct
group of fibro-osseous lesions. Cancer 1995;76:1155-1165.
4
Slootweg PJ, Panders AK, et al. Psammomatoid ossifying fibroma of
the paranasal sinuses. An extragnathic variant of cemento-ossifying
fibroma. J Cran Max Fac Surg 1993;21:294-7.
5
Granados R, Carrillo R, et al. Psammomatoid ossifying fibromas:
immunohistochemical analysis and differential diagnosis with psammomatous meningiomas of craniofacial bones. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 2006;101:614-9.
Case n. 2
Myxoid liposarcoma of the foot
L. Roncati, A. Maiorana
Integrated Department of Laboratory Services, Pathologic Anatomy
and Forensic Medicine, Section of Pathologic Anatomy, University of
Modena and Reggio Emilia, Modena, Italy
Background. A 52 years-old woman underwent surgical
excision of a nodular, mobile, fatty neoformation of the right
big toe, which had been present for 2 years and had gradually
grown in size. The lesion caused pain on walking. Grossly,
the nodule was soft and well defined, measuring 3.8 × 2.7 ×
2.5 cm. Its cut surface appeared uniformly greyish and translucent.
Methods. Immunohistochemistry was performed on paraffin-embedded sections using a broad panel of antibodies, such
as α-smooth muscle actin (Ventana), desmin (Ventana), pankeratins (MoAb MNF 116 – Dako), S100 protein (Ventana),
CD34 (Ventana). Interphase F.I.S.H. was performed on formalin-fixed paraffin-embedded tissue using the CHOP Dual
Color Break Apart Rearrangement Probe (Abbott).
Results. Histologically, the neoplasia was composed of
spindle cells, immersed in abundant myxoid stroma, with focal marked mucinous accumulation. A vascular crow ‘s feet
pattern of thin-walled branching vessels was present, together
with a focal component of round cells. The tumor reached
the margin of resection. Immunohistochemical reactions for
α-smooth muscle actin, desmin, pankeratins, S100 protein,
CD34 were negative. Interphase F.I.S.H. revealed the presence of the t(12;16) (q13;p11) translocation with FUS-CHOP
fusion gene. The diagnosis of “myxoid liposarcoma with focal
round cell component” (5-10% of tumor cell population) was
rendered. Since tumor removal had been incomplete, re-excision was necessary followed by radiotherapy (10 Gy focused
on the surgical site). The patient is alive and well after a 3 year
follow-up period.
Discussion. Approximately 75% of myxoid liposarcomas
arise in the deep soft tissue of the buttocks and lower limbs, in
particular thigh, groin and popliteal region, giving rise to neurovascular and muscular compressive symptoms. Rare cases
localize in the feet. Isolated reports were described in unusual
locations 1, such as breast, ovary, scrotum, spermatic cord,
vulva and thyroid. The tumour may be seldom accompanied
by a paraneoplastic neurological syndrome (subacute complete
ophtalmoplegia), asssociated with anti-Hu antibody 2. Grossly,
pure myxoid liposarcomas are multinodular gelatinous masses
with a variable yellow tint, whereas predominantly round cell
liposarcomas show a white fleshy appearance. The small size
of the tumor (< 5 cm) may make the clinical diagnosis of
malignancy difficult, since the findings of large size (> 5 cm)
and rapid enlargement are the major clinical indicators for
malignancy. Presurgical superficial (U/S-Scan) and deep
(MRI-Scan) radiological investigation are useful to determine
the size, shape, outline and, in particular, presence of typical
cystic zones. Histologically, the differentiated myxoid component shows a low cellularity (paucicellular myxoid liposarcoma), composed of spindle/round cells scattered in a myxoid
matrix of hyaluronic acid (Alcian blue-positive), sometimes
with a microcystic pattern or lace-like configuration (pooling
phenomenon). The occurrence of a plexiform capillary vascular network and signet ring lipoblasts in different stages of
maturation are important diagnostic clues. Mitotic figures are
tipically rare or absent. As myxoid liposarcoma loses its differentiation, it assumes a round cell appearance, characterized
by the progressive accumulation of primitive round cells with
high nuclear/cytoplasmic ratio and prominent nucleoli, growing in sheets and accompanied by vascular texture attenuation
(cellular myxoid liposarcoma). The amount of round cells correlates with the development of distant metastases and should
be always estimated in a well-sampled specimen (one section
per centimetre tumor diameter). In more than 95% of myxoid/
round cell liposarcomas, a classical t (12;16) (q13;p11) or t
(12;22) (q13;q12) translocation can be found, giving rise, respectively, to chimeric FUS-CHOP or EWSR1-CHOP genes.
The identification of such translocations in lipomatous tumors
is a powerful tool that aides in the correct identification of
myxoid/round cell liposarcomas.
A comparative study 3 of 16 cases of tumors previously diagnosed as primary myxoid/round cell liposarcoma of the retroperitoneum and 18 cases of myxoid/round cell liposarcoma of
the extremities disclosed that FUS-CHOP or EWSR1-CHOP
fusion genes were present only in tumors of the limbs, being absent in retroperitoneal tumors. The findings suggested
that apparent primary myxoid/round cell liposarcomas of the
retroperitoneum are actually well-differentiated (or dedifferentiated) liposarcomas with morphological features mimicking myxoid/round cell liposarcomas. As a consequence, the
detection of FUS-CHOP or EWSR1-CHOP fusion genes in an
apparent myxoid/round cell liposarcoma of the retroperitoneal
area should lead to the suggestion of metastasis and prompt
search for a primary localization outside the retroperitoneum.
Similarly, a further study carried-out in 15 cases of presumed
“multifocal” myxoid/round cell liposarcoma 4 was able to
evidence (using LOH and RT-PCR for determination of the
FUS-CHOP and EWSR1-CHOP breakpoints) the existence
of a clonal relationship in the different tumors arisen in the
same patient, supporting the “metastatic” nature of the apparent “multifocal” myxoid/round cell liposarcoma, with
obvious consequences on therapeutic strategies. Cytogenetics
may also be helpful in the routine differential diagnosis of
myxoid liposarcoma. Potential mimics 5 6 include a wide range
219
Lectures
of benign or malignant subcutaneous (myxolipoma, myxoid
nodular fasciitis, superficial angiomyxoma, aggressive angiomyxoma, myxoid dermatofibrosarcoma protuberans, myxoid
neurofibroma, dermal nerve sheath myxoma) and deep-seated
myxoid soft tissue tumors (low grade myxoid malignant fibrous histiocytoma, myxofibrosarcoma, extraskeletal myxoid
chondrosarcoma, myxoid synovial sarcoma, myxoid leiomyosarcoma, myxoid malignant peripheral nerve sheath tumour).
Intratumoral hemorrhage is a common finding in myxoid
liposarcoma and, if present, can simulate a vascular neoplasia.
The main histological parameters to follow in the differential
diagnosis of myxoid soft tissue tumors are the architectural
pattern, vascular pattern, cellularity and cytological aspects.
Immunohistochemistry and histochemical reactions for mucosubstances may provide additional useful information. When
pure myxoid liposarcoma loses its differentiation and assumes
a round cell appearance, the differential diagnosis moves
towards other round cell neoplasias, such as rhabdomyosarcoma, poorly differentiated synovial sarcoma, Ewing’s sarcoma/
primitive neuroectodermal tumor, lymphoma, melanoma and
carcinoma, making the correct identification of myxoid soft
tissue tumors a continuous diagnostic challenge.
Pure myxoid liposarcoma exhibits high risk of local recurrence and a 20% rate of distant metastases. On the other side,
round cell liposarcoma gives rise to metastases in approximately 70% of cases. Both tumors show an unusual pattern of
spread, since metastases arise mainly in extrapulmonary sites
(2/3 of cases), such as soft tissues (mediastinum, retroperitoneum, thorax, distant extremity), bone (spinal cord, ribs),
liver and serous membranes, lung metastases being observed
in approximately 30% of cases. The average survival rate is
80% at 5 years and 50% at 10 years, being mainly related to
the quality of local excision. The time interval of appearance
of the first metastasis is on average 68 months 7. According to
standard surgical procedures, wide excision with safety histopathological margins of at least 1 cm, should be performed
each time the presence of a near-by neurovascular axis can
allow it. Surgery is usually followed by radiotherapy and,
sometimes, in biologically aggressive tumors, by adjuvant
chemotherapy (Doxorubicine, Ifosfamide, Dacarbazine), following the indications of histopathological examination (size
> 7.5 cm, number of round cells > 25%, p53 overexpression
at immunostaining, R1 or R2 initial resection margins) 8. The
tumor is higly chemo- and radiosensitive, when compared
with other soft tissue sarcomas, and its sensitivity to radiation
treatment appears mainly to be related to the occurrence of
the typical vascular crow’s feet pattern, since ionizing radiations can induce vascular damage with consequent hypoxic
death of tumor cells. The chemo-radiosensitivity of myxoid
liposarcoma and the possibility of using new drugs, such as
trabectedin (ET-743; Yondelis), the first marine-derived anticancer product, able to induce the detachment of FUS-CHOP
protein from the promoters of target genes and reduce neoplastic growth 9, allow a more conservative surgical approach
(limb-sparing surgery) in cases of large tumors affecting the
limbs.
References
1
Weiss SW, Goldblum JR. Liposarcoma. In: Enziger & Weiss’s soft
tissue tumors, V ed. Mosby Elsevier 2008;16:477-516.
2
Chan JW. Subacute complete ophthalmoplegia: an anti-Hu paraneoplastic manifestation of myxoid liposarcoma. Clin Experiment Ophthalmol 2007;35(5):491-2.
3
De Vreeze RS, de Jong D, Tielen IH, et al. Primary retroperitoneal myxoid/round cell liposarcoma is a nonexisting disease: an
immunohistochemical and molecular biological analysis. Mod Pathol
2009;22(2):223-31.
4
5
6
7
8
9
De Vreeze R, de Jong D, Nederlof P, et al. Multifocal myxoid liposarcoma--metastasis or second primary tumor?: a molecular biological
analysis. J Mol Diagn 2010;12(2):238-43.
Ninfo VV, Montesco MC. Myxoid tumors of soft tissues: a challenging
pathological diagnosis. Adv Clin Path 1998;2(2):101-15.
Graadt van Roggen JF, Hogendoorn PC, et al. Myxoid tumours of soft
tissue. Histopathology 1999;35(4):291-312.
Kempson RL, Fletcher CDM, Evans HL, et al. Lipomatous tumors. In:
Tumors of the Soft Tissues, III ed. AFIP 2001;4:187-238.
Loubignac F, Bourtoul C, Chapel F. Myxoid liposarcoma: a rare
soft-tissue tumor with a misleading benign appearance. World J Surg
Oncol 2009;22;7:42.
Germano G, Frapolli R, Simone M, et al. Antitumor and anti-inflammatory effects of trabectedin on human myxoid liposarcoma cells.
Cancer Res 2010;70(6):2235-44.
Case n. 3
Peripheral primitive neuroectodermal tumor
(pPNET) of the small bowel
M. Milione, A. Testi, F. Perrone, F. Melotti, S. Pilotti,
G. Pelosi
Dipartimento di Patologia Diagnostica e Laboratorio, Fondazione
IRCCS Istituto Nazionale dei Tumori, Milano
Clinical History. A 43-year-old man was admitted, on March
2010, to Tivoli City Hospital with acute abdomen and intermittent abdominal pain of 5 months’ duration. His family history
was non-contributory. Routine laboratory data on admission
were within normal limits. Tumor markers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA
19-9) showed normal values; Computed tomographic (CT)
scan of the abdomen showed ascitic fluid and an 80-mm diameter mass in ileo-cecal region. Laparotomy was conducted.
The tumor consisted of polycystic components, and part of its
surface adhered tightly to the initial portion of the jejunum
whereas ascending colon, and greater omentum were normal.
The tumor exhibited movability and could be easily dislocated
from the abdominal cavity. An en-bloc resection of the tumor,
including the ascending colon and proximal jejunum (each
20 cm in length), and greater omentum, was performed. Endto-end jejunojejunostomy and colono-colonostomy were also
carried out. Macroscopically, the resected tumor was 10 cm
in major size, and 290 g in weight; it was elastic, and soft
in consistency. The cut surface of the tumor showed mostly
polycystic and, partially, solid features. There were bleeding
and necrotizing parts inside the tumor. The tumour ulcerated
the mucosa. Mucosal surfaces away from the ulcerated area
were normal.
After surgery no lesion were detected with Octreoscan study.
Plasmatic chromogaranin levels were normal.
Discussion. Microscopically, the specimens showed a sheetlike proliferation of spindle-to-polygonal cells with large
vesicular nuclei with thin vascular stroma. Focally the tumor
formed ribbon-like or rosette structures. Moderate mitosis
was noted (10/50 high-power field) No invasive growth or
metastasis to lymph nodes was noted. Immunohistochemestry showed the tumour cells to stain focal positively with:
cytokeratin (CK) AE1/AE3, CAM 5.2, synaptophisin (SYN),
chromogranin A (CgA) and CD 117, strongly diffuse positively with vimentin, CD 99, FlI-1and negatively for epithelial
membrane antigen (EMA), S100, leucocyte common antigen
(LCA), CD 34, smooth muscle actine (SMA), carcinoembryonic antigen (CEA), desmin an d WT-1. Proliferative index
of neoplasia valued with MIB-1/Ki-67 immunostaining was
about 30%. P53 was hyperexpressed. Based on histology and
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
immunohistochemistry findings, the tumor was diagnosed as
a extraskeletal ES/PNET, suspected of the small bowel, rather
than an epitheliod GIST, or neuroendocrine poorly differentiated carcinoma (PDEC). Primitive neuroectodermal tumor
(PNET) arises in soft tissue, and is thought to be of neural
crest origin. This tumor, which usually develops in the chest
wall and the extremities of children and adolescents 1-9, is very
rare, and is highly aggressive and malignant, and is characteristic of small round-cell tumours (SRCT). Pathology diagnosis in the present case was difficult because ES/PNET is
extremely rare among tumors of the small intestine. Diagnosis
required the exclusion of similar tumors showing undifferentiated small round cell morphology, including neuroblastoma,
malignant lymphoma 9, rhabdomyosarcoma, gastrointestinal
stromal tumor (GIST), and desmoplastic small round cell
tumor (DSRCT). Lymphoma could be excluded because the
tumor cells did not express leukocyte common antigen (LCA).
DSCRT could be excluded because the tumor lacked desmoplastic stromal reaction, WT-1 and EMA immunoreactivity,
but expressed CD99 and CD117/c-kit. Neuroblastoma, rhabdomyosarcoma, could be excluded based on microscopy and
immunohistochemical data CD99 is expressed in ES/PNET
and other malignancies such as lymphoblastic lymphoma,
rhabdomyosarcoma, DSRCT, synovial sarcoma, and solitary
fibrous tumors. Small Bowel PDEC was favoured by clinical
presentation (age, site, occlusive syndrome with peritonitis
and ascitis) and was sustained by IHC positivity for cytokeratins, chromogranin A and synaptophysin, but morphology (no
intratumoral necrosis, no perivascular glomeruloid pattern
and abundant pseudorosette formations) and MIB 1/Ki-67
value were against this diagnosis. CD117 was originally a
marker for c-kit, a transmembrane tyrosine kinase normally
expressed by Cajal’s interstitial intestine cells and now known
to be a stem cell marker. CD117 is expressed in a variety of
cancers including gastrointestinal stromal tumors, malignant
melanoma, mastocytosis, acute myelocytic leukemia, anaplastic lymphoma, germinoma, and ES/PNET. The EWS gene
rearrangement in 22q12 demonstration facilitates a prompt
discrimination between ES/PNET and other morphologically
similar round cell tumors as more than 90% of all ES/PNET.
Molecular analysis for C-KIT gene were performed on
formalin fixed paraffin embedded material, genomic DNA
amplification has shown wild type exons 9, 11,13 and 17.
According to the exclusive diagnosis, the present case was
ultimately diagnosed an extraskeletal ES/PNET. Clinically,
peripheral primitive neuroectodermal tumor (pPNET) may
occur anywhere in the body 10 11. Systematic revision of 54
cases of extracranial pPNET encountered at Memorial SloanKettering Cancer Center over a 20-year period and found that
the primary sites were thoraco-pulmonary (25 cases), pelvis
(12 cases), retroperitoneum or abdomen (10 cases), limb (five
cases), neck (one case) and unknown (one case) 1. Isolated
cases of pPNET have been reported in various visceral sites,
including the pancreas 12, heart 13, kidney 14, ovary 15, uterus,
testis, urinary bladder and parotid gland 1. Despite combined
therapy with surgery, chemotherapy and irradiation, the prognosis of pPNET is poor. Kushner et al. indicated that only
25% of patients with tumors greater than 5 cm were alive at
24 months 1. In conclusion, we have documented a rare case of
pPNET arising from the small bowel with perforation at onset.
The perforation was considered to be caused by massive invasion of the tumor cells with prominent tumor necrosis and/or
local ischemic changes. It is important for both surgeons and
pathologists to remember that intraabdominal pPNET may
present with acute abdomen.
References
1
Kushner BH, Hajdu SI, Gulati SC, et al. Extracranial primitive neuroectodermal tumors. Cancer 1991;67:1825-9.
2
Marina NM, Etcubanas E, Parham DM, et al. Peripheral primitive
neuroectodermal tumor (peripheral neuroepithelioma) in children.
Cancer 1989;64:1952-60.
3
Harper PG, Pringle J, Souhami RL. Neuroepithelioma-a rare malignant
peripheral nerve tumor of primitive origin. Cancer 1981;48:2282-7.
4
Askin FB, Rosai J, Sibley RK, et al. Malignant small cell tumor of the
thoracopulmonary region in childhood. Cancer 1979;43:2438-51.
5
Dehner LP. Primitive neuroectodermal tumor and Ewing’s sarcoma.
Am J Surg Pathol 1993;17:1-13.
6
Mor Y, Nass D, Raviv G, Neumann Y, et al. Malignant peripheral
primitive neuroectodermal tumor (PNET) of the kidney. Med Pediatr
Oncol 1994;23:437-40.
7
Furman J, Murphy WM, Jelsma PF, et al. Primary primitive neuroectodermal tumor of the kidney. Am J Clin Pathol 1996;106:339-44.
8
Horn LC, Fischer U, Bilek K. Primitive neuroectodermal tumor of the
cervix uteri: a case report. Gen Diagn Pathol 1996/97;142:227-30.
9
Horie Y, Kato M. Peripheral primitive neuroectodermal tumor of the
small bowel mesentery: a case showing perforation at onset. Pathol Int
2000;50:398-403.
10
Enzinger FM, Weiss SW. Primitive neuroectodermal tumors and related lesions. In: Soft Tissue Tumors, 3rd ed. New York: Mosby 1995,
pp. 929-64.
11
Deb RA, Desai SB, Amonkar PP, et al. Primar primitive neuroectodermal tumour of the parotid gland. Histopathology 1998;33:375-8.
12
Danner DB, Hruban RH, Pitt HA, et al. Primitive neuroectodermal
tumor arising in the pancreas. Mod Pathol 1994;7:200-4.
13
Charney DA, Charney JM, Ghali VS, et al. Primitive neuroectodermal tumor of the myocardium: A case report, review of the literature, immunohistochemical, and ultrastructural study. Hum Pathol
1996;27:1365-19.
14
Marley EF, Liapis H, Humphrey PA, et al. Primitive neuroectodermal
tumor of the kidney. Another enigma: A pathologic, immunohistochemical, and molecular diagnostic study. Am. J. Surg. Pathol.
1997;21:354-9.
15
Kawaguchi S, Fukuda T, Miyamoto S, et al. Peripheral primitive neuroectodermal tumor of the ovary confirmed by CD99 immunostaining,
karyotypic analysis, and RT-PCR for EWS/FLI-1 chimeric mRNA. Am
J Surg Pathol 1998;22:1417-22.
Case n. 4
Pleomorphic lobular carcinoma in situ of breast
G. Falconieri, V. Angione, S. Pizzolitto
Struttura Operativa Complessa di Anatomia Patologica, Azienda
Ospedaliero Universitaria, Udine, Italy
Clinical History. A quadrant biopsy is performed in a 55-yearold woman who has mammographic evidence of suspicious
microcalcifications and a core needle biopsy suspicious for
“ductal” carcinoma in situ with comedonecrosis. Specimen inspection reveals poorly demarcated white–gray consolidations
that, on cut surface, express yellowish comedo-like material.
Tissue material is fixed in formalin and routinely processed.
A panel of antibodies was applied to paraffin sections directed
against estrogen (ER) and progesterone (PR) receptors, p63,
E-cadherin, low- and high-molecular weight keratins. Hematoxylin-eosin stained sections feature gland units irregularly
distended by a population of medium-sized to large epithelial
cells with highly atypical nuclei. Central necrosis and microcalcifications are noticed. Tumor cells are discohesive, with
amphophilic to slightly eosinophilic cytoplasm. No infiltrative
component is recognized. Tumor cells are positive for highmolecular-weight keratins as well as ER/PR; they are negative
for E-cadherin. Cells positive for p63 regularly decorate the
basal layers of the affected units. The combined features were
consistent with high-grade pleomorphic lobular carcinoma in
situ (PLCIS) with comedonecrosis.
Lectures
Discussion. In most cases, recognition of classic lobular
carcinoma in situ (LCIS) is prompted by a number of histologic and cytologic features, including distention of terminal
tubulolobular units by fairly uniform, small to medium-sized
round epithelial cells involving more than 50% of the acini
within a terminal duct lobular unit. Tumor cells are discohesive and show mild nuclear atypia with an increased nuclear
to cytoplasmic ratio; mitoses are rare. LCIS is positive for
both ER/PR and negative for e-cadherin; it tends to express
a greater amount of high-molecular-weight keratins. The
diagnosis of LCIS has several clinical implications: notably, it is considered a marker of increased risk for invasive
breast cancer, either ipsi- or contralaterally, but its incidental
recognition in core needle biopsy specimens is not generally considered an indication for further surgery. In fact,
unlike ductal carcinoma in situ (DCIS), LCIS documented
at resection margins is managed conservatively by means of
tamoxifen and/or follow-up. Variants of LCIS have been reported, mirroring a number of architectural and/or cytologic
changes. Tumour cells may be of larger size, show increased
variation of cell shape and size, and may exhibit variable
nuclear pleomorphism (e.g., two- to threefold variation in
nuclear size), an increased nuclear/cytoplasmic ratio, and
nucleoli. These lesions are also referred to as pleomorphic
LCIS (PLCIS). In addition, tumor-cell necrosis (so-called
comedonecrosis) is often encountered. Tumor cells may also
feature a relatively abundant, stainable, or “apocrine” cytoplasm. Because of their different clinical implications, these
variants of LCIS must be distinguished from high-grade (or
grade III) DCIS, in particular when they are associated with
comedonecrosis. On a morphologic ground, it should be
pointed out that PLCIS still maintains the basic microscopic
features of conventional lobular neoplasia and that several
features militate against DCIS. Conventional areas of LCIS
may be present next to the PLCIS foci suggesting that the
two conditions are closely related. Although at a first glance
necrosis, microcalcification, and high-grade nuclei suggest
intraductal carcinoma, discohesion of tumor cells is a clue to
lobular neoplasia. On the other hand, comedonecrosis is not
a distinctive feature of DCIS, since it can be seen in several
other proliferative conditions of the breast, such as classic LCIS or florid papillomatosis. Like common LCIS, the
high-grade variant is also consistently positive for ER/PR,
although in a lesser amount, and negative for E-cadherin,
whereas a reverse immunostaining pattern is often seen in
high-grade DCIS. The apocrine variant of PLCIS characteristically shows much less ER/PR content and several cytogenetic alteration including 16p gain and several losses (11q,
13q, 17p). In the multistep pathway of lobular neoplasia, it is
also postulated that apocrine PLCIS is the potential precursor of apocrine infiltrating lobular carcinoma. The proliferative ki67 index of PLCIS is significantly higher. At times,
the distinction between DCIS and LCIS may be problematic
due to “packing” of tumor cells in LCIS and heterogeneous
e-cadherin staining in some DCIS. It is also possible that
some such cases might represent true mixed tumor, thus
indicating that the diagnoses of DCIS and LCIS are not
mutually exclusive. It is suggested that in situ tumors with
a mixed phenotype be treated as DCIS. On the other hand,
the management of patients with PLCIS is still controversial,
with informed opinions recommending either a conservative
221
approach (along to the lines commonly adopted for classic
LCIS) or more effective surgical measures. A number of
drawbacks (including the paucity of series, limited data, lack
of prospective and clinically validated studies) preclude firm
conclusions. In addition, it is not known whether the breast
cancer risk (level and laterality) associated with this lesion
is comparable with that of conventional LCIS. However,
infiltrating lobular carcinoma may be associated with PLCIS
in as much as 45% of cases, especially in post-menopausal
women. Furthermore, an increased phenotypic aggressiveness characterizes PLCIS-related invasive lobular carcinoma
as a high nuclear grade and the overexpression of Her2, p53
and c-myc oncogenes indicate. For these reasons, accountable experts recommend that PLCIS treatment should be
probably more “DCIS-tailored”, including surgical excision
of the entire lesion or a quadrant biopsy in cases of PLCIS diagnosed on core needle biopsy. Given the increased
chance of an associated invasive component the opportunity
of a sentinel lymph node biopsy should be also considered.
References
Barsky SH, Bose S. Should LCIS Be Regarded as a Heterogeneous Disease? Breast J 1999;5:407-12.
Bentz JS, Yassa N, Clayton F. Pleomorphic lobular carcinoma of
the breast: clinicopathologic features of 12 cases. Mod Pathol.
1998;11:814-22.
Cangiarella J, Guth A, Axelrod D, et al. Is surgical excision necessary
for the management of atypical lobular hyperplasia and lobular carcinoma in situ diagnosed on core needle biopsy?: a report of 38 cases
and review of the literature. Arch Pathol Lab Med 2008;132:979-83.
Chen YY, Hwang ES, Roy R, et al. Genetic and phenotypic characteristics of pleomorphic lobular carcinoma in situ of the breast. Am J Surg
Pathol 2009;33:1683-94.
Chivukula M, Haynik DM, Brufsky A, et al. Pleomorphic lobular carcinoma in situ (PLCIS) on breast core needle biopsies: clinical significance and immunoprofile. Am J Surg Pathol. 2008;32:1721-6.
Fadare O. Pleomorphic lobular carcinoma in situ of the breast composed
almost entirely of signet ring cells. Pathol Int 2006;56:683-7.
Fadare O, Dadmanesh F, Alvarado-Cabrero I, et al. Lobular intraepithelial neoplasia [lobular carcinoma in situ] with comedo-type
necrosis: A clinicopathologic study of 18 cases. Am J Surg Pathol
2006;30:1445-53.
Hanby AM, Hughes TA. In situ and invasive lobular neoplasia of the
breast. Histopathology 2008;52:58-66.
Jacobs TW, Pliss N, Kouria G, et al. Carcinomas in situ of the breast with
indeterminate features: role of E-cadherin staining in categorization.
Am J Surg Pathol 2001;25:229-36.
Koerner F, Maluf H. Uncommon morphologic patterns of lobular neoplasia. Ann Diagn Pathol 1999;3:249-59.
Maluf HM. Differential diagnosis of solid carcinoma in situ. Semin Diagn
Pathol 2004;21:25-31.
Maluf HM, Swanson PE, Koerner FC. Solid low-grade in situ carcinoma
of the breast: role of associated lesions and E-cadherin in differential
diagnosis. Am J Surg Pathol 2001;25:237-44.
Middleton LP, Palacios DM, Bryant BR, et al. Pleomorphic lobular carcinoma: morphology, immunohistochemistry, and molecular analysis.
Am J Surg Pathol 2000;24:1650-6.
Reis-Filho JS, Simpson PT, Jones C, et al. Pleomorphic lobular carcinoma of the breast: role of comprehensive molecular pathology in
characterization of an entity. J Pathol 2005;207:1-13.
Schnitt SJ, Morrow M. Lobular carcinoma in situ: current concepts and
controversies. Semin Diagn Pathol 1999;16:209-23.
Sneige N, Wang J, Baker BA, et al. Clinical, histopathologic, and biologic features of pleomorphic lobular (ductal-lobular) carcinoma in
situ of the breast: a report of 24 cases. Mod Pathol 2002;15:1044-50.
Wahed A, Connelly J, Reese T. E-cadherin expression in pleomorphic
lobular carcinoma: an aid to differentiation from ductal carcinoma.
Ann Diagn Pathol 2002;6:349-51.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Case n. 5
Pulmonary metastasis of rhabdoid melanoma
G. Falconieri, M. Rocco, S. Pizzolitto
Struttura Operativa Complessa di Anatomia Patologica, Azienda
Ospedaliero Universitaria, Udine, Italy
Clinical History. A peripheral nodular opacity is documented
in a 49 year-old, otherwise healthy woman during a routine
plain chest-X ray film. About 24 years previously a diagnosis
of invasive melanoma, no further specified, had been done on
excisional skin biopsy. A wedge-shaped lung biopsy is carried out. The lung specimen reveals a 2 cm, sub-pleural grey
nodule. A panel of antibodies is applied to paraffin sections
directed against broad spectrum keratins, TTF1, leucocyte
common antigen, S100 protein, HMB45, Melan A, tyrosinase
and MITF-1. Hematoxylin-eosin stained sections feature a
discohesive proliferation of epithelioid cells. Cytoplasm is
relatively abundant, brightly eosinophilic to glassy, sometimes pushing the nucleus at the periphery and imparting a
plasmacytoid appearance to tumor cells. Nuclei are vescicular, with finely dispersed chromatin and prominent nucleoli.
Mitotic activity is brisk. Tumor cells are positive for s100
protein, HMB45, melan A, MITF1 and tyrosinase and negative for the other markers. The overall features are consistent
with metastatic melanoma, with rhabdoid cells. Follow-up at
3 years is uneventful.
Discussion. Traditionally, rhabdoid cells are defined as variably sized elements featuring abundant, eosinophilic and
fibrillary cytoplasm recalling rudimentary skeletal muscle
differentiation. A number of malignant tumors may exhibit a
rhabdoid phenotype, including carcinoma, melanoma, large
cell lymphoma, mesenchymal malignancies such as nerve
sheath sarcoma or synovial sarcoma. Intrathoracic tumors featuring rhabdoid cells raise additional interpretive challenges.
Rhabdoid carcinomas are rare in the lung. Current review of
the literature shows that less than 40 cases of rhabdoid pulmonary carcinoma have been compiled in the American and
English literature. This carcinoma is considered aggressive if
compared with the more common non-small cell carcinoma.
The rhabdoid component may be focal and associated with a
conventional large cell carcinoma or adenocarcinoma. In the
series by Tamboli et al., 11 cases of lung rhabdoid carcinomas
were described: the patients were all adults and presented
mostly in advanced stage (III or IV) carcinoma. History of
cigarette smoking could be elicited in more than half of the
patients. However, the rhabdoid cells were inconsistently
observed. In addition, in all cases an epithelial differentiation
could be recognized while in four cases, features of glandular
differentiation were seen. No specific immunohistochemical profile was detected, although most tumors reacted for
keratins and vimentin. Interestingly, no immunostaining was
reported for TTF-1, an antibody that frequently reacts with
conventional adenocarcinoma of lung. Metastatic deposits
had a predominance of rhabdoid cells. The prognosis for
pulmonary rhabdoid carcinoma is generally poor; however,
long-term survivors are apparently not exceptional. In the case
reported by Hiroshima the patient was a 70-year-old woman
with a stage I tumor. Recurrence was detected 6 years after
thoracic surgery and was comparable with the primary lesion.
The survival time noted in this case, which is even longer for
the more common non-small cell carcinoma, suggests that
localized diseases may have a greater chance of curability.
Similarly, more than 5-year survival was observed in the case
reported by Kaneko et al., who described a 59-year-old man
presenting with a lung-confined tumor. The patient developed
an adrenal metastasis 3 years after lobectomy. On the other
hand, in their study of 14 patients, Shimazaki et al. found
that rhabdoid cell-rich tumors entailed a poorer prognosis
regardless of tumor stage, and their observation was also
validated by a statistical analysis suggesting than the number
of rhabdoid cells may be a significant prognosticator. Interestingly, if the compiled cases of rhabdoid lung carcinoma are
considered, lymph node metastases have been documented in
13 of 33 cases, the proportion of lung tumors without local
metastases at presentation being roughly two thirds. Also, we
are not aware of cases in which the metastases were found
prior to the lung tumor. Immunohistochemistry and electron
microscopy suggest an epithelial lineage, given the consistent
immunoreactivity for keratins and ultrastructural features
such as paranuclear intermediate filaments. Tumor size and
stage did not appear to have any predictive means.
Notably, the differential diagnosis of extra-renal rhabdoid
neoplasm may be a difficult task because of its numerous
microscopic mimickers. In the lung, the matter is further
compounded by the existence of site-specific simulators: for
the sake of brevity, only some of such lesions will be briefly
discussed. The differentiation may be difficult on pure morphologic grounds and must be clinically driven, first. Then,
the judicious use of a limited antibody panel may suffice for
reliably segregating these lesions and singling out those that
may benefit from specific treatments. Large cell lymphoma
may occur as a primary pulmonary and/or mediastinal lesion.
Tumor cells are often immunopositive for LCA and lymphoid
antigens such as CD3, CD20, or CD30 may be expressed.
Specific pulmonary lymphoproliferative disorders such as
lymphomatoid granulomatosis may be recognized by virtue
of clinical and pathologic features, although resorting to immunohistochemistry is useful to rule out other conditions.
Melanoma, as the case at issue indicates, can be virtually
indistinguishable from rhabdoid tumors as well as from any
other malignancy inasmuch as rhabdoid changes are often
detected in recurrent or metastatic lesions; immunoreactivity
for S100 protein, in absence of staining for other antigens, is,
however, expected in most cases of melanoma. Epithelioid angiosarcoma may rarely but not exceptionally occur in the lung
as a primary tumor, either alone or associated with extensive
pleural involvement. Poorly differentiated forms may lack
evidence of diagnostic clues such as a freely anastomosing
vascular channel pattern or intracellular lumina. In addition,
tumor cells may have a rich stainable cytoplasm, recalling that
seen in rhabdoid tumors, and epithelioid angiosarcoma cells
may react to antibodies against keratins, thus suggesting a
tumor of true epithelial lineage. However, clues such as abortive vessels or intracellular lumina may be often recognized;
notably, angiosarcoma is often positive for factor VIII-related
antigen, CD31, CD34 and fli-1.
References
Attems JH, Lintner F. Pseudomesotheliomatous adenocarcinoma of the
lung with rhabdoid features. Pathol Res Pract 2001;197:841-6.
Cavazza A, Colby TV, Tsokos M, et al. Lung tumors with a rhabdoid
phenotype. Am J Clin Pathol 1996;105:182-8.
Chetty R. Combined large cell neuroendocrine, small cell and squamous
carcinomas of the lung with rhabdoid cells. Pathology 2000;32:20912.
Chetty R, Bhana B, Batitang S, et al. Lung carcinomas composed of rhabdoid cells. Eur J Surg Oncol 1997;23:432-4.
Falconieri G, Moran CA, Pizzolitto S, et al. Intrathoracic rhabdoid carcinoma: a clinicopathological, immunohistochemical, and ultrastructural study of 6 cases. Ann Diagn Pathol 2005;9:279-83.
Lectures
Hiroshima K, Shibuya K, Shimamura F, et al. Pulmonary large cell carcinoma with rhabdoid phenotype. Ultrastruct Pathol 2003;27:55-9.
Kaneko T, Honda T, Fukushima M, et al. Large cell carcinoma of the
lung with a rhabdoid phenotype. Pathol In. 2002;52:643-7.
Rubenchik I, Dardick I, Auger M. Cytopathology and ultrastructure of
primary rhabdoid tumor of lung. Ultrastruct Pathol 1996;20:355-60.
Shimazaki H, Aida S, Sato M, et al. Lung carcinoma with rhabdoid cells:
a clinicopathological study and survival analysis of 14 cases. Histopathology 2001;38:425-34.
Tamboli P, Toprani TH, Amin MB, et al. Carcinoma of lung with rhabdoid features. Hum Pathol 2004;35:8-13.
Wick MR, Ritter JH, Dehner LP. Malignant rhabdoid tumors: a clinicopathologic review and conceptual discussion. Semin Diagn Pathol
1995;12:233-48.
Case n. 6
Small cell neuroendocrine carcinoma
with myofibroblastic and skeletal muscle
differentation
G. Pelosi
Dipartimento di Patologia Diagnostica e Laboratorio, Fondazione
IRCCS Istituto Nazionale dei Tumori, Milano
Clinical history. A 76-year-old Caucasian man, former smoker since 30 years (previously he smoked 30 cigarettes/day for
at least 20 years), underwent left upper lobectomy for Aspergillus infection (fungus ball) in 1980. The patient experienced
hemoptysis in 2003, when a granulomatous inflammation was
found in the left main bronchus (likely related to the previous surgery) that was treated with LASER therapy. In May
2007, the patient began to complain of hemoptysis again and
a chest X ray examination showed a huge tumor mass in the
right upper lobe, measuring 6-7 cm in diameter. A total body
CT scan investigation confirmed the presence of this tumor
mass in the right upper lobe, sized 67 × 48 × 50 mm, which
compressed the lobar bronchus and apparently infiltrated the
azygos vein but was not associated with pleural effusion or
distant metastases. As PET scan examination did not reveal
distant metastases, a right upper lobectomy with a complete
hilar-mediastinal lymphadenectomy was performed at the end
of May 2007. The postoperative clinical course was uneventful, and the patient was discharged ten days later in good
general conditions. The tumor measured 7 cm in its greatest
dimension, was located in the pulmonary upper lobe, attained
the visceral pleura but with no azygos vein infiltration and
showed necrosis and hemorrhage with friable tissue on cut
section. Histopathologic examination revealed a biphasic tumor composed of 1) a high-grade neuroendocrine carcinoma
component arranged in nests, trabeculae or solid aggregates
with finely granular chromatin and inconspicuous nucleoli,
and 2) a spindle to pleomorphic cell component with abundant
collagen deposition resembling high-grade sarcoma. The two
components were intimately intermingled with each other, but
a slight prevalence of the sarcoma-like component (55-60%)
was noted. The immunohistochemical study revealed a strong
and diffuse positivity for cytokeratins (AE1-AE3) in the epithelial-like component and for CD56 in all tumor cells, and
a more variable immunoreactivity for S-100 protein, GFAP,
synaptophysin, sarcomeric actin, neurofilaments, TTF-1,
smooth-muscle actin, calponin, caldesmon and CD10. Co-expression of cytokeratins, desmin and myogenin was localized
in the same aggregates of tumors cells exhibiting epithelial
features, suggesting dipartite differentiation. Ki-67 labeling
index was higher in the epithelial-like component (80 to 90%)
than in the sarcoma-like population (30 to 40%). Electron
223
microscopy study showed myofibroblastic differentiation
in the spindle cell component, whereas neuroendocrine differentiation in the epithelial cell-like component shows and,
less frequently, coexisting bundles of contractile filaments
containing abortive Z bands reminiscent of skeletal muscle
differentiation, suggesting co-localized rhabdomyoblastic and
neuroendocrine differentiation. Tumor staging was pT3N1M0
because of a single peribronchial lymph node metastasis. After adjuvant chemotherapy, the patient is alive and well with
no sign of metastatic disease.
Discussion. The case here reported is a combined small-cell
carcinoma with skeletal muscle differentiation and spindle
cell sarcoma component of myofibroblastic type. Combined
small-cell carcinoma variant makes up about 10 to 30% of
all small cell carcinomas of the lung. It refers to the variable
admixture of small-cell and non-small cell carcinoma elements, the latter usually including squamous cell carcinoma,
adenocarcinoma and/or large-cell carcinoma 1, and much more
uncommonly spindle cell 2 3 or giant cell carcinoma 4 5. Other
exceedingly rare combinations in the theme of neuroendocrine carcinomas of the lung include associations of atypical
carcinoid and rhabdomyosarcoma 6, small cell carcinoma
plus adenocarcinoma and spindle-shaped cell tumor 7, small
cell carcinoma plus squamous cell carcinoma and spindle
cell carcinoma 8, small cell carcinoma plus sarcomatoid carcinoma with either spindle cell or giant cell carcinoma 9, and
carcinoid and adenocarcinoma 10 11. Moreover, occurrence of
small cell carcinoma with skeletal muscle differentiation has
been described in the larynx 12, as well as in the skin, nasal
cavity and urinary bladder 13, and combination of high-grade
neuroendocrine carcinoma and alveolar rhabdomyosarcoma is
also on record in the anorectal junction 14. Although intimate
intermingling of small cell carcinoma elements with scattered
rhabdomyoblastic cells 13 and even tripartite differentation in
individual cells with concurrent epidermoid, glandular and
neuroendocrine features 15 or dipartite differentiation with
rhabdomyogenous and cytokeratin expression within the same
mesenchymal tumor cells of carcinosarcomas 16 have been described in the literature, the current case is worth of mention
because of dipartite differentiation of small cell carcinoma
and rhabdomyosarcoma coexisting with spindle cell sarcoma,
probably derived from a common protoepithelial stem cell. It
has been demonstrated that additional genetic alterations may
be found in the mesenchymal component of sarcomatoid carcinomas of the lung, which were lacking in the epithelial one,
suggesting mesenchymal transformation during epithelial carcinogenesis 17. In our case, the spindle cell component lacked
any epithelial or rhabdomyogenous differentiation, probably
because of a complete myofibroblastic/smooth muscle transdifferentiation of carcinomatous cells or early divergence during tumor progression of the same ancestor lesion.
References
1
Travis W, Brambilla E, Muller-Hermelink H, et al. Tumours of the
lung, pleura, thymus and heart. Edited by Cancer IAfRo. Lyon: IARC
Press 2004, p. 344.
2
Tsubota Y, Kawaguchi T, Hoso T, et al. A combined small cell and
spindle cell carcinoma of the lung. Report of a unique case with immunohistochemical and ultrastructural studies. Am J Surg Pathol
1992;16:1108-15.
3
Niho S, Yokose T, Nagai K, et al. A case of synchronous double
primary lung cancer with neuroendocrine features. Jpn J Clin Oncol
1999;29:219-25.
4
Bégin P, Sahai S, Wang N. Giant cell formation in small cell carcinoma of the lung. Cancer 1983;52:1875-9.
5
Müller K, Fisseler-Eckhoff A. Small cell bronchial cancer--pathologic
anatomy. Langenbecks Arch Chir Suppl Kongressbd 1991:534-543.
224
6
7
8
9
10
11
12
13
14
15
16
17
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Rainosek D, Ro J, Ordonez N, et al. Sarcomatoid carcinoma of the
lung. A case with atypical carcinoid and rhabdomyosarcomatous
components. Am J Clin Pathol 1994;102:360-4.
Hsiao H, Tsai H, Liu Y, et al. A rare case of combined small-cell
lung cancer with unusual soft tissue metastasis. Kaohsiung J Med Sci
2006;22:352-6.
Gotoh M, Yamamoto Y, Huang C, et al. A combined small cell carcinoma of the lung containing three components: small cell, spindle cell
and squamous cell carcinoma. Eur J Cardiothorac Surg 2004;26:10479.
Fishback N, Travis W, Moran C, et al. Pleomorphic (spindle/giant
cell) carcinoma of the lung. A clinicopathologic correlation of 78
cases. Cancer 1994;73:2936-45.
Sen F, Borczuk AC. Combined carcinoid tumor of the lung: a combination of carcinoid and adenocarcinoma. Lung Cancer 1998;21:53-8.
Cavazza A, Toffanetti R, Ferrari G, et al. Combined neoplasia of the
lung: description ofa acase of adenocarcinoma mixed with typical
carcinoid. Pathologica 2001;93:216-220.
Doglioni C, Ferlito A, Chiamenti C,et al. Laryngeal carcinoma showing multidirectional epithelial neuroendocrine and sarcomatous differentation. ORL J Otorhinolaryngol Relat Spec 1990;52:316-26.
Eusebi V, Damiani S, Pasquinelli G, et al. Small cell neuroendocrine
carcinoma with skeletal muscle differentation. Am J Surg pathol
2000;24:223-30.
Roncaroli F, Montironi R, Feliciotti F, et al. Sarcomatoid carcinoma
of the anorectal junction with neuroendocrine and rhabdomyoblastic
features. Am J Surg Pathol 1995;19:217-23.
McDowell EM, Trump BF. Pulmonary smell cell carcinoma showing
tripartite differentiation in individual cells. Hum Pathol. 1981;12:28694.
Wick MR, Ritter JH, Humphrey PA. Sarcomatoid carcinomas of the
lung: a clinicopathologic review. Am J Clin Pathol 1997;108:40-53.
Dacic S, Finkelstein SD, Sasatomi E, et al. Molecular pathogenesis of
pulmonary carcinosarcoma as determined by microdissection-based
allelotyping. Am J Surg Pathol 2002;26:510-6.
Case n. 7
Hepatocellular carcinoma with unusual
endocrine features
M. Milione, F. Melotti, A. Carbone *, G. Pelosi
Dipartimento di Patologia Diagnostica e Laboratorio, Fondazione
IRCCS Istituto Nazionale dei Tumori, Milano; * IRCCS - C.R.O. Centro di Riferimento Oncologico di Aviano, Udine
Clinical History. During a follow-up visit for chronic hepatitis C in a 36-years old man, a nodular lesion measuring around
1 cm in diameter was detected in the S5 liver segment using
abdominal ultrasonography. Serum a-fetoprotein (AFP) was
25.3 ng/ml. Two months later a new nodule measuring just
about 3,3 mm in diameter was found in the S6 liver segment.
Three months later the S6 nodule grew up rapidly and was approximately 2.9 cm in diameter. The tumor in S5 grew slowly
and also achieved approximately 2.3 cm. AFP levels were
elevated to 3787.0 ng/ml. Hepatitis B surface antigen and antibody were negative. Carcinoembryonic antigen (CEA) was
within normal limits. Using computed tomography (CT) the
S6 tumor was enhanced in the early phase and the S5 tumor
showed peripheral enhancement in the early phase. Lymph
nodes were not distinguished. In abdominal ultrasonography
(US) the S5 and S6 tumors demonstrated a mosaic pattern.
At our institution, a liver core biopsy was performed on both
nodules.
Discussion. Histologically the S6 tumor was composed of
round tumor cells with moderately represented eosinophilic
cytoplasm and round nuclei showing a trabecular organization. The tumor was reliable with moderately differentiated HCC, but was intermingled with small round cells with
scarce cytoplasm, which resembled those found in endocrine
carcinoma (EC). This population shaped solid nests along
with the trabecular arrangement intermitted with pseudoacinar areas and rosette formation. It stained positively for
chromogranin-A, synaptophysin, CD56, strongly for CK19,
alpha phetoprotein (AFP) and Glypican 3; and it resulted
negative for Hep Par1, CK7, CK20, TTF1, serotonin, insulin,
and glucagon. MIB-1 immunostain demonstrated high proliferative activity (70-80% of the cells were positive). p53 was
positive in 60% of neoplastic cells. The diagnosis of HCC
with Endocrine features was thus established. The S5 nodule
showed morphologic and immunophenotipic features of classic well differentiated (G1) HCC. On rare occasions endocrine
character appear within a Hapatocelluar Carcinoma (HCC)
nodule. Origin of hepatic endocrine tumors is vague, the most
interesting hypotheses planned are: A) derivation from the endocrine cells nearby in the intrahepatic bile duct epithelium 1
and B) endocrine differentiation of a distinct malignant stem
cell originator of additional hepatic malignant tumors 2. The
growth of a lot of hepatic carcinoids consisting of uniform tumor cells, in a noncirrhotic liver without necrosis or degeneration, wired the former notion. Carcinomas of the extrahepatic
biliary system showed a high rate of endocrine differentiation,
and had a poor prognosis 3. Intrahepatic cholangiocarcinoma
and HCC have also been reported to go through endocrine
differentiation. Positivity on immunohistochemistry for neuroendocrine markers in some cases of HCC was described 4 5.
These findings support the second hypothesis. It is also well
known that in HCC, a less well-differentiated tumor clone,
arises within the original tumor and proliferates, eventually
replacing the latter. As a result, a clone undergoing endocrine
differentiation may proliferate, replacing the entire tumor, and
form a complete EC. In the present case, the primary hepatic
EC microscopically resembled an HCC. The occurrence of
this EC on a background of hepatitis C cirrhosis and its coexistence with another HCC strongly suggest that this primary
hepatic EC did not arise de novo from the endocrine system in
the hepatic parenchyma, and leads us to speculate that one of
the HCCs underwent endocrine differentiation and thereafter
transformed into a EC. In general, endocrine carcinomas grow
rapidly and have a poor prognosis. Endocrine differentiation
in other organ cancers has more malignant behavior (3,5) In
the present case the S6 tumor with neuroendocrine carcinoma
showed more rapid growth than a typical HCC of S5. In addition, labeling index of p53 and Ki-67 of the small round
endocrine component were significantly higher than those of
the HCC component, and suggesting that the former has more
abnormalities of p53 and higher proliferative activity. HCC
with endocrine appearance has higher proliferative activity
and malignant potential than ordinary HCC. Few authors have
described primary endocrine tumors in the liver combined
with hepatocellular carcinoma, but these represented differentiation of the malignant liver cells into a endocrine tumor 6 7.
Separately from the collision and combined types, endocrine
tumors can also arise in an isolated manner primarily in the
liver in the shape of carcinoids or highgrade tumors represented by small cell carcinomas 1 8. The distinction between primary and metastatic endocrine tumors is important in making
the diagnosis of primary hepatic EC, as the liver is the most
common site of metastasis for these tumors. We investigated
the full body, principally the lung, pancreas, and gastrointestinal tract, for additional primary lesions by CT, MRI, and
endoscopy, but were incapable to find any primary lesions
outside the liver. It is imperative to distinguish carcinoid
tumors and EC clinic pathologically as EC is more malignant
and has poorer prognosis 2. Even though various hormones
Lectures
such as serotonin and gastrin have been frequently established
in primary hepatic carcinoid, such positive staining is hard to
identify in primary liver EC because of poor differentiation.
On the other hand, some collective immunohistochemical
features may origin misunderstanding, and care should hence
be taken in the diagnosis of EC.
References
1
Pilichowska M, Kimura N, Ouchi A, et al. Primary hepatic carcinoid
and neuroendocrine carcinoma clinicopathological and immunohistochemical study of five cases. Pathol Int 1999;49:318-24.
2
Gould VE, Banner BF, Baerwaldt M. Neuroendocrine neoplasms in
unusual primary sites. Diagn Histopathol 1981;4:263-77.
3
Hsu W, Dezidel DJ, Gould VE, et al. Neuroendocrine differentiation and prognosis of extrahepatic biliary tract carcinomas. Surgery
1991;110:604-10.
4
Artopoulos JG, Destuni C. Primary mixed hepatocellular carcinoma
with carcinoid characteristics: a case report. Hepato- Gastroenterology 1994;41:442-4.
5
Alpert LI, Zak FG, Werthamer S, et al. Cholangiocarcinoma: a
clinicopathologic study of five cases with ultrastructural observations.
Hum Pathol 1974;5:709-28.
6
Barsky SH, Linnoila I, Triche, TJ, et al. Hepatocellular carcinoma
with carcinoid features. Hum Pathol 1984;15(9):892-4.
7
Yamaguchi R, Nakashima O, Ogata T, et al Hepatocellular carcinoma with an unusual neuroendocrine component. Pathol Int
2004;54(11):861-5.
8
Rückert RI, Rückert JC, Dörffel Y, et al. Primary hepatic neuroendocrine tumor: successful hepatectomy in two cases and review of the
literature. Digestion 1999;60(2):110-6.
Case n. 8
Intrahepatic cholangiocarcinoma with thyroidlike features
V. Eusebi
Dipartimento di Ematologia e Scienze Oncologiche “L. e A. Seragnoli”, Osp. Bellaria, Anatomia Patologica, Bologna
Clinical history. A 52-year-old male suffering from abdominal pain for several months was admitted to hospital. Family
history was not relevant. A CT scan of the abdomen revealed
a homogeneous enhancing lesion of the right hepatic lobe of
18 cm in greatest axis. No other lesions were present in the
chest, head and neck and urogenital apparatus. Laboratory
data, including thyroid function tests, were within normal
range. A core biopsy of the liver mass was obtained which led
to the diagnosis of cholangiocarcinoma. This was followed by
hepatic lobectomy. Eighteen months after surgery the patient
is alive with no evidence of recurrence or metastatic disease.
Histology from pre operative biopsy as well as from surgical
specimen was identical. At low power the lesion was circumscribed by a thin fibrous capsule and showed a remarkable
follicular architecture. Follicles were of various sizes, ranging
from small to large. The content of follicles closely resembled
colloid being pale eosinophilic with occasional vacuoles at
the interface with the epithelium. The neoplastic cells were
mostly cuboidal with granular eosinophilic cytoplasm. Nuclei
were round to ovoid in shape, with scanty chromatin and
inconspicuous nucleoli. Some nuclei were clear and showed
neat membrane that was occasionally grooved. Mitoses were
1/10 hpf (x400). The eosinophilic luminal content of follicles
was rich in mucosubstances as evidenced by positivity for Alcian blue pH 2.5 and PAS after diastase digestion. Neoplastic
cells were positive for CK7, CK19, CAM 5.2 and CK AE1
225
and consistently negative for CEA, CK20, CD 56, hepatic
specific antigen, thyroglobulin, TTF-1, CD56, synaptophysin
and chromogranin.
Discussion. The case here reported was a large hepatic
tumour showing spongy cut surface with occasional bloodfilled cystic spaces. Histologically a remarkable follicular
architecture was evident. Neoplastic cells were mostly cuboidal with regular nuclei showing grooves and clearing of
the chromatin. The macroscopic and histological features
are similar to a follicular variant of papillary carcinoma of
the thyroid. Metastasis to the liver from well-differentiated
carcinomas of the thyroid is a well known, although rare phenomenon 1. An additional remote possibility would be that of
ectopic normal thyroid tissue in the liver as the case reported
by Strohschneider et al. 2 Nevertheless in the present patient
the neoplastic cells were all negative for thyroglobulin and
TTF-1 at the variance with well differentiated thyroid neoplasms and ectopic normal thyroid tissues wherever they are
found. Finally no evidence of a thyroid lesion of any kind was
found by clinical investigations, ultrasonography, CT scan
and laboratory tests. As no other primary was found after 13
months, including breast, kidney and pancreas, the cholangiocellular nature of the hepatic neoplasm was then favoured
for the presence of mucosubstances and further supported by
immunohistochemistry that evidenced CK7, CK19 and CAM
5.2 positivity along with CK20 and hepatic specific antigen
negativity. In differential diagnosis bile duct adenoma and
biliary cystoadenomas of the serous variant have to be taken
in consideration. It seems that a similar case had been reported
by Foucar et al. 7 who described an unusual variant of peripheral well-differentiated cholangiocarcinoma in a 27 yr-old
pregnant patient with histological features very similar to the
present case. Neoplastic lesions having thyroid-like features
have been reported in the breast and in the kidney 1 3-6. In the
breast the several cases described were superimposable to the
tall cell variant of papillary carcinoma of thyroid 1 3. The cases
reported in kidney were very similar to follicular carcinoma
of thyroid 4-6. Therefore it seems that thyroid- like features can
occur in tumours located in different organs and liver has to
be added to the list. To be aware of the existence of tumours
in the liver histologically mimicking follicular carcinomas
of thyroid can avoid diagnostic erroneous interpretation and
more cases are needed to establish the biological behaviour.
References
1
Eusebi V, Damiani S, Ellis IO, et al. Breast tumor resembling the
tall cell variant of papillary thyroid carcinoma. Am J Surg Pathol
2003;27:1114-8.
2
Strohschneider T, Timm D, Worbes C. Ectopic thyroid gland tissue in
the liver. Der Chirurg 1993;64:751-3.
3
Tosi AL, Ragazzi M, Asioli S, et al. Breast tumor resembling the tall
cell variant of papillary thyroid carcinoma: report of 4 cases with
evidence of malignant potential. Int J Surg Pathol 2007;5:14-9.
4
Jung SJ, Chung JI, Park SH, et al. Thyroid follicular carcinoma-like
tumor of the kidney: a case report with morphologic, immunohistochemical and genetic analysis. Am J Surg Pathol 2006;30:411-5.
5
Sterlacci W, Verdofer I, Gabriel M, et al. Thyroid follicular carcinoma-like renale tumor: a case report with morphologic, immunophenotypic, cytogenetic and scintigraphic studies. Virchows Arch
2008;452:91-5.
6
Amin MB, Gupta R, Ondrej H, et al. Primary thyroid like follicular
carcinoma of the kidney: report of six cases of a histologically distinct
adult renal epithelial neoplasm. Am J Surg Pathol 2009;33:393-400.
7
Foucar E, Kaplan LR, Gold JH, et al. Well differentiated peripheral
cholangiocarcinoma with unusual clinical course. Gastroenterlogy
1979;77:347-53.
226
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Problems in neuropathology
Moderators: F. Giangaspero (Roma), G. Cenacchi (Bologna)
Glioneuronal tumors with leptomeningeal
dissemination
Marina P. Gardiman, Matteo Fassan.
Department of Diagnostic Medical Sciences and Special Therapies,
Pathology Unit, University of Padova, Padova (PD), Italy
Background. Glioneuronal tumors are a group of primary
brain neoplasm of relatively recent acquisition in the World
Health Organization (WHO) Classification of Central Nervous
System (CNS) tumors which has been recently expanded with
new recognized entities such as rosette-forming tumor of the
fourth ventricle, the papillary glioneuronal tumor and rosetted glioneuronal tumor/glioneuronal tumor with neuropil-like
islands 1. Glioneuronal tumors are characterized by a biphasic
neurocytic and glial population. The neuronal component consists of synaptophysin-positive neurocytes with round nuclei
and clear cytoplasm occasionally intermingled with neurons
and intermediated-size “ganglioid” cells, whereas the glial
component exhibits features of glial fibrillary acidic protein
(GFAP) positive astrocytes. The histogenesis of these tumors
is unclear, but an origin from multipotent precursors capable
of divergent differentiation has been suggested 2.
Leptomeningeal dissemination in glioneuronal tumors is very
rare, but the incidence in low grade gliomas (a name for a
wide variety of neoplasm of glial or mixed glial-neuronal
origin) is estimated at 5% at diagnosis and 7-10% at progression. Among neoplasm of astrocytic origin, it is well known
that pilocytic astrocitoma can disseminate 3 but also a new
codified glial neoplasm in the 2007 WHO Classification of
tumors of the SNC, such as pilomyxoid astrocitoma, shows a
characteristic high tendency to disseminate.
The spreading along the subarachnoidal spaces of cerebrospinal fluid (CSF)of glioneuronal neoplasms has been reported
in the last few years more frequently than in the past probably
related to the more diffuse use of magnetic resonance imaging (MRI) in tumor staging and follow-up. Well-established
examples of glioneuronal tumors with leptomeningeal dissemination include ganglioglioma and pleomorphic xantoastrocitoma 4.
In diagnostic practice is still possible to encounter glioneuronal tumors that cannot be placed into any of the well-defined
WHO categories despite this growing list of entities. We have
recently published four pediatric cases of diffuse leptomeningeal tumors which cannot easily be classified in the currently
used CNS WHO classification, but have the histological and
immunohistochemical criteria to be considered as glioneuronal tumors.
Methods. Cases were retrieved from the Institutional files of
the authors. One case had been previously reported as spinal
low-grade neoplasm with diffuse leptomeningeal dissemination 5. Pathology reports and the histological slides were reviewed. Immunohistochemical analysis was performed using
the standard avidin-biotin-peroxidase method. Fluorescence
in situ hybridization (FISH) was performed on formalin-fixed,
paraffin-embedded tissues of one of the considered cases.
Clinical findings: the children were all admitted to the Padova University Hospital with the symptoms and sign of
hydrocephalus (morning headache, vomiting and increased
cranial circumference). Neuroradiological findings: tumors
were characterized by similar radiological appearance, i.e.
contrast-enhanced head and spine magnetic resonance images
revealed a tetraventricular communicating hydrocephalus, a
diffuse cerebral and spinal leptomeningeal enhancement, a
marked progressive cortical and subcortical cystic involvement of the cerebellum, basal temporal and frontal lobes,
brainstem and spinal cord without evidence of a primary
intraparenchymal mass.
Results. In all cases a dural biopsy was performed. Minute
samples characterized by a pearly opacified surface and
an increased consistence were obtained. The histological
samples showed thickened desmoplastic leptomeninges with
sclerohyaline bands and enlarged capillary-sized blood vessels diffusely infiltrated by a monotonous population of cells
arranged in straight lines or in small lobules within a compact
to loosely fibrillary stroma. Cells were characterized by round
to oval nuclei with finely granular dispersed chromatin, inconspicuous nucleoli with clear oligodendrocyte-like features
with perinuclear haloes. No mitosis, necrosis, calcifications,
lymphoid infiltration, myxoid changes or endothelial vascular
proliferation were observed. No Rosenthal fibers, nor rosettes
or pseudorosettes were detected.
Tumor cells showed diffuse immunoreactivity for synaptophysin and S100, patchy reactivity for GFAP and negative for
neurofilaments or epithelial membrane antigen. Proliferation
index, as percentage of MIB1-positive cells [MIB1 labeling
index (MIB1 L.I.)], was always less than 1%.
Only in case #3, after a first dural biopsy (performed in
2002), we obtained a significative sample of tissue from the
lesion appeared on the inner surface of the lateral ventricle
frontal horn (2007). The analyzed sample was composed by
a biphasic architecture. The more differentiated part of the
tumor was abutting in the ventricle lumen and composed by
uniform small cuboidal cells with round nuclei and scant clear
cytoplasm intermingled with “ganglioid” cells occasionally
arranged in perivascular pseudorosettes or pseudopapillary
structures.
Additional features include fibrillary areas mimicking neuropil and rare foci of microvascular proliferation of the capillary-sized blood vessels. The inner part of the tumor showed
anaplastic histological features with increased cellularity and
a diffuse honeycomb pattern of growth. The neoplastic oligodendrocyte-like cells, diffusely infiltrating the brain parenchyma and showed mild polymorphism with hyperchromatic
nuclei. Endothelial proliferation in the branching capillaries
was evident. No tumoral necrosis was observed. An increased
mitotic activity (three mitotic figures (10 high-power field)
with an MIB1 L. I. higher than 5% was detected. FISH
analysis revealed deletion of 1p, whereas 19q was intact. A
significant number of nuclei were immunopositive for Neu-N.
Tumor cells were also diffusely synaptophysin positive. Scattered cells were GFAP positive.
Discussion. The main histological characteristics of these
tumors affecting our four pediatric patients deserve special
considerations. On microscopic examination, these tumors
were composed by cells characterized by round to oval nuclei
with inconspicuous nucleoli with clear oligodendrocyte-like
cytoplasm. These histological findings might have favored the
diagnosis of oligodendrogliomas and oligodendrogliomatosis
in some of the similar cases presented in the Literature 6 7.
227
Lectures
Moreover, the diagnosis of oligodendrogliomas was achieved
only on cytologic criteria, that is, clear cytoplasm and round
nuclei caused by the lack of specific markers for oligodendrogliomas. As previously described in a case of diffuse leptomeningeal oligodendroglioma 7, we found the deletion of 1p
in one of our cases. This deletion is neither pathognomonic
of oligodendroglioma 8 nor particularly frequent in pediatric
cases that usually do not show 1p/19q co-deletions 9. In oligodendrogliomas, synaptophysin immunoreactivity is usually
caused by residual parenchyma and is frequently seen at the
infiltrating tumor borders. In our cases, the constant immunohistochemical profiles observed (i.e.: the positive reactivity
for synaptophysin and Neu-N) strongly suggest a glioneuronal
commitment of the neoplasm.
Also, neurocytomas and dysembrioplastic neuroepithelial
tumor (DNT) show similar histological/immunophenotypical profile 10, but in our cases a common characteristic was
the absence of a primary neoplastic mass in contrast with the
pathological evidence of the other glioneuronal tumors (i.e.:
DNT, extraventricular neurocytoma, papillary glioneuronal
tumor and rosette-forming glioneuronal tumor) 1.
A possible explanation about the origin of these diffuse leptomeningeal tumors could be isolated groups of glioneuronal
progenitor cells entrapped in the context of the leptomeninges during the primitive migration. These embryonal cells
could be able of divergent differentiation with neuronal,
oligodendroglial and astrocytic features 11. In fact, cases of
morphologically classic oligodendroglioma with neurocytic
rosettes or neurocytomas arboring 1p/19q deletion have been
described 11 12. The description of these entities suggests a
histogenetic overlap between oligodendroglioma and extraventricular neurocytoma 11, and further supports the existence
of a new “superfamily” of tumors with oligodendroglial and
neurocytic potential in which our series of diffuse leptomeningeal glioneuronal tumors could be included.
Interestingly, in the other similar cases presented in the literature, but considered as diffuse leptomeningeal oligodendrogliomas, the immunohistochemical profiles are quite variable
and sometimes inconsistent which could be related to the
glioneuronal nature of the described neoplasms, and further
indicate the difficulty to classify these types of tumors.
Three of four patients are alive up to 2 years of follow-up, following minimal to no clinical intervention, and these data suggest these tumors as neoplasm with a slow progressive and quite
indolent course. However, in case #3, the subsequent appearance
of a bulking neoplastic intraventricular lesion with anaplasia,
high mitotic index and focal vascular endothelial proliferation
suggests a potential aggressive biological transformation.
In conclusion, we hypothesized that the tumors affecting the
children we described represent a new nosological entity characterized by: i) intense enhancement of subarachnoidal space
with cystic lesions; ii) diffuse leptomeningeal infiltration by
glioneuronalcells without a primary mass; and iii) quite indolent course.
For these reasons, this group of neoplasms could be descriptively named “diffuse leptomeningeal glioneuronal tumors.”
Further studies and larger validation are needed to test our
hypothesis and to consider “leptomeningeal glioneuronal
tumors” as a distinct nosological entity in the SNC tumor
classification.
References
1
Louis DN, Ohgaki H, Wiestler OD, et al. WHO Classification of Tumours of the Central Nervous System, 4th ed. Lyon: IARC:2007.
2
Allende DS, Prayson RA. The Expanding Family of Glioneuronal
Tumors. Adv Anat Pathol 2009;16:33-9.
3
4
5
6
7
8
9
10
11
12
Kreiger PA, Okada Y, Simon S, et al. Losses of chromosomes 1p
and 19q are rare in pediatric oligodendrogliomas. Acta Neuropathol
2005;109:387-92.
Passone E, Pizzolitto S, D’Agostini S, et al. Non-anaplastic pleomorphic xantoastrocitoma with neuroradiological evidences of leptomeningeal dissemination. Childs Nerv Syst 2006;22:614-8.
Perilongo G, Gardiman M, Bisaglia L, et al. Spinal low-grade neoplasms with estensive leptomeningeal dissemination in children.
Childs Nerv Syst 2002;18:505-12.
Armao DM, Stone J, Castillo M, et al. Diffuse leptomeningeal oligodendrogliomatosis: radiologic/pathologic correlation. Am J Neuroradiol 2000;21:1122-6.
Bourne TD, Mandell JW, Matsumoto JA, et al. Primary disseminated
leptomeningeal oligodendroglioma with 1p deletion. J Neurosurg
2006;105:465-9.
Brandes AA, Tosoni A, Cavallo G, et al. Correlation between O6methylguanine DNA methyltransferase promoter methylation status,
1p and 19q deletions and response to temozolomide in anaplastic
and recurrent oligodendroglioma: a prospective GICNO study. J Clin
Oncol 2006;24:4746-53.
Kreiger PA, Okada Y, Simon S, et al. Losses of chromosomes 1p
and 19q are rare in pediatric oligodendrogliomas. Acta Neuropathol
2005;109:387-92.
Yamamoto T, Komori T, Shibata N, et al. Multifocal neurocytoma/
gangliocytoma with extensive leptomeningeal dissemination in the
brain and spinal cord. Am J Surg Pathol 1996;20:363-70.
Perry A, Scheithauer BW, Macaulay RJB, et al. Oligodendrogliomas
with neurocytic differentiation. A report of 4 cases with diagnostic and
histogenetic implication. J Neuropathol Exp Neurol 2002;61:947-55.
Perry A, Fuller CE, Banerjee R, et al. Ancillary FISH analysis for 1p
and 19q status: preliminary observations in 287 gliomas and oligodendroglioma mimics. Front Biosci 2003;8:a1-9.
Prognostic factors in meningiomas
V. Barresi
Department of Human Pathology, University of Messina, Italy
Meningiomas account for approximately 24-30% of primary
intracranial tumors; they occur most commonly in middleaged and elderly patients and show a female predominance 1.
According to the WHO classification system, meningiomas
are classified into several histotypes, the most common of
which are represented by meningothelial, fibrous and transitional meningiomas 1. Moreover, three histological grades
of increasing malignancy are recognized for these tumours 1,
with most of meningiomas falling into grade I and presenting
as indolent neoplasias 1.
The main prognostic questions regarding meningiomas involve prediction of recurrence and, for malignant variants,
prediction of survival. The most important clinical factor
in recurrence risk is represented by the extent of surgical
resection, which is influenced by tumor site, extent of invasion and by the attachment to vital intracranial structures.
According to Simpson’s scale 2, the degree of surgical resection is commonly classified into five grades (grade 1: macroscopically complete removal, including dura and bone;
grade 2: macroscopically complete removal, with apparently
reliable coagulation of dural attachments; grade 3: macroscopic complete excision of the solid tumor, but insufficient
dural coagulation or bone excision; grade 4: partial removal
of the tumor; grade 5: simple decompression), displaying
increasing recurrence risk. A major issue relates to the recurrence of totally resected (Simpson’s grade 1) meningiomas
which, in spite of total macroscopic removal including dura
and bone, display a recurrence rate around 10% 2. It has been
hypothesized that the development of recurrences of these
meningiomas may be related to the presence of microscopic
clusters of neoplastic cells left in the dura mater or in the
228
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
arachnoid membrane 3 4 in relationship to their biological
activity.
In the last years a number of studies has been carried out
in order to evidence histo-prognostic factors able to predict
the clinical course of meningiomas. At now, the histological
grade and the proliferation index are considered to be the
most powerful histological prognosticators for the outcome
of these neoplasias 5. Indeed, grade I meningiomas display
recurrence rates of 7-25%, atypical meningiomas recur in
29-52% of cases and anaplastic variant at rates of 50-94% 5.
Moreover, a mitotic index higher than 4 mitoses/10 HPF has
been significantly associated with 8-fold higher recurrence
rates of meningiomas 6; similarly, Ki-67 labeling index has
been shown to significantly correlate with the prognosis of
these tumors 5 7.
The absence of progesterone receptors expression is also regarded as a significant prognostic factor for the development
of recurrences in meningiomas 8, but only in association with
high mitotic index and histological grade. High vascularity
and peri-tumoral vasogenic oedema have been also proposed
as significant prognostic factors correlated with adverse
clinical course of meningiomas 5. In recent years our group
has evaluated the prognostic role of neo-angiogenesis and
its regulators on the outcome of meningiomas, showing that
a high quantity of tumour neo-angiogenesis, reflected by a
high microvessel density (MVD) appears to be significantly
associated with a shorter overall survival and with the development of recurrences in totally resected neoplasias. In
addition, according to our findings, a high ratio between the
concentrations of the pro-angiogenic vascular endothelial
growth factor (VEGF) and the anti-angiogenic semaphorin3A
(SEMA3A) in the microenvironment of the tumour behaves
as a negative predictor of recurrences in meningiomas. We
may hypothesize that neo-angiogenesis is blocked or stimulated depending on the prevalence of VEGF or SEMA3A and
that microscopic not-removed neoplastic foci may grow and
give rise to recurrent tumours for their higher capability to
stimulate proliferation and neo-angiogenesis in relationship to
VEGF/SEMA3A balance in favour of VEGF.
Finally, in our Department the prognostic role of proteins involved in the regulation of neoplastic growth and progression
has been also tested in meningiomas. Basing on our results,
high expression of caveolin-1, a 22 KDa protein which stimulates the proliferation of neoplastic cells, or that of matrix
metalloproteinase-9, an enzyme involved in the invasive potential of tumour cells, appear as negative prognostic factors
for meningiomas. On the other hand, a low expression of the
CAAT-enhancer binding protein δ (CEBP/δ) seems to be associated with lower recurrence risk of these neoplasias.
References
1
Perry A, Louis DN, Scheithauer BW, et al. Meningiomas. In: Louis
DN, Ohgaki H, Wiestler OD, et al (eds). WHO Classification of
Tumors of the Central Nervous System. Lyon: IARCC press 2007,
pp. 164-72.
2
Simpson D. The recurrence of intracranial meningiomas after surgical treatment. J Neurol Neurosurg Psychiatry 1957;20:22-39.
3
Kamitani H, Masuzawa H, Kanazawa I, et al. Recurrence of convexity
meningiomas: tumour cells in the arachnoid membrane. Surg Neurol
2001;56:228-35.
4
Kinjo T, al-Mefty O, Kanaan I. Grade zero removal of supratentorial
convexity meningiomas. Neurosurgery 1993;33:394-9.
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Perry A, Stafford SL, Scheithauer BW, et al. “Malignancy” in meningiomas: a clinico-pathological study of 116 patients with grading
implications. Cancer 1999;85:2046-56.
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Perry A, Stafford SL, Scheithauer BW, et al. Meningioma grading: an
analysis of histological parameters. Am J Surg Pathol 1997;21:145565.
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Perry A, Stafford SL, Scheithauer BW, et al. The prognostic significance of MIB-1, p53, and DNA flow cytometry in completely resected
primary meningiomas. Cancer 1998;82:2262-9.
Perry A, Cai DX, Scheithauer BW, et al. Merlin, DAL-1 and progesterone receptor expression in clinico-pathologic subset of meningioma:
a correlative immunohistochemical study of 175 cases. J Neuropathol
Exp Neurol 2000;59:872-9.
Molecular alterations in embryonal tumors
of central nervous system
M. Gessi
Inst. of Neuropathology, University of Bonn Medical Center, Bonn,
Germany
Embryonal tumors of the CNS are described as malignant
small, round cell tumors with possible divergent patterns of
differentiation. According to the World Health Organization
classification (2007), this group of tumors includes: medulloblastoma, the most common embryonal tumor subtype,
the central nervous system primitive neuroectodermal tumor (CNS-PNET), and the atypical teratoid/rhabdoid tumor
(ATRT). Although they could share common light microscopy features, embryonal tumors appear to evolve by alterations
of a wide spectrum of genetic pathways.
The largest part of molecular research on embryonal tumors
has been focused on medulloblastoma biology, and over the
years, many aspects of signalling pathways regulating the
biology of this tumor, have been revealed. The most common
genetic alteration in medulloblastoma is the loss of chromosome 17p, often in association with isochromosome 17q:
i(17)(q10), occurring in 30-50% of cases. Although the precise role in medulloblastoma oncogenesis and its prognostic
significance are not known, the region 17p13.2-13.3 harbors
many tumor suppressor genes, including TP53. However,
while sporadic TP53 mutations are uncommon in medulloblastoma, germline mutations of TP53 gene, resulting in the
Li–Fraumeni syndrome, have been related to an increased
medulloblastoma incidence.
Numerous investigations have also demonstrated the pivotal
role of the sonic hedgehog (SHH) signaling pathway in medulloblastoma pathogenesis. Molecular alterations in components of the SHH pathway (i.e. the inactivating mutations of
PTCH1 and SUFU and/or activating mutations of SMO), have
been found in 15-20% of sporadic medulloblastomas. Moreover, patients with Gorlin’s syndrome (harboring germline
alterations in PTCH1 gene) present also an high incidence of
several tumor types, including medulloblastoma.
Alterations of the WNT signaling pathway have also been
implicated to the development of medulloblastoma: approximately 15 to 20% of sporadic medulloblastoma present
mutations in genes, including APC, AXIN-1, AXIN-2 or
CTNNB1-β-catenin, all members of the WNT pathway.
Genomic amplifications of n-Myc and c-Myc are commonly
encountered in medulloblastoma and characterize a subset of
clinically aggressive tumors, frequently with large cell/anaplastic histological features. Other molecular pathways, including the tyrosin-kinase family receptors Erbb, insulin-like
growth factor 1 receptor (IGF1R) and PDGFR, have been also
directly implicated in medulloblastoma pathogenesis.
Molecular genetic investigations and transcriptional expression profile analyses have shown that atypical teratoid/rhabdoid tumors (ATRT) are distinct from other embryonal
tumors. Inactivating deletions or mutations of the tumor suppressor gene hSNF5/INI-1, located in the chromosomal region
22q11.2, encoding a subunit of the SWI/SNF family of chro-
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matin-remodelling complexes, have been found in more than
75% of cases. Although its specific tumor suppressor function
remains still unknown, the alteration of the hSNF5/INI-1 is
now considered as a crucial step in the pathogenesis of most
ATRT and today its recognition is a powerful diagnostic tool
for the diagnosis of these tumors.
In contrast to ATRTs and medulloblastomas, various molecular pathways have been hypothesized to play a role in
the CNS-PNET pathogenesis but, due to the rarity and the
heterogeneity of these tumors, only a limited number of studies on large series of cases have been made. Alterations affecting genes of various molecular pathways (i.e. WNT, Tp53,
RASFF1A, n-Myc and c-Myc) have been also described in
CNS-PNET cases. Recently, new findings demonstrating the
implication of microRNAs (miR-517c and miR-520g) in the
biology of CNS-PNET and ependymoblastomas have been
reported.
In conclusion, many progresses in the molecular characterization of medulloblastoma and other embryonal tumors have
been made. However, these biological data are still the subject
of intensive translational research in order to define new tools
for the improvement of patients risk stratification procedures
as well as their management.
Surgical pathology of epilepsy
G. Marucci
Section of Pathology, Department of Haemathology and Oncological Sciences Section of Pathology, Bellaria Hospital, University of
Bologna, Italy
Background. Surgical approach has become a useful alternative to treat refractory epilepsy, with a postoperative favorable
outcome that in temporal lobe epilepsy accounts for about
70% of patients. Cases must be studied with a multidisciplinary approach that involves pathologists, neurosurgeons,
neurologists and neuroradiologists. First of all pathologist
should be present in operation room to better understand
the adopted surgical strategy and the correct orientation of
removed specimens. A large series of histopathological pictures may be found in such tissues: hippocampal sclerosis
(HS), focal cortical dysplasia (FCD), mild cortical dysplasia,
hamartomas, vascular lesions, low-grade glioneuronal tumors,
scars or gliotic lesions. Although numerous histochemical and
immunohistochemical stains have been proposed in literature,
in everyday practice Nissl, Kluver and anti-NeuN antiserum
could be considered the most easy and useful tools in histological diagnosis.
Methods. The lesions that typically are encountered in epilepsy surgery are represented by HS, FCD and heterotopias.
Neuropathological classification of HS proposed in 1992 by
Wyler et al. was based on a semiquantitative evaluation of cell
loss in the Ammon Horn subfields, resulting in a distinction
of five grades, although in routine classic and severe Ammon
Horn Sclerosis are the most frequent reported diagnosis. In
2007 Blümcke et al. recognized five patterns of HS adopting
a computerized cluster analysis, and applied the term of MTS
(mesial temporal sclerosis) 1A to histological pictures similar
to the classic Ammon Horn Sclerosis and the term of MTS 1B
to the severe Ammon Horn Sclerosis. Recently it has emerged
a growing interest in evaluating also the presence of alterations in Dentate Gyrus, in particular the so called granular cell
dispersion.
Presence of FCD are usually assessed following the scheme
proposed by Palmini et al., who distinguished type IA (iso-
lated architectural abnormalities), type IB (architectural abnormalities plus giant or immature neurons), type IIA (architectural abnormalities with dysmorphic neurons but without
balloon cells) and type IIB (architectural abnormalities with
dysmorphic neurons and balloon cells).
The term of heterotopia is applied to alterations of cortical
development in which apparently normal brain tissue is mislocated in abnormal sites. The most common subtype is represented by nodular heterotopia characterized by the presence of
heterotopic islands of grey matter into the white matter.
The other lesions found in these patients are not specific of
epilepsy surgery setting: regarding low-grade tumors a comparison between tumors operated with the so called tailored
resection (characterized by anterior-mesial temporal resection
along with amygdalohippocampectomy) and with simple lesionectomy was performed.
Finally in some cases a fresh 0,5 cm3 tissue sample from
Dentate Gyrus of the hippocampus has been collected immediately after removing for tissue culture.
Results. Between April 2001 and April 2010 110 patients
(52 males and 58 females) with drug resistant temporal lobe
epilepsy underwent epilepsy surgery in Bellaria Hospital,
Bologna. Histological examination has evidenced 15 cases of
hippocampal sclerosis, 20 cases of focal cortical dysplasia,
41 cases of hippocampal sclerosis associated to focal cortical
dysplasia (dual pathology), 27 cases of low grade tumor, 2
cases of nodular heterotopia, 3 cases of vascular lesions and
2 cases of encephalocele. A favorable post-surgical epilepsy
outcome was achieved in 77% of cases: in particular in 61%
of cases it was obtained a complete disappearance of seizures
(Engel Class IA). Furthermore it has been demonstrated a better seizure outcome for temporo-mesial glioneuronal tumors
associated with epilepsy in patients who underwent tailored
resection rather than simple lesionectomy. Finally surgical
approach makes available hippocampi not only for routine
histological examination but also for further studies. Adult
neural stem cells are undifferentiated cells that are present
in the adult brain and are capable to divide and differentiate
into astrocytes, oligodendrocytes and neurons. These cells are
present in the subgranular zone (SGZ) of the Dentate Gyrus of
the hippocampus and it has been demonstrated the possibility
to generate neurosphere from the SGZ.
Inflammatory myopathies
G Cenacchi
Dipartimento Clinico di Scienze Radiologiche e Istocitopatologiche,
“Alma Mater Studiorum” Università di Bologna, Italy
Background. The inflammatory myopathies (IM) are an
heterogeneous group of acquired disorders of skeletal muscle
with undefined etiology and pathogenesis. Inflammatory
myopathies can be subdivided in two main groups: infectious
myositis and immunogenic myositis. The autoimmune myopathies include polymyositis (PM), dermatomyositis (DM),
overlap syndromes, and inclusion body myositis (IBM). Recent findings have confirmed that PM is an uncommon, but
frequently misdiagnosed disorder: PM mimics many other
myopathies and remains a diagnosis of exclusion. Muscle
biopsy is the gold standard for the diagnosis; the histological
cornerstone is the identification of cellular infiltrates in muscle
tissue, however infiltrates are not always present. Induction of
Major Histocompatibility Complex class I (MHC-I) antigen
in muscle fibres precedes inflammatory infiltrates, persists
in chronic phase, and is unaffected by immunosuppressive
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
therapy so it is considered a good marker of IM. Many Authors consider only the sarcolemmal MHC-I staining even if
evidences that a reticular pattern of internal MHC-I reactivity
in fibres of myositis patients are reported.
Methods and Results. We revised 64 adult muscle biopsies
from a file of the Pathology Department of the Azienda Ospedaliera-Universitaria S. Orsola-Malpighi to evaluate the
diagnostic role of both immunohistochemistry for MHC-I
stain (samples were scored by two independent and blinded
investigators and an average of 580 fibres were evaluated
for each biopsy. The percentage of MHC-I internal labelled
fibres was determined and interobserver reproducibility was
evaluated) and transmission electron microscopy. The positive muscle fibres displayed MHC-I staining of the cytoplasm
rather than of the sarcolemma. Positive fibres were observed
in all samples (21 IM cases and 43 controls). Interobserver reproducibility was moderate (K = 0,568). The specificity of the
test was of 100% when the percentage of the internal labelled
fibres was higher than 50%, as mean of the two observers. Ultrastructural studies are necessary in many cases, especially in
IBM, by screening other myopathies with inflammation, such
as dystrophies, toxic and metabolic myopathies. IBM or dystrophies, especially without a positive family history, can be
diagnosed as PM and can therefore be treated unsuccessfully
and unnecessarily for many years, thereby exposing patients
to the long-term side-effects of prednisolone and immunosuppressant. In sporadic IBM, in addition to autoimmune
inflammation, there are degenerative features characterized
by vacuolization and accumulation of stressor and amyloid-related molecules. The diagnosis of IBM rests on morphological
criteria including inflammatory infiltrates with mononuclear
cell invasion of non-necrotic muscle fibers, rimmed vacuoles
and either intracellular amyloid or inclusions consisting of 1518 nm filaments at the ultrastructural level. In PM, multifocal
lymphocytic infiltrations invade healthy muscle fibers: in addition to primary inflammation there are vacuolated muscle
fibers containing lamellar membranous residues and amorphous electron dense material. When the intramuscular blood
vessels show endothelial hyperplasia with tubuloreticular
profiles, fibrin thrombi and obliteration of capillary lumina,
the diagnosis may be DM.
Conclusions. This review outlines the fundamentally different pathology between different IM as evolved the past few
years, provides a critical analysis of the diagnostic markers,
and summarizes the most significant developments on their
pathogenesis as relate to therapeutic strategies.
Slide seminar: Epatic pathology
Moderators: G. Faa (Cagliari), L. Terracciano (Basilea)
Colangite sclerosante e pancreatite
autoimmune
L. Terracciano
Department of Patology, University Hospital, Basel, Switzerland
Case history. A 51 year-old man was referred to University
Hospiatl Basel, Switzerland,with abdominal pain, jaundice
weight loss and diarrhea as presenting symptoms. Laboratory
examinations showed an increase of transaminases and cholestatic parameters, A liver biopsy was performed and a diagnosis of sclerosing cholangitis was rendered. Two months later
because of persisting abdominal pain and diffuse enlargement
of the pancreas on imaging, pancreas carcinoma was suspected. The patient underwent a duodeno-pancreatic resection. Histology was consistent with autoimmune pancreatitis.
4 months later a further liver biopsy showed the full-blown
picture of autoimmnue sclerosing cholangitis.
Primary sclerosing cholangitis is a cholestatic disease characterized by patchy inflammation, fibrosis, and stricturing of
the intrahepatic and/or extrahepatic bile ducts.1 The diagnosis
of primary sclerosing cholangitis is based on characteristic
cholangio-graphic findings, in combination with clinical, biochemical, and histological features.
The disease lacks a definitive etiological factor, although a
strong association with inflammatory bowel disease is well
recognized.
Autoimmune pancreatitis (AIP) is a recently recognized clinicopathological entity, which was first described by Sarles in
1961 as a “chronic inflammatory sclerosis of the pancreas”
of possible autoimmune pathogenesis associated with hypergamma-globulinemia. The disease has been gaining new
attention for the last two decades, and the term “autoimmune
pancreatitis”, coined by Yoshida in 1995, has only recently
been widely accepted in the scientific literature. Due to the
possible involvement of the biliary tract, the term autoimmune
pancreatocholangitis (AIPC) has been introduced. The main
reasons for the rising interest in investigating AIPC reside in
its increasing frequency, partly due to an increased awareness
of the disease but also due to a potentially increased incidence
in the last 20-30 years, its not yet clarified aetiology and
pathogenesis and ist still undefined clinical spectrum. The
coexistence of AIPC with other autoimmune-related diseases,
such as Sjo¨gren’s syndrome, inflammatory bowel diseases
(IBD)
and rheumathoid arthritis, the presence of immunologic abnormalities in subsets of patients (hypergammaglobulinemia,
elevated serum IgG4 levels, presence of auto-antibodies),
and the association with a specific HLA-haplotype in the
Japanese population, represent the main pieces of evidence
of an autoimmune pathogenesis of the disease. All lesions
incorporated into the spectrum of the disease, including the
pancreatic manifestations are characterized by a plasma cellrich, often mass-forming inflammatory process with numerous IgG4-positive plasma cells. Altough very similar to primary sclerosding cholangitis, IgG4 sclerosing cholangitis not
rarely show peculiar histological features. As in the pancreas,
biliary involvement by IgG4-related autoimmune disease can
be diffuse or localized, producing either a generalized but irregular thickening or a tumefactive lesion. The histological
appearance is similar in both situations: lymphoplasmacytic
inflammation, fibrosis (often with a swirling or storiform arrangement) and obliterative phlebitis. Despite the dense periluminal inflammation, the biliary epithelium is usually intact.
This is in distinct contrast to PSC, which often produces mucosal erosion. In another contrast with PSC, the inflammatory
process is often more dense at the periphery of the duct. This
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phenomenon is partly due to dense inflammation in the walls
of periductal vein branches, but lymphoplasmacytic inflammation around nerve twigs and forming nodular infiltrates in
periductal soft tissue are also characteristic features of IAC.
While lymphocytes and plasma cells predominate, eosinophils
can be numerous and are occasionally numerically dominant.
Neutrophils, commonly seen in PSC, are not a feature of
IgG4 cholangitis. Immunohistochemical staining for IgG4 is
a useful tool for confirming the diagnosis of IgG4 cholangitis.
Accumulating evidence suggests that the bile duct lesions and
the concomitant pancreatitis in patients with IgG4 cholangitis
improve with corticosteroid treatment which distinguishes
IgG4 cholangitis from PSC.
Post liver transplant complications (PLTC):
recurrence of hepatitis (RH) or cellular rejection
(CR)?
E. David
Anatomia Patologica, II Az. Osp. Molinette Torino
PLTC are constituted by a various group of diseases that are
crucial for the clinical management of patients in liver transplantation (LT).
Liver biopsy (LB) is the gold standard for diagnosis of rejection. The search of clinico-pathological correlations, advantaging of the sequential evaluation of follow-up biopsies,
represent the most valuable working method.
Frequently, different pathologic processes are present on the
same LB. As a matter of fact, most chronic liver diseases can
recur in the graft, and LBs may display features that require
differential diagnosis between (acute or chronic) CR and de
novo conditions such as de novo autoimmune hepatitis, drug
toxicity and vascular lesions.
We recommend the slides to be initially examined by the
pathologist blindly to clinical data, a diagnosis or possible
differential diagnoses being formulated, then histology to be
compared with the available clinical data and the final diagnosis to be discussed with the physicians. Pathologists must be
familiar with atypical presentation of PLTC and aware to recognize the primary process that has to be primarily treated.
PLTC are traditionally distinguished as early and late events,
but both RH and CR can occur early.
We propose to distinguish three broad categories of PLTC:
RH, CR and de novo diseases.
Rejection can be distinguished in early acute CR, late CR
with“atypical” features, chronic rejection and antibody-mediated rejection. Acute CR is the commonest cause of early graft
dysfunction, its incidence ranging from 24 to 80%. A RAI
(Rejection Activity Index) may be used, with an histological
scoring system from 0 to 9.
HCV related- cirrhosis represents a very common indication
for LT and unfortunately the recurrence of HCV infection is
universal and immediate. HCV RH occurs in up to 90% of
patients at 5 years from transplantation, nevertheless some
patients will present an indolent course, whereas others will
rapidly progress to cirrhosis. Compared to non-transplanted
HCV patients who develop cirrhosis at a rate of less than 5%
over 5 years, the course of post-transplant recurrent HCV is
accelerated with up to 20- 40% progressing to cirrhosis within
5 years. Factors influencing the prevalence and severity of
disease recurrence include: the virus genotype,the host immunogenetic background and the immunosoppressive treatment.
Both hepatitis B and D relapse, but prophylactic measures
have significantly decreased their recurrence.
Differential diagnosis between acute CR and RH is crucial on
LB because immunosuppresive treatment is associated with
an increased risk of allograft cirrhosis and mortality.
Histologically, acute CR is characterized by a various combination of features of predominantly mononuclear (including
blastic or activated lymphocytes, neutrophils and eosinophil)
portal inflammation, of subendothelial inflammation in portal
and/or terminal hepatic veins, and of bile duct inflammation
and damage.
Lobular necroinflammatory activity and interfacie inflammation with ductular reaction is usually more prominent in RH
than in CR. But
CR and RH may coexist, and in such cases it is mandatory to
identify the predominant process to be treated.
In protocol LB, (hepatitis-like) necroinflammatory lesions
occur in 40% of adult LT recipients after 12 months from
transplantion and in 60% of pediatric patients after 10 years,
whit normal serological tests. The possible causes of this
idiopathic post transplantation hepatitis include: atypical
rejection, de novo autoimmune hepatitis and infection from
unknown agents. A significant percentage of these patients
may show progression to cirrhosis without significant liver
test abnormalites.
In conclusion, LB may represent a diagnostic challenge for
the pathologists, nevertheless a systematic approach to morphological analysis of liver lesions can satisfactorily identify
or give the clue for diagnosis of clinical syndromes such as
immuno reactions (rejection), hepatitis, cholestasis, drug toxicity, and for prognostic staging of evolutive PLTC.
A focal liver lesion in a young body-builder
M. Roncalli, L. Di Tommaso, A. Destro *, E. David **,
L. Terracciano ***
Department of Pathology University of Milan School of Medicine &
IRCCS Humanitas Clinical Institute, Rozzano, Milan, Italy; * Molecular Genetic Laboratory, IRCCS Humanitas Clinical Institute, Rozzano, Milan, Italy; ** Anatomia Patologica II° Azienda Ospedaliera
Molinette, Torino, Italy; *** Institute of Pathology, University Hospital
Basel, Basel, Switzerland
Clinical history. A 35 years old asymptomatic man with a
history of anabolic steroid intake (body builder) underwent
a surgical resection of a 6.5 cm. focal liver lesion located in
II-III liver segments. The lesion was incidentally discovered
after a routine US of the liver. Grossly the lesion appeared as a
greenish, unencapsulated nodule of 6.5 cm, with well-defined
margins and located in the context of an otherwise unremarkable parenchyma. The nodule margins were close to those of
the surgical resection. Microscopical examination revealed a
well differentiated hepatocellular proliferation composed by
hepatocytes with focally increased N/C ratio and organized in
trabecular and small acinar structures.
The main diagnostic issue was hepatocellular adenoma vs
well differentiated hepatocellular carcinoma. A number of
histochemical, immunocytochemical and molecular studies
were carried out to address the diagnostic issue.
The patient is alive and well 6 years after the original diagnosis.
The discussion will focus on the differential diagnosis and on
the possible pathogenetic links between liver cell adenoma
and carcinoma.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Wednesday, September 22nd, 2010
Molecular diagnosis of solid tumours.
A practical approach for organ pathologies
Molecular diagnosis in colorectal cancer
A. Scarpa
Department of Pathology and ARC-Net Research Center, Verona University Hospital, Verona, Italy
Colorectal cancer is a disease whose moleclar basis are clearer
than those of many other cancers. Besides a precise histologic
diagnosis and pathological staging, the pathologist can provide a molecular characterization of a colorectal neoplasia,
thus permitting to 1) unveil the existence of a hederitary
syndrome, 2) help assessing prognosis, 3) predict response
to therapy.
Hereditary cancer syndromes account for 1-5% of all colorectal neoplasms. The main forms are the familial adenomatous
polyposis (FAP) in its classic variant, due to mutations in the
APC gene, and its attenuated form with biallelic mutations of
MYH gene (MAP) and the nonpoliposyc forms as HNPCC
due to mutations in mismatch repair genes (MLH1, MSH2,
MSH6, PMS2). The cost/benefit of sequencing of entire genes
is too high to permit the analysis of patients with colorectal
cancer. One of the main objectives is therefore to stratify
colorectal cancer patients according to different levels of
genetic risk using a stepwise procedure. The first approach to
help identifying hereditary syndromes is clinical and resides
in the study of the phenotype and the genealogic tree that is of
great help for FAP and MAP. For HNPCC a major help also
comes from the analysis of the tumor samples for the presence
of the molecular phenomenon called microsatellite instablity
(characterizing the vast majority of HNPCC cancers) and/or
for the immunohistochemical expression of mismatch repair
proteins. The lack of expression of one of the proteins suggests the presence of a somatic homozygous mutation in cancer cells and indicates which gene is to be sequencedd to find
a germline mutation. Finding a germline mutation defines the
the follow-up for the patient and the surveillance programme
for the family.
Microsatellite instabilty is also observed in 10-15% of sporadic colorectal cancers and is due to the somatic inactivation of
a mismatch repair gene, more frequently MLH1. Patients with
sporadic colorectal cancers of this molecular phenotype show
a longer survival especialy in Stages II and III. Wheter or not
these patients benefit from adjuvant therapies remains to be
determined. An additional prognostic indiocator in colorectal
cancer is the presence of P53 mutations that seems to be associated with a worse prognosis, higher risk of metastasis and
resistance to chemio and radiotherapy.
The recent development of targetted drugs specifically inhibiting the receptor of the epidermal growth factor (EGFR) as
cetuximab or panitumab is giving new hopes for the treatment
of metastatic colorectal cancer (mCRC) resistent to standard
chemotherapy. Various studies have unequivocally shown
that KRAS mutational status is able to predict response to
anti-EGFR therapy in patients with mCRC. Recently the
American Society of Clinical Oncology (ASCO) and the
Agenzia Italiana del Farmaco (AIFA) have suggested that
all patients with mCRC who are candidates for anti-EGFR
therapy must have the KRAS status assessed and in the case
a mutation in codons 12 and 13 should not be subjected to
therapy. The sequencing analysis of DNA prepared from
paraffin embedded tissues is highly efficient pending a cancer
cell enrichment to more than 60%. In our experience on the
latest 375 patients with mCRC, we observed that 163 (43.5%)
had a KRAS mutation (162 in codons 12 and 13, 1 in codon
22), 5 (1.5%) were not PCR amplifiable and 207 (55%) had a
wild type KRAS sequence.
Analysis of genes frequently mutated in CRC is also effective in selecting candidate patients for treatment with antiEGFR drugs cetuximab and panitumumab. Approximately
40% of CRC patients harbour a K-Ras mutation conferring
resistance to anti-EGFR drugs. Of the remaining 60% with
a wild type K-Ras tumor, 5-10% carry the B-Raf activating
mutation V600E, which also negates response to these agents
and dictates a very poor prognosis. This prompted the search
and discovery of a novel selective B-Raf inhibitor targeting
tumors with V600E mutation. An additional 20% of K-Ras wt
CRC patients are resistant to anti-EGFR drugs, due to activating mutations in exons 9 and 20 of PIK3CA, thus providing
the rationale to test novel agents targeting PI3K/Akt pathway.
More recently, a signature of 6 genes among a 57 gene set
was associated with response to cetuximab among Ki-Ras wt
CRC patients.
In the same fashion, in gastric cancer the identification of
genetic lesions such as PIK3CA mutations or HER2 amplifications, reported in 15% of tumors, may allow treatment with
targeted agents not otherwise indicated for this disease.
In pancreatic cancer, a recent global genomic analysis revealed 12 cell signalling pathways altered in 67-100% of
tumors, including among the others, genes such as Hedgehog,
TGFß and Wnt/Notch, for which novel targeted agents are
now available. In addition, the identification of BRCA2 mutations, which hamper DNA repair efficiency, provides the
opportunity of a synthetic lethality therapeutic approach by
combining PARP inhibitors with DNA-damaging agents such
as platinum derivatives.
The large body of information emerging from cancer gene/
protein expression profiles is making a major contribution in
the clinically efficient sub-classification of cancers. This will
further help in the selection of patients, through the use of
reliable biomarkers, who may benefit from specific targeted
agents or chemotherapeutics.
In several types of GI cancers, particularly colorectal cancer
(CRC), analysis of a limited set of genes currently allows
more tailored treatment. Along with the formerly reported Oncotype DX colon, a novel 38 gene signature named Coloprint,
can predict prognosis in stage II and III CRC patients, identifying those more likely to benefit from adjuvant treatment.
Finally, genotyping profiles are providing useful information,
to more specifically predict activity and toxicity of several
previously available chemotherapeutics currently used in GI
cancer.
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Thursday, September 23rd, 2010
Colon neoplasms
Deficit of DNA mismatch repair:
diagnostic algorithm and clinical implications
G. Lanza, I. Maestri, L. Ulazzi, R. Gafà
Sezione di Anatomia Patologica, Università di Ferrara, Ferrara, Italia
Nearly 15% of colorectal carcinomas (CRCs) display microsatellite instability (MSI or MSI-H, high frequency MSI)
caused by impairment of the DNA Mismatch Repair (MMR)
system. The distinction between MMR-proficient (MMRp)
and MMR-deficient (MMRd) tumors represents a fundamental step in the molecular classification of CRC with important
clinical implications.
Most MSI-H CRCs (70%) are sporadic and in these tumors
inactivation of the MMR system is determined by somatic promoter methylation of the MLH1 gene. The remaining MSI-H
CRCs are hereditary (Lynch syndrome) and produced by germline mutations of a MMR gene (MLH1, MSH2, MSH6, and
more rarely PMS2) with somatic inactivation of the second wild
type allele. It has been consistently demonstrated that inactivation of the MMR genes is associated with immunohistochemical loss of expression of the corresponding protein. In addition,
as MMR proteins work as heterodimers, abnormalities of the
obligatory partners (MSH2 and MLH1) will result in degradation of their dimers and concurrent loss of expression of both
the obligatory and secondary partner proteins (MSH2/MSH6
and MLH1/PMS2). Conversely, abnormalities in genes of the
secondary partner proteins (MSH6 and PMS2) will determine
selective loss of MSH6 and PMS2 expression, respectively, as
their function is compensated by other proteins. Therefore, immunohistochemical analysis of MMR proteins espression represents a rapid and reliable test for the identification of MMRd
colorectal tumors, also indicating the gene that is most likely
inactivated. Many studies demonstrated an excellent correlation of the results obtained by immunohistochemistry and MSI
analysis. Only a small fraction of hereditary cases with missense mutations (generally of MLH1) leading to nonfunctional
proteins with maintained antigenicity might result MSI-H with
normal expression of the MMR proteins.
Lynch syndrome accounts for 2-3% of all CRCs. MSI testing and immunohistochemical analysis of MMR proteins
expression are worldwide employed for the identification of
colorectal cancer patients with presumptive Lynch syndrome,
to be tested for MMR genes germline mutations. It is recommended that MSI or MMR protein expression analyses should
be carried out on tumors from patients clinically at high risk or
selected on the basis of the revised Bethesda guidelines. However, molecular screening investigations performed on large
series of unselected surgically removed colorectal cancers
indicated that a large fraction of Lynch syndrome cases should
be unrecognized using common criteria of selection. These
data suggest that screening of all CRCs for MSI or abnormal
MMR protein expression should be a more effective approach
for the identification of hereditary cases.
Recent studies showed that sporadic MSI-H MLH1-negative
tumors frequently harbour BRAF V600E gene mutations.
Conversely, BRAF mutations have not been detected in
MSI-H MLH1-negative tumors from patients with Lynch syndrome. Also in our experience BRAF gene mutation analysis
could be employed as an aid for discriminating hereditary
from sporadic MLH1-negative MSI-H carcinomas.
MMR status has been clearly demonstrated to be an independent prognostic indicator in colorectal cancer. Patients with
stage II and III MSI carcinomas display higher survival rates
with respect to patients with non-MSI tumors. In addition,
emerging data suggest that patients with MSI tumors don’t
have significant benefit from adjuvant 5-fluorouracil-based
chemotherapy. Although the use of MMR status assessment
as a prognostic and predictive test has not yet been validated
and incorporated into clinical practice, it is advisable to perform this analysis in stage II colon cancer patients. Owing to
the favourable outcome and lack of benefit of current standard
treatment, patients with stage II MSI-H colon cancers should
not receive adjuvant chemotherapy.
In conclusion, accumulated evidence indicates that MMR
status evaluation is of great importance in the management
of CRC patients. Pathologists have an essential role in MMR
status testing.
234
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Friday, September 24th, 2010
Slide seminar: Lymphoma surgical pathology
Moderators: V. Franco (Palermo), S. Pileri (Bologna)
Grey zones of hodgkin lymphoma: report of
a case with features intermediate between
primary mediastinal B-cell lymphoma, classical
hodgkin lymphoma and nodular lymphocytepredominant Hodgkin lymphoma
A. Zamò1, G. Todeschini2, R. Zanotti2, F. Benedetti2, F.
Menestrina1
1
2
Department of Pathology and Diagnostics, University of Verona;
Department of Medicine, University of Verona
Background. Hodgkin lymphoma (HL) was one of the first
lymphomas to be defined as an entity on morphological and
clinical grounds, and diagnostic criteria seem straightforward 1.
Yet, accurate morphological evaluation coupled to the use of
extensive immunohistochemical panels have highlighted the
presence of several “grey zones” (GZ). A GZ can be defined
in several ways: as a morphological overlap, as a composite
morphology with or without a transition area, as an aberrant
immunophenotype, or as a mixture of these conditions.
In brief, GZ of HL include only one WHO-defined entity,
called “B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical
Hodgkin lymphoma” 2, and other non-WHO-defined GZ,
namely between T-cell rich diffuse large B-cell lymphoma
and nodular lymphocyte predominant HL, between classical
HL and anaplastic large cell lymphoma and also other composite HL and non-Hodgkin lymphoma, not included in the
previous categories.
We report a case showing features intermediate between
primary mediastinal B-cell lymphoma (PMBL), classical HL
(cHL) and nodular lymphocyte-predominant Hodgkin lymphoma (N-LPHL).
Case description. A 37-year old male presented in another
hospital in January 2009 with dyspepsia, night fever and
sweats. In February the left arm became swollen, and a chest
X-ray was taken, showing a mediastinal enlargement. CT
scan confirmed the presence of a 90 x 60 mm mass as well as
multiple lymphadenopathies, including subcarinal, supraclavicular and axillary (bilateral). The patient was classified as
stage IIB bulky mediastinal.
Laboratory analyses showed the follwing values: Hemoglobin 14.1 g/dl, Platelets 290x109/L, Leukocytes 6.8x109/L, Neutrophils 5.4x 109/L, Lymphocytes 0.53x109/L, ESR 36 mm,
normal beta2-microglobulin and LDH values.
A first lymph node biopsy was taken, and the patient was
diagnosed with nodular sclerosis cHL.
After two cycles of ABVD chemotherapy, PET-scan showed
an increased SUV in paratracheal region, and persistence of
supraclavicular lymphadenopathies. CT scan also showed a
decrease of the mediastinal mass (38 x 18 mm). A second
biopsy (consisting of two supraclavicular lymph node fragments) was taken and sent to our Department.
Histopathological examination showed two different pictures
in the two fragments.
In one of the fragments, the lymph node structure was partly
preserved, with several reactive follicles present. Focally large
atypical cell were present, showing a morphology reminiscent
sometimes of LH cells, sometimes of HRS cells. These cells
were mostly positive for CD20, PAX5, BOB1, OCT2, p63,
MUM1/IRF4, partially for CD79a and CD30; BCL6 was expressed only focally and EBER was negative. The microenviroment was suggestive of N-LPHL, including B-cell nodules
composed mostly by small cells, that embedded the large
cells, although these were found also outside the nodules.
The second fragment showed a diffuse infiltration of lymphoid cells, showing marked polymorphism, where the dominant cells were medium to large-sized, frequently with a clear
cytoplasm; often neoplastic cells showed a “pop-corn” or
more rarely “sternbergoid” appearance. Compartmentalizing
sclerosis was focally present. Neoplastic cells were diffusely
positive for CD20, PAX5, BOB1 and OCT2, variably positive
for CD79a, CD30, MDC, MUM1/IRF4, BCL6, p63, focally
positive for CD23, negative for EBER. Ki-67 marked around
50% of cells.
The final diagnosis was “B-cell lymphoma, unclassifiable,
with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma” (grey zone lymphoma) with a comment explaining the peculiarity of the case.
The patient underwent one R-CHOP cycle, followed by one
R-DHAP, and then sequential high-dose therapy associated
with four infusion of Rituximab and stem cell reinfusion after
high dose of Cytarabine and after Mitoxantrone and high dose
of Melphalan, (completed on the 29th of January this year).
After therapy, CT scan showed a further reduction of the
mediastinal mass (4mm), while PET-scan was completely
negative. At the last follow-up (19th of July 2010) the patient
was in complete remission.
Discussion. B-cell lymphoma, unclassifiable, with features
intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma has been recognized as an entity in
the 2008 WHO classification 2. However, the introduction of
this category has stirred some discussion, mostly concerning
the acceptance by clinicians, who might face a problem in
deciding the most appropriate therapeutic regimen.
This category is heterogeneous by definition, comprising a
mixture of features (both morphological and immunophenotypical) of PMBL and HL. Adding even more complexity,
composite PMBL and cHL lymphoma, are also mentioned
in a very short paragraph of the WHO blue book, and were
included in the “B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and
classical Hodgkin lymphoma category” by EAHP panellists
at the last EAHP lymphoma workshop (Bordeaux 2008). This
approach is also reflected in the ICD-O code chosen for this
entity, that is 9596/3, corresponding to “composite Hodgkin
and non-Hodgkin lymphoma”.
The clinical presentation of this lymphoma is usually similar
to PMBL, although a male prevalence has been reported 3 4.
The most common morphology is similar to PMBL, but with
greater cellular heterogeneity, including many HRS-like cells,
sometimes with the presence of areas that closely resemble
cHL.
Lectures
The immunophenotype is positive for B-cell markers (CD20,
CD79a, PAX5), even in HRS-like cells (whereas in cHL
CD20 is usually weak or negative, and CD79a usually negative) although some aberrant markers may be present, like
CD15 or diffuse CD30 expression (focal CD30 expression is
very common in PMBL). EBER is usually negative.
Recently Hoeller et al. have published a work trying to spot
the most significant immunophenotypic differences between
PMBL and HL 5. The authors proposed a diagnostic algorithm
based on BOB1, CD79a and cyclin E. They also confirmed
p63 (TP73L) as a useful and highly reproducible marker of
PMBL as previously reported by our group 6.
To our knowledge the case we describe is the first mediastinal
lymphoma with features intermediate between PMBL, cHL
and N-LPHL. These intermediate features were mostly evident
comparing the two different fragments, one showing intermediate features between cHL and N-LPHL, and one between
cHL and PMBL. Intermediate features were both morphological and immunophenotypical. Classical HL features included
a large nucleolus in many large cells, partial CD30 expression
and very strong MDC expression. N-LPHL features included
several cells resembling LH cells, diffuse positivity for CD20,
partial positivity for CD79a as well as p63, BOB1, OCT2 and
BCL6 expression, and a PTGC-like microenvironment in one
of the fragments. PMBL features included the presence in one
of the fragments of clusters of medium to large cells with clear
cytoplasm, showing CD20, CD79a, p63, BOB1, OCT2 and
BCL6 expression, as well as partial CD23 expression.
Our personal interpretation is that the first fragment might
represent an initial lesion of PMBL. Should only one biopsy
have been taken, the patient might have been misdiagnosed.
Several cases of synchronous or metachronous PMBL and HL
have been reported; more commonly relapses show features of
PMBL, raising the suspicion that this component might have
been present ab initio and relapsed because of inadequate
therapy.
235
Conclusion. The experience gained from this and other similar cases supports two conclusions:
a)in case of a mediastinal mass, the largest possible biopsy
should be taken; needle biopsies should be completely
avoided;
b)more studies are needed to understand the nature of GZ
lymphomas, but the hypothesis that most grey zone cases
should be considered inside the morphologic and phenotypic spectrum of PMBL seems sound (also for therapeutic
purposes).
References
1
Stein H. Hodgkin lymphoma. In: Swerdlow SH, Campo E, Harris NL,
Jaffe ES, Pileri SA, Stein H, et al., eds. WHO Classification of Tumours of Haematopoietic an Lymphoid Tissues. Lyon, France: IARC
2008, pp. 322. [Bosman FT, Jaffe ES, Lakhani SR, Ohgaki H (Series
Editor): World Health Organization Classification of Tumours].
2
Jaffe ES, Stein H, Swerdlow SH, Campo E, Pileri SA, Harris NL.
B-cell lymphoma, unclassifiable, with features intermediate between
diffuse large B-cell lymphoma and classical Hodgkin lymphoma. In:
Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H,
et al., eds. WHO Classification of Tumours of Haematopoietic an
Lymphoid Tissues. Lyon, France: IARC 2008, pp. 267-268. [Bosman
FT, Jaffe ES, Lakhani SR, Ohgaki H (Series Editor): World Health
Organization Classification of Tumours].
3
Garcia JF, Mollejo M, Fraga M, Forteza J, Muniesa JA, Perez-Guillermo M, et al. Large B-cell lymphoma with Hodgkin’s features. Histopathology 2005;47:101-10.
4
Traverse-Glehen A, Pittaluga S, Gaulard P, Sorbara L, Alonso MA,
Raffeld M, et al. Mediastinal gray zone lymphoma: the missing link
between classic Hodgkin’s lymphoma and mediastinal large B-cell
lymphoma. Am J Surg Pathol 2005;29:1411-21.
5
Hoeller S, Zihler D, Zlobec I, Obermann EC, Pileri SA, Dirnhofer S,
et al. BOB.1, CD79a and cyclin E are the most appropriate markers to
discriminate classical Hodgkin’s lymphoma from primary mediastinal
large B-cell lymphoma. Histopathology 2010:56:217-28.
6
Zamo A, Malpeli G, Scarpa A, Doglioni C, Chilosi M, Menestrina F.
Expression of TP73L is a helpful diagnostic marker of primary mediastinal large B-cell lymphomas. Mod Pathol 2005;18:1448-53.
Pathologica 2010;102:237-383
P16INK4a is a useful marker in uterine
adenocarcinoma classification
M.A. Caponio, T. Addati, S. Petroni, O. Popescu, G. Giannone,
R. Di Girolamo, V. Rubini, A. Kardashi, G. Simone
Pathological Anatomy Unit, NCI “Giovanni Paolo II”, Bari, Italy
Background. Endocervical adenocarcinomas (ADCs) are increasing each year. Determining primary site of uterine ADC
can be problematic due to the overlapping morphology of endocervical and endometrial ADCs. The same problem can regard
metastatic ADCs of extra-uterine origin. P16INK4a is a molecular
biomarker potentially useful in discriminating endocervical ADC,
diffuse positive (P), from endometrial negative (N) or focally
positive (FP) and metastatic ADCs of extra-uterine origin and
from reactive glandular cells. The aim of this study was to investigate p16 expression in endocervical, endometrial and metastatic
ADCs of extra-uterine origin.
Methods. We observed 43 cervical biopsies of uterine ADC.
P16INK4a (CINtec p16 Histology Kit) has been investigated in all
histological samples.
Results. On 43 cervical biopsies the following diagnosis were
performed: 5 endocervical ADCs, 8 endometrioid-type endometrial ADCs, 5 endometrial serous-papillary ADCs, 12 extra-uterine ADCs, 11 NOS ADCs and 2 endocervical biopsies whitout
atypia. Three out of 5 endocervical ADCs were p16 P, 1 FP and
1 case was p16 N. One endometrioid ADCs resulted p16 P, 4
FP and 3 N. Three endometrial serous-papillary ADC have been
classified p16 P and 2 FP. On NOS ADCs, 3 resulted p16 P, 5 FP
and 3 N. Of the 7 ADCs of ovarian origin, 4 resulted P and 3 N.
In 2 out four cases from large intestine FP was detected, whereas
2 resulted N. One case of breast origin was N such as 2 endocervical samples without atypia.
P16 positive was prevalent in endocervical and serous papillary
ADCs of endometrial or ovarian origin, whereas endometrioid
ADCs, such as metastatic non ovarian lesions generally presented
only focal or negative immunostaining. Some bias in diagnostic
use of p16, leading to disagreement between bioptic and surgical
sample could be due to sampling problems and neoplastic heterogeneity. P16 seems to be an useful marker in ADCs particularly
in reclassifying NOS ADCs.
Predictors of recurrence or progression in
pituitary adenomas differ according to tumour
subtypes: a classification-tree approach
1,2)A. Righi, 3)P. Agati, 4)G. Frank, 5)M. Faustini-fustini, 6)R.
Agati, 4)D. Mazzatenta, 1)A.Farnedi, 6)F. Menetti, 1)G. Marucci,
1)M.P. Foschini
1)Anatomia patologica, Dipartimento ematologia-oncologia, Università di
Bologna, Ospedale Bellaria, Bologna, Italia; 2)Scienze biomediche e oncologia umana, Molinette, Torino, Italia; 3)Dipartimento di statistica “P. Fortunati”, Università di Bologna, Bologna, Italia; 4)Centro di chirurgia dei tumori
ipofisari, Ospedale Bellaria, Bologna, Italia; 5)Unità di endocrinologia, Dipartimento di medicina, Ospedale Bellaria, Bologna,Italia; 6)Dipartimento
di Neuroradiologia, Ospedale Bellaria, Bologna, Italia
Background. It is difficult to evaluate the recurrence and progression potential of pituitary adenomas (PA) at presentation.
The World Health Organization Classification of Endocrine
Tumors suggests that invasion of the surrounding structures,
size at presentation, Ki67 labelling index (LI) higher than
3%, and extensive p53 expression are indicators of aggressive behaviour. Nevertheless, Ki67 and p53 LIs evaluation is
subject to inter-observer variability and their cut-off value is
controversial.
Methods. Aim of the present study is to analyse the prognostic
value of age, invasion, size, Ki67, and p53 protein LIs (evaluated
using a digital image analysis) in a series of 166 pituitary adenomas with a minimum follow-up of 6 years using the receiver
Oral communications and Posters
operator characteristic (ROC) curve and the classification and
regression tree analysis (CART).
Results. In the un-stratified dataset, the commonly used threshold
index of 3% has a high specificity (93.2%) but a very low sensitivity (27.8%); p53 LI, even if slightly higher in PA with progression or recurrence, is not significant using ROC curve and CART
analyses. On the contrary, the CART-derived tree evidences that
each PA subtype has its specific prognostic factors. In cases of
PRL and ACTH type PA, the Ki67 LI has the main prognostic
value. Specifically, a cut-off of 4.40% shows the highest accuracy
in the PRL type of PA, while the cut off in the group of ACTH
is 1.70%. Invasion as evaluated by MRI emerges as the most
important prognostic factor in cases of non-functioning PA since
it identifies 5 of 6 (83.3%) cases with recurrence or progression.
In the non-invasive subgroup, the Ki67 LI is useful in identifying
patients at risk of recurrence/progression. On CART analysis, GH
adenomas show different prognostic features since patient age
and sex appear to be the most useful.
Conclusions. In conclusion, the CART algorithm generates decision trees which appear to be useful to identify PA with high risk
of recurrence.
Pilomatrix carcinoma arising in a pilomatrixoma
L. Alessandrini, R. Salmaso, R. Cappellesso, A. Fassina
Dipartimento di Scienze Medicodiagnostiche e Terapeutiche, Università
di Padova, Italia
Background. Pilomatrix carcinoma is a rare malignant tumor of
hair matrix differentiation, occurring most frequently in elderly
patients on posterior neck, pre- and retro-auricolar area. It can be
distinguished from its benign counterpart mainly for the presence
of necrosis, islands of basaloid cells with high mitotic index and
true capsular infiltration.
Methods. 81-year-old patient presented an ulcerated lesion in
the left lower eyelid which was excised and routinely processed
for H&E, PAS, and for immunohistochemistry for cytokeratins
(CKs), Epithelial Membrane Antigen, Carcinoembryonic antigen,
S-100 and MIB-1.
Results. The lesion was a symmetric dermal nodule with an overlying ulcerated epidermis, characterized by peripheral lobules of
basaloid cells arranged in sheets and nests and a central area with
“ghost” cells, and pale eosinophilic cytoplasm. In most basaloid
nests, cells had hyperchromatic nuclei and prominent nucleoli, with
high mitotic rate (4-8 mitoses/HPF), as confirmed by MIB-1 reaction (40%), with pushing margins with islands of infiltration of the
surrounding capsule. Squamous differentiation was demonstrated
by positive and strong staining for CKs, negative in “ghost” cells.
Foci of necrosis and a patchy lymphocytic infiltrate were present,
whereas vascular and perineural invasion was not detected. Besides
the histological features of malignancy, benign aspects were identified: few basaloid lobules composed of uniformly sized, typical
cells with low mitotic rate and “ghost” cells formation towards the
centre of the tumor. Only 50 cases of pilomatrix carcinoma have so
far been reported, which usually arises de novo, and only occasionally in a pilomatrixoma: neither molecular biology nor immunohistochemistry are helpful in distinguishing the two entities, and their
distinction remains uniquely on the histological recognition.
PIK3CA gene mutations in lung neuroendocrine
tumors
A. Capodanno, G. Alì, L. Boldrini, G. Riccardo, A. Servadio,
M.I. Rotondo, G. Fontanini
Surgery, Santa Chiara Hospital, Pisa, Italy
Background. Lung neuroendocrine tumors represent about 20%
of all lung carcinomas and comprise a large spectrum of tumors
that share structural, morphologic, immunohistochemical, and
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
ultrastructural features. Lung neuroendocrine tumors are classified into four main groups with different biologic aggressiveness:
typical carcinoids (TCs), atypical carcinoids (ATs), large-cell
neuroendocrine carcinomas (LCNCs), and small-cell lung carcinomas (SCLCs). Recently, somatic mutations in the phosphatidylinositol-3-kinase (PI3K) catalytic subunit (PIK3CA) gene
have been reported in several human cancers. The cancer-associated PIK3CA mutations lead to an enhanced enzymatic activity,
the upregulation of the downstream signalling cascade, and the
oncogenic cell transformation. In this study, we investigated the
PIK3CA gene status in lung neuroendocrine tumors.
Methods. Mutations in the helical and kinase domains of the
PIK3CA gene were determined by direct gene sequencing analysis in 189 lung neuroendocrine tumors, including 80 TCs, 17
ACs, 17 LCNCs, and 75 SCLCs.
Results. The frequency of PIK3CA gene mutation in lung neuroendocrine tumors was 52/189 (27.5%). The mutation distribution
was approximately twice in the kinase domain (37/52) compared
with the helical domain (15/52). The most prevalent PIK3CA
gene anomalies were the H1047R and G1049S mutations in the
kinase domain and the E542K and E547K mutations in the helical domain. No significant associations were observed between
PIK3CA gene status and age, sex, or lymph node status of the
patients. However, we found a significant association between
PIK3CA gene status and lung neuroendocrine tumor histology
(p=0.029) with PIK3CA mutations that were more frequent in
more aggressive AC, LCNC, and SCLC histotypes. Our study is
the first report of PIK3CA gene mutations in lung neuroendocrine
tumors and the high frequency of mutations suggests an important
role of PIK3 kinase in tumorigenesis of these tumors.
Role of glucocorticoides and matrix
metalloproteinases in the pathogenesis of pelvic
organ prolapse: a clinicopathological study of 34
cases
1)A.M. Altavilla, 2)D. Caliandro, 2)M. Politano, 1)L. Carluccio,
2)L. Milano
1)U.O. di Anatomia Patologica, Pia Fondazione “Card .G. Panico”,
Azienda Ospedaliera, Tricase (Le), Italia; 2)U.O. di Ostetricia e Ginecologia, Pia Fondazione “Card. G. Panico”, Azienda Ospedaliera, Tricase
(Le), Italia
Background. Pelvic organ prolapse(POP) is a debilitating disorder for women. Risk factors are known but the pathogenesis is
unclear. Imbalance between metalloproteinases(MMPs) and their
inhibitors TIMPs plays an important role in connective remodelling process. Many data suggest that glucocorticoides(GC) excess
damage matrix homeostasis. The aim of our study is to evaluate in
incontinent women connective tissue alterations and the immunohistochemical expression of MMP2/TIMP2, in uterosacral ligament, a pelvic support, compatibly with changes of cortisol levels
and with an Hypothalamic-Pituitary-Adrenal axis activation.
Methods. We analyzed the uterosacral ligaments specimen of
16 incontinent women and as controls from 18 women who
underwent benign gynaecologic surgery. Histochemistry for trichrome and elastic stain and immunohistochemistry for MMP2
and TIMP2 were performed on paraffin embedded sections. The
slides were scored by the pathologist blinded to diagnoses. GCs
profile was evaluated on the response of basal Cortisol-ACTH
ratio before and after a dexamethasone-suppression test(0.5mg).
Statistic analyses: mean±SD, Spearman’s rank, chi-squared test,
p-values of < 0.05 significant.
Results. There was no difference(ns) in parity, menopausal status
and age between groups, the only significant datum was stress
incontinence(p < 0.05). In POP group GCs levels were increased
compared to controls, excluding effects of age and parity. Women
with POP compared to those without revealed a decrease of
collagen cellularity. The score dispersion rate of elastin did not
show difference between groups. POP group revealed an important higher MMP2 expression than non-POP group(p < 0.01).
The ratio of MMP2/TIMP2 was higher in the POP-group than
in controls. These data are consistent with increased collagen
break-down as a pathologic aetiology of prolapse with a laxity
of collagen content due to connective degradation rather than a
decrease of collagen synthesis and with GCs influence.
Nasal seromucinous hamartoma (microglandular
adenosis): a morphological and molecular study
of five cases
A. Ambrosini Spaltro, L. Morandi, D.V. Spagnolo *, A. Cavazza **, M. Brisigotti ***, S. Damiani, V. Eusebi
Sezione di Anatomia Patologica, Dipartimento di Oncologia ed Ematologia, Università di Bologna, Ospedale Bellaria, Bologna, Italia; * Department of Anatomical Pathology, PathWest Laboratory Medicine, Queen
Elizabeth II Medical Centre, Nedlands, Western Australia; ** Unità Operativa di Anatomia Patologica, Arcispedale S. Maria Nuova, Reggio Emilia, Italia; *** Unità Operativa di Anatomia Patologica, Ospedale Infermi,
Rimini, Italia;
Background. Seromucinous hamartoma (SH) is a rare glandular
lesion of the sinonasal tract and nasopharynx. Cases reported in
the literature are limited and there are few follow-up data.
Methods. The clinicopathological features of five cases of nasal
SH were analyzed. Immunoreactivity for alpha-smooth muscle
actin (α-SMA), calponin, desmin, p63, CK 14, laminin, collagen IV, S100 protein, Ki-67 and EMA was assessed. Molecular
analyses for clonality using mtDNA (mitochondrial DNA) were
conducted. The mtDNA of five cases with normal nasal mucosa
obtained by turbinate resection was used as the “normal” counterpart for genetic analysis.
Results and comments. Patients (3 F and 2 M) ranged from 49
to 66 yrs in age. All lesions were located in the nasal cavity. In 4
cases with follow-up there was no recurrence.
In all cases the lamina propria exhibited a proliferation of small
seromucinous glands embedded in a dense, fibrotic stroma. Neither mitotic activity nor nuclear atypia were observed.
Around the small proliferating seromucinous glands, no immunoreactivity for p63 and CK 14 was detected, but expression of laminin (2 of 5 cases) and collagen IV (all cases) was observed. Glandular epithelial cells were positive for S100 protein in all cases, for
EMA in 4/5 cases and Ki-67 positivity ranged from 1% to 2%. The
immediately periglandular stromal cells were reactive for calponin
in all cases, for α-SMA in 4/5 cases and focally for desmin in 2/5
cases, while the intervening stroma was completely negative.
In the 5 cases with normal nasal mucosa the mean mutation rate
was 0.83% (0.23% homoplasmy, 0.67% heteroplasmy), while the
lesional cases showed a higher mutation rate, especially in heteroplasmy (0.52% homoplasmy, 2.02% heteroplasmy).
These features indicate that this unusual glandular proliferation is a
hyperplastic lesion both at morphological and molecular levels. It also
shares some similarities with microglandular adenosis of the breast.
Endoscopic mucosal resection and endoscopic
submucosal dissection as an alternative treatment
for dysplastic lesions and early cancer of the
stomach
M.R. Ambrosio, M. Onorati, B.J. Rocca, V. Mourmouras,
M. Mario *, L. Barbagli, F. De Luca, C. Vindigni
Department of Human Pathology and Oncology-Anatomic Pathology Section, Santa Maria delle Scotte, Siena, Italy; * Gastroenteric Unit, Santa
Maria Delle Scotte, Siena, Italy
Background. Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have been developed as
treatment for gastric dysplastic lesions and early gastric cancer in
Japan and in the Western world.
239
Oral communications and Posters
Methods. During the period 1998-2009, 34 EMR and 10 ESD for
a total of 44 lesions, histologically diagnosed as dysplasia or early
cancer, were performed in the Hospital of Siena.
Results. 24 cases (55%) were piecemeal resections and 20
(45%) were en bloc resections. The lesions were mostly located
in the body (19 cases) and the size ranged from 0.8 to 3.5 cm. According to the endoscopic Paris Classification, 19 cases were type
I (Is = 18, Ip = 1), 11 type II, prevalently IIa (10 cases) and 14
mixed types, prevalently IIa+IIc (11 cases). Complications were
represented by two cases of perforations and two cases of late
onset of bleeding, readily treated and resolved. According to the
histological Vienna classification, 16 cases were type 3, 8 were
4.1, 14 were 5.1 and 6 were 5.2. Complete (lateral and deep margins free) and incomplete resection was confirmed histologically
in 32 (76%) and 10 cases (24%) lesions respectively; in two cases
margin involvement was not evaluable. Seven patients underwent
surgical treatment because of the involvement of the cut ends. In
one of these cases the diagnosis was that of high grade dysplasia,
three cases were T1, in two cases the cancer involved the muscular layer (T2) and in one case there was no cancer. All cases were
N0. The median follow-up period was 30 months (range 3-135).
In three cases of lesion 3, one recurred after 9 months and two
after 60 months; a case of 5.1 lesion recurred after 12 months.
Conclusions. Our data suggest that EMR may provide an alternative treatment to surgery for selected cases of preneoplastic and
superficial neoplastic gastric lesions.
Cortical thymic epithelial tumors have an
increased risk of developing additional
malignancies: lack of immunologic surveillance?
M.R. Ambrosio, B.J. Rocca, F. Granato *, D. Spina, S. Lazzi,
L. Leoncini
Human Pathology and Oncology-Anatomic Pathology Section, Santa Maria delle Scotte, Siena, Italy; * Cardiothoracic and Vascular Surgery, Thoracic Surgery Unit, Santa Maria delle
Background. The increased risk of developing an additional malignancy (AM) before or after a thymic epithelial tumors (TET)
has not yet been fully examined and no relations with histologic
pattern of thymic neoplasma was found.
Methods. 52 patients who underwent surgical excision for TET
were studied. Based on the WHO classification, the tumors were
classified as A, AB (B1 and B2-like) and B thymoma, and thymic
carcinoma (C). A control population was provided by the creation
of a further database comprising 114 patients with colorectal
cancer (CC).
Results. Patients with TET showed a statistically significant
higher risk of developing AM compared to patients with CC
(12/52 vs 11/114 patients, p = 0.0374). The association between
TETs and AM was related to the TET histotype. B2, B3, AB
(B2-like) and C were histotypes more correlated with the onset
of an AM. Taking into consideration the histogenesis of these
thymomas prevalently from cortical epithelial cells (cTECs),
cases were divided into two sub-groups. Sub-group 1 included
29 patients with A, AB (B1-like) and B1 thymomas, sub-group 2
comprised 23 patients with AB (B2-like), B2, B3 thymomas and
C. Sub-group 2 showing a statistically significant higher risk of
developing an AM (p = 0.008). The time interval (TI) between
the appearance of the first and second tumor in TET group was
significantly shorter than those in CC group (p = 0.014), with
TETs following AM in many cases (n = 10).
Conclusions. Patients affected by TETs have a significantly
higher risk of developing AM and this risk is considerably greater
in tumors exhibiting a prevalent cortical origin. This may be
related to the role of cTECs in presenting foreign antigen. The
generally low TI values between TETs and other malignancies
suggest the potential presence of an immunological impairment
that often appears prior to evidence of TET.
Aberrant expression of TFR1/CD71 in thyroid
carcinomas identifies a novel potential diagnostic
marker and therapeutic target
1)A. Torrisi, 1)P. Amico, 2)I. Cataldo, 3)R. Parenti, 1)G.M. Vecchio, 4)R. Perris, 1)G. Magro
1)Dipartimento “G.F. Ingrassia” - Università di Catania, Azienda Ospedaliero-Universitaria –Policlinico Vittorio Emanuele, Catania, Italia;
2)Dipartimento di Patologia, Università di Verona, Verona, Italia; 3)Dipartimento di Scienze Fisiologiche, Università di Catania, Catania, Italia; 4)Comt, Università di Parma, Parma, Italia
Background. Type I receptor for transferrin (TfR1/CD71) is
overexpressed in several malignant tumors. We investigated the
expression of TfR1/CD71 in benign and malignant thyroid tissues.
Methods. Tissue samples, including benign lesions and follicular-derived carcinomas, from 241 patients and a total of 35 benign
and malignant fresh specimens were assayed for TfR1/CD71 expression by RT-PRC, Western blot and immunohistochemistry.
Results. We found that transcription of TfR1/CD71 gene is constitutive in thyroid epithelia, but the mRNA is differently translated in benign and malignant tissues. In benign tissues low levels
of TfR1/CD71 were found, whereas most carcinomas exhibited
overexpression of the receptor, predominantly in the cytoplasm
of neoplastic cells. The highest expression level was detected in
primary and metastatic papillary carcinomas and anaplastic carcinomas, with a positivity ranged from 86% to 100% of the cases.
Our findings suggest that altered expression of TfR1/CD71 is a
marker of malignancy in thyroid tissues where it is useful in distinguishing PTC from benign lesions with PTC-like cyto-architectural alterations and follicular variant PTC from benign follicularpatterned lesions. The present observations support the rationale
for the use of radiolabeled transferrin/transferrin analogs and/or
anti-TfR1/CD71 antibodies for diagnostic and/or radiotherapeutic
purposes in TfR1/CD71-expressing thyroid tumors.
Comparison of three different methods for the
analysis of codon G12 and G13 of the KRAS gene
1)Andreozzi MC. 2)Bihl MP. 3)Foesrster A. 4)Rufle A. 5)Tornillo L. 6)Terracciano LM.
1)Institute of pathology, University of basel, Basel, Switzerland 2)Institute of pathology, University of basel, Basel, Switzerland 3)Institute of
pathology, University of b, Basel, Switzerland 4)Institute of pathology,
University of basel, Basel, Switzerland 5)Institute of pathology, University
of basel, Basel, Switzerland 6)Institute of pathology, University of basel,
Basel, Switzerland
Aims and Methods. KRAS mutation screening has been achieved
high importance in selecting the right therapy for patients with
colorectal cancer and non-small-cell lung cancer especially in
metastatic disease stage. Screening for KRAS mutations in these
patients provide additional information on optimizing treatment
options with targeted drugs. Paraffin embedded tissue from 100
colon carcinomas were randomly selected to include a wide spectrum of KRAS mutations. A comparison of three different methods for the analysis of the KRAS gene of codon G12 and G13
using the same DNA preparation for all methods was performed.
For the molecular analysis the following methods were used:
Pyrosequencing. First step: Amplification of the sequence for
analyzation by PCR. Second step: Enzymatic reaction cascade
during the synthesis of the previously amplified sequence that
converts the specific nucleotide incorporation into light.
INFINITI®: The analyzer is designed to measure fluorescence
signals of labelled DNA target hybridized to BioFilmChip® microarrays. The analyzer automates the assays and integrates all
the discrete processes of sample (PCR amplicons) handling, reagent management, hybridization, detection, and result analysis.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Dideoxysequencing. The DNA to be sequenced is amplified by
PCR.
Results. The following mutations were detected in codon 12 and
13 as follows: G12A 5x, G12C 2x, G12D 16x, G12S 3x, G12V
15x, G13C 1x, G13D 17x, G13R 1x and 40 samples were wild
type.
Conclusions. This is the first study, which analyzed three different methods in a comparative matter. All these 3 methods are
suitable for detecting hot spot mutations in the Kras oncogene.
Infinity technology seems to be more sensitive in samples contaminated with high amounts of non mutated cells or in samples
of low DNA quality. Wrong results were all a problem of sensitivity (1% false negative for Infinity and Dideoxysequencing
technology, respectively).
Vegfa amplification in different neoplastic
entities: tissue microarray analysis on 2292 tissue
samples
1)Andreozzi MC. 2)Vlajnic T. 3)Zlobec I. 4)Bihl MP. 5)Tornillo
L. 6)Schneider S. 7)Lugli A. 8)Terracciano LM.
1)Institute of pathology, University of basel, Basel, Switzerland 2)Institute
of pathology, University of basel, Basel, Switzerland 3)Institute of pathology, University of basel, Basel, Switzerland 4)Institute of pathology, University of basel, Basel, Switzerland 5)Institute of pathology, University of
basel, Basel, Switzerland 6)Institute of pathology, University of basel, Basel, Switzerland 7)Institute of pathology, University of basel, Basel, Switzerland 8)Institute of pathology, University of basel, Basel, Switzerland
Aims. Angiogenesis plays an important role in progression of
several tumor types. Evidence from preclinical and clinical studies indicates that vascular endothelial growth factor (VEGFA) is
the predominant angiogenic factor. The aim of this study was a
systematic investigation of VEGFA amplification in a large survey of solid human tumors in tissue microarray format.
Methods. FISH analysis of the VEGFA gene was performed
in a multi tumor array (n = 2292) including 132 different tumor
categories and 31 normal tissue types. Additionally VEGFA gene
amplification was evaluated in a further large series of sporadic
CRC resections (n = 1280) and the obtained data were compared
to relevant clinico-pathological features.
Results. VEGFA amplification was detected in carcinoma of
colon (n = 39; 3%), gall bladder (n = 5; 13.2%), pancreas (n = 3;
6.5%), prostate (n = 6; 15.8%), stomach (n = 6; 14.3%), testis
seminoma (n = 4; 8.5%) and colon adenoma (n = 7; 9.2%). VEGFA amplification in CRC significantly correlated with higher
T stage and higher tumor grade, presence of vascular invasion,
right sided location and with worse survival in univariate and
multivariable analysis.
Conclusions. Albeit rare, VEGFA amplification can be detected
in several different tumor entities. In CRC it highlights a small
subset of CRCs with aggressive phenotype. Additional studies are
needed to evaluate its significance in other neoplastic entities.
Extraskeletal Ewing sarcoma in a 78-years-old
woman: a case report
U.F. Angelotti, R. Scamarcio, A. Scivetti, A. Colagrande,
C. Traversi, A. Cimmino, L. Resta
Anatomia patologica, Policlinico, Bari, Italia
Background. Extraskeletal Ewing sarcoma (EES) is a rare soft tissue tumour morphologically indistinguishable from the more common Ewing Sarcoma of bone. It must be differentiated from other
small, blue round cell tumours, including primitive neuroectodermal tumour and neuroblastoma. The age at the time of diagnosis,
unlike its osseous counterpart, has a wide range, from infancy to
the elderly, and has a slight predominance in male patient.
Methods. We present a case of EES in the retroperitoneum of a
78-year-old woman which in 1993 has been diagnosed of Ewing
sarcoma of the upper third of right arm. The history clinical is
silent until April 2000 when it occurs disease recurrence in the
distal third of the right arm, followed in February 2003 by intervention of right axillary lymph nodes metastatic dissection and
in November 2007 by secondary localization of Ewing sarcoma
at level of the fifth hepatic segment. For about four months, the
patient complains of pain in the epi-mesogastrial area, anorexia,
fatigue and malaise; the computed tomographic scan shows solid
expansive mass, suggestive of peritoneal metastasis, in the mesogastrial median area.
Results. The tumour lacks of immunoreactivity for epithelial,
lymphoid, vascular, neuroendocrine, neural and muscle markers.
Immunohistochemically, the tumor was positive for vimentin,
CD99, slightly positive for CD45LC, but negative for CKpool, CD117, CK-20, MPO. Extraskeletal Ewing sarcoma was
confirmed by electron microscopy, which showed a prominent
nucleus with marginated chromatin, few organelles and abundant
glycogen. Primitive neuroectodermal tumour was excluded because of the lack of neural differentiation by histologic analysis,
immunohistochemistry and electron microscopy. This case serves
to remind the reader that EES is not a tumour that occurs exclusively in young patients.
The question of reproducibility in cytology,
histology and colposcopy
1) D. Antonini 2) A. Marsico 3) A. Anastasio 4) M. I. Rostan 5)
R. Navone
1) Ospedale degli Infermi, UO di Anatomia Patologica, Biella, Italia 2)
Ospedale Koelliker, UO di Anatomia Patologica, Torino, Italia 3)Dipartimento di Scienze Biomediche e Oncologia Umana dell’Università di Torino (Sez. di Anatomia Patologica), Italia 4) Dipartimento di Scienze Biomediche e Oncologia Umana dell’Università di Torino (Sez. di Anatomia
Patologica), Italia 5) Dipartimento di Scienze Biomediche e Oncologia
Umana dell’Università di Torino (Sez. di Anatomia Patologica), Italia
Background. It is well known that colposcopy has a low specificity (Barrasso, 1998), above all if used as a 1st level test. Indeed,
should a positive Pap test be followed by a colposcopic grade 1
abnormal transformation zone (ATZ), then, 79% of cases will be
histologically positive; without a previous positive cytology, the
values of positivity of histology are very low i.e. in the range of
20% for grade 1 colposcopical ATZ
Methods. We evaluated the correlation amongst cytology, histology and colposcopy in a spontaneous screening group (21,451
cases), where colposcopy was done at the same time as the Pap
test. A biopsy was also carried out in the presence of grade I or
higher ATZ.
Results. A total of 21,451 Pap tests were done along with colposcopies. There were 2,175 abnormal colposcopy results (mostly
grade 1 ANTZ). The colposcopic diagnosis were: white epithelium (1,002 cases), 603 keratosis, 279 punctate, 280 mosaic and
11 carcinoma. The cyto-histological diagnosis of the abnormal
colposcopy results included 210 L-SIL, 56 H-SIL and 11 carcinomas (277 cases). There were 170 abnormal cytology results
(confirmed by histology) (113 L-SIL, 54 H-SIL, 2 endocervical adenocarcinoma in situ (AIS) and 1 carcinoma) in patients
with a normal colposcopy result (G 0). Whilst there were 1.898
abnormal colposcopy results associated to normal cytology and
histology.
Our data showed that 89.1% of the patients had both a negative
colposcopy and Pap test and that 1.3% of the cases were both positive. However, there were 8.8% of patients in whom, although
the colposcopy was positive, the cytology and histology were
negative. Whilst, despite the fact that a 0.8% of the Pap tests and
histology were positive, their colposcopies were negative. The
discordance between the colposcopy and histo-cytology results
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Oral communications and Posters
indicates that colposcopy alone, i.e. without the association of
cytology and histology, is not able to offer a definitive diagnosis.
This is particularly true for abnormal colposcopy results (grade I
ANTZ or higher) that should always have an anatomopathological confirmation.
Neonatal neuroblastoma mimicking
sacrococcygeal teratoma: an autoptic case.
Vincenzo Arena*, Ilaria Pennacchia*, Egidio Stigliano*, Fabio
De Giorgio°, Arnaldo Carbone*, Fabio Maria Vecchio*
*Institute of Pathology; °Institute of Legal Medicine; Catholic University
of Sacred Heart, Roma
Background. Neonatal masses occurring in the sacrococcygeal
region are mostly teratomas. We report herein a case of neonatal
neuroblastoma in a newborn male infant delivered after a normal
pregnancy.
Methods. The neonate was brought to our hospital after a normal
vaginal delivery (38 weeks of gestation), because of an extremely
large sacrococcygeal mass. A RMI showed an enormous neoplastic mass with an undifferentiated and uniform internal structure,
which extended from the sacrococcygeal region to the celiac
region and was pressing on the rectum and the bladder. Multiple
metastatic lesions to the liver were seen too. The general condition of the child rapidly worsened and he died before a biopsy
was performed on the mass. For this reason the autoptic examination was required and revealed the presence of a tumor mass of
12 cm in diameter, which was extremely firm and immobile and
adherent to the rectum. Multiple repetitive lesions were noted
both in the liver and adrenal glands. A macroscopic diagnosis
of “likely” malignant teratoma was made. Interestingly, histopathologic examination of the tumor showed rosette formation
and neuroblastoma cells with small nuclei and fibroid cytoplasm.
The tumor cells were strongly immunopositive for NSE and Sinaptophysin. The histopathological diagnosis was neuroblastoma,
classified as stroma poor, undifferentiated in the Shimada pathological classification
Discussion. The differential diagnosis of sacrococcygeal neoplasms includes several lesions like meningomyelocele, lipoma
and lhymphangioma; however, in newborns they are most often
teratomas. Our case suggests that neuroblastoma should be considered in differential diagnosis by pathologists who perform
perinatal autopsies and confirms once again the role of autopsy
in the correct nosographic definition of diseases.
Chondroma of the hand with osteoid formation
Vincenzo Arena*; Ilaria Pennacchia; Arnaldo Capelli; Arnaldo
Carbone; Fabio Maria Vecchio
Institute of Pathology Catholic University of Sacred Heart Roma (Italy)
Background. Chondroma is the most common bone tumor arising in the hand. Histologically the majority of them consist of
mature hyaline cartilage arranged in a lobular pattern. Frequently
the cartilage has focal or diffuse calcification.
Methods. A 36 year-old woman presented with a swelling of the
proximal phalanx of the 3th finger of the left hand appeared five
months before, with no history of a previous traumatic event. At xray the lesion extended up to the ulnar cortex without evidence of a
pathologic fracture. Histologically the lesion was composed of well
differentiated chondrocytes and mature hyaline cartilage. Areas of
myxoid stroma with scattered cells without any atypical features
were also present. Neither double-nucleated cells nor clusters
of chondrocytes were seen. No mitotic figures were seen [MIB1 < 2%]. Interestingly focal deposits of osteoid within the lesion
were also seen. The patient had no relapse with 1 year follow-up.
Results. In chondroma of the hand. in case the myxoid component is predominant, a myxoid variant of chondroma should
be considered. In this context, the main differential diagnosis
is myxoid chondrosarcoma. In our case, a diagnosis of malignancy was not considered due to the absence of cellular
pleomorphism and because of the hand being a very unusual
site for malignant chondroid neoplasms. As for the unusual
presence of osteoid matrix formation, it is described in both
chondroblastoma and in chondromyxoid fibroma. The lesion
we described did not fullfill all the criteria for a diagnosis of
the above mentioned entities. However, we believe the finding
of a osteoid matrix is stricking in the setting of a chondroma of
the hand. It is common for both benign and malignant cartilage
tumors, to undergo pathologic fracture, making the histology
of new bone formation associated with the cartilage somewhat
complex but in the case presented, neither radiologic nor
pathologic signs of fracture were seen.
Refined Immunohistochemistry scoring criteria for
HER-2 “borderline” breast cancer: study on 230 cases.
Arena Vincenzo, Pennacchia Ilaria, Fabio Maria Vecchio, Arnaldo Carbone
Institute of Pathology, Catholic University of Sacred Heart, Rome
Background. Two methods are used for measuring HER-2 in the
clinical setting: immunohistochemical analysis (IHC) and fluorescence in situ hybridization (FISH). Convenience dictates that IHC
remains the screening test for HER-2 status in patients with breast
cancer, adopting FISH as second-line diagnostic tool in case of
doubtful IHC results. Aim of this study is to investigate IHC criteria for scoring HER2 in order to refine the “2+” category.
Methods. Two hundred thirty cases resulted IHC/2+ (DAKO
scoring system) were subsequently evaluated for HER-2 gene
amplification by FISH. Granularity of signal, linear, even though
not complete, membrane decoration, signal intensity (1+ to 3+
score) and presence of linear paired definite membrane signal
between cells (“track” feature) were blinded evaluated by us and
discordant cases were discussed until an agreement was reached.
Results. A granular staining pattern was seen in 73% of HER-2
FISH negative cases (p = 0.0006). Seventy four percent of FISH
positive cases showed linear membrane decoration of some extent
(p = 0.011). An intense overall IHC signal (3+) was observed in
45% of FISH positive cases (p = 0.0009). Fifty nine percent of
FISH positive cases presented “track” images in > 25% of cell
population (p = 0.054; ns), whereas 68% of FISH positive cases
presented simultaneously “rimming” and strong IHC reactivity
(p = 0.0002).
Conclusions. Combined intensity and linearity of membrane
signal, even though limited (intense partial membrane staining)
resulted the best aid for the pathologist in making final scoring decision in borderline IHC HER-2 tests. In our opinion, the
effort of the pathologist in adding IHC details for refining the
worldwide validated criteria for IHC HER-2 assessment, could
reduce the number of “borderline” cases undergoing FISH with a
significant benefit to economy lab.
Are we losing the value of autopsy? Evidence
from an epidemiological descriptive study
Vincenzo Arena*; Luca Valerio#; Ilaria Pennacchia*; Fabio De
Giorgio§;Bruno Federico°; Arnaldo Capelli*; Fabio Maria Vecchio*
*Institute of Pathology; #Institute of Hygiene; §Institute of Legal Medicine; Catholic University of Sacred Heart, Roma; °Department of Health
and Sport Sciences; University of Cassino
Background. Substantial evidence shows the high accuracy of
autopsy relative to clinical diagnosis in determining the cause of
death. Traditionally, pathologists provide clinicians with a feedback for improvement, by identifying the diseases at greater risk
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
of error. Nevertheless, the number of autopsies has been on the
decrease in all countries over the last twenty years.
Methods. We describe with a statistical analysis the variations in
the characteristics of cases referred to pathologists in Our Hospital between two three-year periods over 20 years, to investigate
the extent of the problem and the possible objective and cultural
causes. Data were derived from the hospital’s Pathology registry,
which includes data on all autopsies and is updated daily.
Results. Autopsies have decreased in number and their composition has changed significantly: in terms of epidemiology, with a
surge of neonatal autopsies; in terms of requesting wards and diseases diagnosed, with heart surgery, emergency and cardiovascular
diseases and their respective diagnoses having increased in importance relative to requests from specialists in infectious diseases.
Discussio. Our data show that such evolution is not consistent
with that of the causes of hospital mortality in Italy over the same
period: the cause of the phenomenon must be elsewhere.
The decision of the clinician to ask for autopsy is mainly dictated
by the latter’s accuracy relative to clinical diagnosis as well as by
the increasing need for defensive medicine.
Conclusion. There seems to be a paradox in the attitude of clinicians towards autopsy diagnosis: while demand for autopsies for
the traditional purposes decreases, more autopsies are requested
for what are likely to be medico-legal reasons. Therefore, accuracy of pathologic diagnosis in the clinicians’ opinion has not
changed, but, for the same reason, what pathology can offer to
clinics, education and research outside defensive medicine cannot
have changed either.
Limits of TIR-3 reporting in Thyroid Fine-Needle
Cytology: 3-Year Experience From A Single
Academic Center
1)Ascoli V. 2)Bosco D. 3)Taffon C. 4)Marinelli L. 5)De Mattia
D. 6)Grillo L. 7)Nardi F.
Anatomia Patologica, Dipartimento di Medicina Sperimentale, Università
La Sapienza, Azienda Policlinico Umberto I°, Roma, Italia
Introduction. The SIAPEC has proposed a 5-tier reporting system for thyroid fine-needle cytology (FNC), which include the
“indeterminate/inconclusive” category (TIR-3). This category
encompasses follicular-patterned lesions. In such cases, only histology (and no cytology alone) can provide a final diagnosis.
Methods. In our laboratory, the 5-tier reporting system has been
used for 7579 thyroid aspirates in the last 3 years (period 2007/
June 2009: 5680; period July-2009/April-2009: 1899). We assessed the distribution of aspirates by the 5 diagnostic categories
in the two periods, and then we focused on TIR-3 cases by evaluating the proportion of surgically treated cases in our hospital and
the histological diagnosis.
Results. A total of 319 cases (4,2%) were interpreted as TIR-3.
Of these 319 cases 125 had surgical follow-up in our hospital
(39,2%). Overall, the surgical yield of malignancy was 22.4% (23
papillary carcinoma, 1 follicular carcinoma, 3 Hürthle cell carcinoma, 1 metastatic renal cell carcinoma); 24% were adenomas
(20 follicular and 10 Hürthle cell adenomas) and the remaining
53.6% were negative (53 nodular hyperplasia, 11 Hashimoto’s
thyroiditis, 3 chronic thyroiditis). Six occult papillary carcinoma
were identified as incidental finding (4 controlateral lobe; 2 omolateral lobe/additional nodule).
Conclusions. Our survey is limited that a major fraction of TIR-3
FNC (60%) had no surgical/histological follow-up in our hospital. Nevertheless, our results are in agreement with the literature
concerning malignancy rate of TIR-3 (22%) and prevalence of
occult thyroid carcinoma; the high fraction of benign nodules
(> 50%) indicate that some TIR-3 cases could rather benefit of a
repeat aspirate after an appropriate interval of observation instead
of unnecessary surgery. For this is crucial the collaboration of pathologists with clinicians and radiologists (ultrasound findings).
Cyclosporine-induced gingival overgrowth is
associated to increased Transglutaminase -2
expression
1)Asioli S. 2)Cassoni P. 3)Righi A. 4)Cassenti A. 5)Maletta F.
6)Carossa S. 7)Navone R.
1)Scienze biomediche e oncologia umana, Molinette, Torino, Italia 2)Scienze biomediche e oncologia umana, Molinette, Torino, Italia 3)Scienze biomediche e oncologia umana, Molinette, Torino, Italia 4)Scienze biomediche e oncologia umana, Molinette, Torino, Italia 5)Scienze biomediche
e oncologia umana, Molinette, Torino, Italia 6)Sezione di riabilitazione
orale e maxillofaciale, Molinette, Torino, Italia 7)Scienze biomediche e
oncologia umana, Molinette, Torino, Italia
Background. Cyclosporine A induced gingival overgrowth,
which is characterized by an extracellular matrix increase, is due
to an altered balance between collagen synthesis and degradation.
Cyclosporine A is a potent immunosuppressant used to prevent
organ transplant rejection and to treat various autoimmune diseases.
Methods. This study proposed to verify if transglutaminase 2, an
enzyme that is thought to be responsible for the assembling and
remodeling of extracellular matrix, played some kind of role in
the pathogenesis of the cyclosporine A-induced gingival overgrowth, its expression in the gingival overgrowth was compared
to normal tissue to evidence any differences.
Cyclosporine A-induced gingival overgrowth tissues were collected from 21 liver transplanted patients and case-controlled
with 20 non-hyperplastic gingival biopsies from healthy patients
who had had previous periodontal treatment. Both the presence
and tissue distribution of transglutaminase 2 was determined in
the two groups by immunohistochemistry and analysed in comparison to the tissue morphology and expression of lymphocyte
related antigens (CD3 and CD20) and a vessel related marker
(CD34).
Results. A significant increase in the transglutaminase 2 expression was observed within the stromal component in the cyclosporine A treated patients compared to controls (p < 0.001). An
increased transglutaminase 2 expression in mesenchymal cells
and/or extracellular matrix in gingival overgrowth suggests
that this molecule has a role in the pathogenesis of the disease.
Further studies will investigate the therapeutic effect of antitransglutaminase 2 drugs (putrescine or 1,4-diaminobutane) in
these patients.
Nuclear membrane decoration by emerin staining
improves cytological detection of papillary
thyroid carcinomas
Asioli S., Maletta F., Pacchioni D., Lupo R., Bussolati G.
Biomedical sciences and human oncology, Molinette, Torino, Italia
Background. The diagnosis of follicular lesions is a grey zone in
thyroid fine-needle aspiration (FNA) cytology. Our study aims to
verify if staining with Emerin is a helpful marker of the follicular
variant of papillary thyroid carcinoma (FVPTC) in the differential
diagnosis of follicular-patterned lesions.
Methods. We designed both a prospective study on smears
and Thin Prep specimens to prove the feasibility of the procedure and a retrospective study on 78 FNA cell-blocks from
cases which, after surgery, turned out to be either benign (34
cases) or malignant lesions (44, of which 31 PTC). From each
sample, we obtained two slides, one stained with Hematoxylin
and Eosin (H&E) and the other with immunohistochemistry
(IHC) for Emerin. In Thy3 cases, HBME-1 and Gal 3 stains
were also done. Two observers gave a judgement in Thy
categories (British Thyroid Association) on H&E, Emerin,
HBME-1 and Gal 3 stained slides.
Results. The prospective study demonstrated that Emerin staining is an effective tool for nuclear membrane decoration and
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amplification of nuclear irregularities. In the retrospective study,
inter-observer agreement proved higher in Emerin-stained slides
(K of Cohen-Fleiss = 0.6890) than H&E-stained slides (K of
Cohen-Fleiss = 0.4878). Sensitivity and overall accuracy were
higher for Emerin (respectively, 77.27% and 84.61%) than H&Estained slides (respectively, 36.36% and 62.82%). Emerin staining proved able to identify all cases of PTC, including all cases
of FVPTC. In Thy3 cases, Emerin’s sensitivity and specificity
(64% and 96%) proved higher than HBME-1’s (60% and 88%),
and Gal3’s (61% and 68%).
Conclusions. Emerin stain, is a useful tool in the cytological
diagnosis of thyroid lesions. It enhances detection of nuclear
irregularities typical of PTC, thus helping to solve inconclusive
FNA cases, mainly in those cases of FVPTC with a reduced
expression of nuclear irregularities in the traditional stains
(H&E).
HER2 overexpression in patients with small tumor
size and node-negative breast cancer: a high risk
group?
1)Petroni S. 1)Asselti M. 2)Giotta F. 3)Quero C. 4)DAamico C.
5)Marzano A.L. 6)Daprile R. 7)Palma F. 8)Simone G.
1)Pathological Anatomy Unit, NCI “Giovanni Paolo II”, Bari, Italia
2)Medical and Experimetal Oncology Department, NCI “Giovanni Paolo
II”, Bari, Italia 3)Pathological Anatomy Unit, NCI “Giovanni Paolo II”,
Bari, Italia 4)Senology Unit, NCI “Giovanni Paolo II”, Bari, Italia 5)Pathological Anatomy Unit, NCI “Giovanni Paolo II”, Bari, Italia 6)Pathological Anatomy Unit, NCI “Giovanni Paolo II”, Bari, Italia 7)Pathological Anatomy Unit, NCI “Giovanni Paolo II”, Bari, Italia 8)Pathological
Anatomy Unit, NCI “Giovanni Paolo II”, Bari, Italia
Background. Approximately 25% of breast cancer is HER2/
Neu overexpressed and/or amplified being both prognostic
and predictive factors associated with worse disease-free and
overall survivals. HER/Neu+ patients with metastatic tumor or
in adjuvant systemic therapies are eligible for treatment with
Trastuzumab according to the results of the major phase III clinical trials that do not include small (pT1a or pT1b), node negative breast cancer. Patients with node-negative breast carcinoma
have a good prognosis but in ~ 20-30%, cases a recurrence of
disease is present. The aim of this study is to evaluate the risk
of recurrence in women with pT1a or pT1b, N0, M0, HER2+
breast cancer.
Methods. We collected 279 women with small invasive breast
cancer, pT1, N0, M0 from 2004 to 2009. 89 out of 279
(32.3%) < 1cm sized (pT1a and b), node-negative (median age:
56 ys) entered the study. In all cases ER, PgR, Ki-67 status
and expression of Her2/Neu, using immunoistochemistry, were
evaluated. Hormonal receptors and Ki-67 were scored according
to St Gallen conference guidelines; expression of Her2/Neu was
detected using HercepTest (DAKO) and scored according to FDA
guidelines.
Results. 18 out of 89 cases were pT1a (20.2%) and 71 pT1b. 68
tumors (76.4%) were ER +, 65 (73.0%) were PgR + and 23 cases
(26%) showed a high proliferative activity (Ki-67 index: > 20%).
10 out of 89 (11.2%), 2 pT1a and 8 pT1b, evidenced Her2/Neu
overexpression: only one case was G1, 2 were ER+/PgR+, 7
showed high expression of Ki-67.
Follow-up data (mean FU: 44.4 months; range 18-156 m.) of
the 10 patients overexpressing HER/Neu were available and
evidenced one relapse (Local and metacronous cancer) in a
woman only treated with radio therapy, in 5 patients treated with
herceptin no relapse occurred. Our results suggest that Her2/Neu
expression could be a significant marker of risk to relapse of disease, being a prognostic and predictive factor also in small breast
carcinoma with pT1a or pT1b, N0, M0.
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Chavez-MacGregor M. HER2-neu positivity in patients with small and
node-negative breast cancer (pT1a,b,N0,M0): a high risk group? Clin
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Colleoni M. Minimal and small size invasive breast cancer with no axillary lymph node involvement: the need for tailored adjuvant therapies.
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Curigliano G. Clinical relevance of HER2 overexpression/amplification
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APC molecular alterations in ileal midgut carcinoid
tumors
1)Azzoni C. 2)Bottarelli L. 3)Pizzi S. 4)D’Adda T. 5)Tamburini
E. 6)Rindi G. 7)Silini EM. 8)Bordi C.
1)Dip patologia e medicina di laboratorio, sez anatomia patologica, Università di Parma, Parma, Italia 2)Dip patologia e medicina di laboratorio, sez anatomia patologica, Università di Parma, Parma, Italia 3)Dip
patologia e medicina di laboratorio, sez anatomia patologica, Università
di Parma, Parma, Italia 4)Dip patologia e medicina di laboratorio, sez
anatomia patologica, Università di Parma, Parma, Italia 5)Dip patologia
e medicina di laboratorio, sez anatomia patologica, Università di Parma,
Parma, Italia 6)Istituto di anatomia patologica, Policlinico universitario
a. gemelli, Roma, Italia 7)Dip patologia e medicina di laboratorio, sez
anatomia patologica, Università di Parma, Parma, Italia 8)Dip patologia
e medicina di laboratorio, sez anatomia patologica, Università di Parma,
Parma, Italia
Background. Classical midgut carcinoids are well-differentiated neuroendocrine tumors arising from lower jejunum, ileum,
caecum and ascending colon. Despite recent advances in the diagnosis and treatment, no etiologic factors have been associated
with these tumors, little is known about their molecular features
and no molecular markers useful for their prognostication have
been identified. A high frequency of cytoplasmic accumulation
or nuclear translocation of β-catenin has been described in gastrointestinal carcinoid tumors but the role of Wnt pathway in the
genesis of ileal carcinoid tumors remains unknown.
Methods. We investigated 30 ileal carcinoid tumors from 14
male and 16 female patients for loss of heterozigosity (LOH) of
the APC gene using the microsatellite markers D5S346 (5q2122) and D5S1965 (5q23) and by direct sequencing of the gene
using four sets of primers amplifying three overlapping portions
of exon 15. All tumors proved to be composed of EC cells by
either serotonin immunostaining or Masson-Fontana argentaffin
reaction. The ileal carcinoids were classified according to the
WHO criteria (all WDEC class) and ENETS grading and staging
(grades G1: 24 cases, G2: 6 cases; stages IIA: 6 tumors, IIIA: 1
tumor, IIIB: 11 tumors and IV: 12 tumors).
Results. LOH was found in 15% of ileal carcinoids not correlating with any clinicopathological feature. APC gene mutations
were detected in 23% of tumors, in 5 of which mutations were
also present in the associated metastatic tissues. Moreover, in
15 (50%) carcinoids the mutational analysis identified a single
nucleotide polymorphism (SNP) in 1493ACG > ACA (T1493T)
as demonstrated also by Pizzi et al. in a series of 60 endocrine
tumours of the gastroenteropancreatic tract.
Our results indicate that the SNP in the codon 1493 of APC gene
is commonly found in ileal carcinoids, a finding that requires
further investigation. The data do not support a relevant role of
the APC pathway in this type of endocrine tumors.
Ultrarapid immunoistochemistry in intraoperative
evaluation of sentinel lymph nodes in breast
cancer
1)Baldin P. 2)Cucchi M.C. 3)Foschini M.P.
1)Dipartimento di Ematologia e Oncologia “L e A Seragnoli” Università
di Bologna Sezione di Anatomia Patologica, Ospedale Bellaria, Bologna,
Italia 2)U.O. di Chirurgia Oncologica, Ospedale Bellaria, Bologna, Italia
3)Dipartimento di Ematologia e Oncologia “L e A Seragnoli” Università
di Bologna Sezione di Anatomia Patologica, Ospedale Bellaria, Italia
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Background. In cases of breast cancer, examination of sentinel
lymph nodes (SNs) is essential to establish the status of regional
lymph nodes. To avoid the need of two separate surgical resections, it is important to develop a reliable method of intraoperative examination (IO) of SNs during the excision of the tumour.
To shorten the technical immunohistochemical procedure it has
been suggested to employ an ultrarapid immunohistochemical
method (UICH) for keratins.
The aim of the present study is to assess whether the use of
ultrarapid cytokeratin stain (UICH) enhances the intraoperative
detection of lymph nodal metastases in breast cancer patients
compared with routine frozen (RF) sections.
Methods. A consecutive series of 70 cases of IO examination
of SNs was evaluated with RF and UIHC at the same time. The
protocol described in Cancer (2005;104:14-9) was followed. In
addition 100 consecutive cases of SNs were evaluated with RF
only. All RF sections were compared with the related paraffin
embedded sections which were subsenquently obtained. Major
discordance was considered when the difference between intraoperative and definitive examination led to a delayed axillary
dissection.
Results. In the case group (70 patients), SNs showed tumoral
involvement in 18 cases (27.7%) (12 macrometastases, 2 micrometastases and 4 ITCs). In 65 cases (92,8%) the IO diagnoses
were similar to those of paraffin sections. In 5 cases paraffin
sections showed additional neoplastic cells, leading in 3 patients
(4.2%) to a delayed axillary dissection. The consecutive group
of 100 patients displayed neoplastic cell in SN in 27 cases (27%)
(23 macrometastases and 4 micrometastases) with 9 major discordances (9%). No false positive cases were detected (100%
specificity).
In conclusion UHIC allows a more accurate IO evaluation of SNs
leading to a lower number of delayed axillary dissections.
Survey on the quality perceived by internal
customers on pathology service
1)Baldoni C. 2)Bondi A. 3)Muraro L.
1) Anatomia Patologica, Dipartimento Oncologico, Ospedale Maggiore,
Bologna, Italia 2) Dipartimento Oncologico, Ospedale Maggiore, Bologna, Italia 3)Staff aziendale, Ospedale Maggiore, Bologna, Italia
Background. Evaluation of customer satisfaction in health care
is a recommended tool in the path of accreditation of healthcare
facilities. For this reason we develop a questionnaire that would
allow to highlight the quality of services perceived by customers
/ users who come to our service.
Methods. The survey was conducted by administering a questionnaire published on the intranet, which could be completed
online within a month. Respondents were contacted via email
with a message containing instructions and a link to open the
questionnaire. Were taken into consideration the following areas:
access to services, waiting time for answers, information, quality
and relational aspects. The results of the survey, which was attended by hospital and territory doctors and nurse coordinators,
were collected and processed statistically by a member of OU
Quality Company.
Results. The most positive aspects from the perspective of internal customers were the quality of services provided and availability of pathologists to participate in study groups, clinical audits,
surveys and general willingness to cooperate shown by all staff.
The most critical were the response time for histological diagnosis, followed by the response time of cytological diagnosis and
the timing and way of delivery of reports / references.
Conclusion. This type of check was particularly significant as it
provided useful information on the expectations, needs and evaluation of clinical services allowing to understand and identify critical points and to activate the process of continuous improvement
in the delivery performance.
Hepatocellular carcinoma induce abnormal
vascular organization: study on the role of their
tumor-infiltring stromal cells
1)Balzarini P. 2)Benetti A. 3)Benerini gatta L. 4)Berenzi A.
5)Dessy E. 6)Portolani N. 7)Giulini SM. 8)Grigolato P. 9)Alessandri G.
1)2nd department of pathology, school of medicine, P.le spedali civili di
brescia, Brescia, Italia 2)2nd department of pathology, school of medicine, P.le spedali civili di brescia, Brescia, Italia 3)2nd department of pathology, school of medicine, P.le spedali civili di brescia, Brescia, Italia
4)2nd department of pathology, school of medicine, P.le spedali civili di
brescia, Brescia, Italia 5)2nd department of pathology, school of medicine, P.le spedali civili di brescia, Brescia, Italia 6)Department of medical
and surgical sciences, P.le spedali civili di brescia, Brescia, Italia 7)Department of medical and surgical sciences, P.le spedali civili di brescia,
Brescia, Italia 8)2nd department of pathology, school of medicine, P.le
spedali civili di brescia, Brescia, Italia 9)Cellular neurobiology laboratory, department of ce, Fondazione neurological institute “carlo besta”,
Milano, Italia
Backgruond. Hepatocellular Carcinoma (HCC) is one of the
most common neoplasms worldwide. Unfortunately, conventional therapy is not effective and a possible explanation for this
failure is the abnormal architectural organization of the HCC
vasculature, which causes poor blood flow and blood stagnation, leading to inadequate delivery of chemotherapeutic drugs
to cancer cells. The aim of this work was to study the phenotypic
and functional features of stromal cells (StCs) infiltrating HCC
(HCC-StCs) both in vivo and in vitro and their relationship in
HCC-induced abnormal neovascularization.
Methods. Neoplastic and normal liver tissue was obtained from
20 patients who underwent curative resection of HCC. Histologic and immunostaining was performed on formalin fixed and
embedded paraffin tissue. Cultures of StCs were obtained from
fresh tissue, neoplastic and adjacent not neoplastic liver sample.
Monoclonal antibodies (mAbs) used for this study were antiCD105, CD44, CD54, NG2, TGFβ1, TGFβ2, TGFβ3, Smooth
Muscle Actin (SMA) and PDGF. We analyzed, also, the presence
of endothelial cells (ECs) markers such as CD31, CD34, Ve-Cad,
Ang-1 and Ang-2 to evaluate vascular tumor infiltration.
Results. We defined the immunopathological features of HCC
biopsies, in particular the grade of malignancy and the rate of
microvascualr density (MVD). By immunohistochemistry, we
found that, compared to adjacent not neoplastic liver counterpart,
HCC-StCs have a reduced expression of mural cell markers NG2
and SMA both in vitro and in vivo. Moreover, HCC-StCs showed
a lower expression of the adhesion molecule NCAM, resulting
in a lower capacity of HCC-StCs to adhere on ECs by using an
adhesion test in vitro. We conclude that HCC-StCs have lost the
capacity to interact with ECs and this may concur to produce the
vascular abnormalities observed in HCC-infiltrating vessels.
Significance of egfr expression in de novo and
progressed atypical and anaplastic meningiomas:
an immunohistochemical and fluorescence in situ
hybridization study
1) Barbagallo G.M. 2) Albanese V. 3)Castaing M. 4) Lanzafame S.
1)Dipartimento di Neurochirurgia, Azienda Ospedaliero-Universitaria
Policlinico OVE, Catania, Italia 2)Dipartimento di Neurochirurgia, Azienda Ospedaliero-Universitaria Policlinico OVE, Catania, Italia 3) Dipartimento G.F. Ingrassia Istituto di Igiene, Catania, Italia 4) Dipartimento
G.F. Ingrassia Anatomia Patologica, Azienda Ospedaliero-Universitaria
Policlinico-OVE, Catania, Italia
Background. The gene encoding EGFR is located on chromosome 7. It encodes a 170 kD protein, which is a transmembrane
receptor responsible for sensing its extracellular ligands, such
as EGF and TGF-α and for transducting this proliferation sig-
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Oral communications and Posters
nal. The purpose of this study is to assess the EGFR protein
expression and the EGFR gene amplification in meningiomas
of different grade. We investigated whether there is a difference
in the EGFR protein expression and the EGFR gene amplification between the so called de novo malignant meningiomas and
meningiomas with malignant progression. We also assessed the
prognostic value of the EGFR expression on overall survival in
different groups of meningiomas.
Methods. All cases of meningiomas diagnosed from year 2000
to 2009 at the Pathology Department of the University of Catania
were reviewed. Five meningiomas with recurrences and progression were selected. They were compared with fifteen meningiomas without recurrences.
Results. The group of G I-II meningiomas without progression
showed a tendency to a better survival than the group of G I-II
meningiomas with recurrences and progression. The group of
G III meningiomas without progression showed a tendency to a
better survival than the group of G III meningiomas with recurrences and progression. The comparison between EGFR expression at baseline and after progression have showed an increased
expression of EGFR protein in the last group. The progression
from benign to atypical or anaplastic meningiomas may be due to
the increased expression of EGFR protein. However there was no
difference in the EGFR expression between the group of G I-II
de novo meningiomas and the group of G I-II progressed meningiomas. The comparison between the group of G III de novo and
progressed meningiomas and EGFR expression was not statistically significant. Our FISH study demonstrated an increase in the
number of EGFR gene copies in only 1 of the 20 meningiomas.
EGFR molecular expression, evaluated by
Immunohistochemistry (IHC) and In Situ
Fluorescent Hybridization (FISH), in lung
carcinomas
1)Baron L. 2)Postiglione M. 3)Trombetta C. 4)Maiello F.M.
5)Quarto F.
1)S.o.c. di anatomia ed istologia patologica e citop, P.o. san leonardo,
Castellammare di stabia, Italia 2)S.o.c. di anatomia ed istologia patologica e citop, P.o. san leonardo, Castellammare di stabia, Italia 3)S.o.c. di
anatomia ed istologia patologica e citop, P.o. san leonardo, Castellammare di stabia, Italia 4)S.o.c. anatomia patologica, Ospedale dei pellegrini,
Napoli, Italia 5)S.o.c. di anatomia ed istologia patologica e citop, P.o. san
leonardo, Castellammare di stabia, Italia
Background. There is an increasing knowledge of underlying
molecular mechanisms involving EGFR for targeted lung cancer
therapies.
Materials and methods. EGFR overexpression by IHC and
EGFR gene copy number by FISH were performed on surgical
histological samples from 49 primary not small cell lung carcinoma (NSCLC): 32 adenocarcinomas(ADC),17 squamous cell
carcinomas(SCC), obtained by casistic of other institution (§).
Comparisons of the proportions of variables within pathologic
characteristics were assessed by using χ2 test.
Results. We were able to detect EGFR overexpression in 18.3%
of the analyzed tumors (9 cases) and it was more frequent in SCC
(5 cases, 29.4%) than in ADC (4 cases, 12.5%).
Non statistically significant differences in degree of differentiation, lymph-node status or pathologic stage were seen.
EGFR amplification by FISH was found in 14.3%, according to criteria suggested by Varella-Garcia(Diagnostic Pathology,2006). The presence of amplification didn’t correlate with
any of morphologic parameters analyzed.
Instead some variables, such as number of EGFR gene copies per
cell and ratio of EGFR gene to chromosome 7, determined by
FISH, could be associated to tumor differentiation, lymph-node
status and tumor stage.
Out of 7 cases with gene amplification (4 ADC and 3 SCC), 4
cases (2 ADC and 2 SCC) showed EGFR overexpression by IHC
too, and 3 cases (2ADC and 1SCC),negative by IHC, had gene
amplification.
The level of agreement for EGFR overexpression by IHC and
EGFR gene amplification by FISH demonstrated a K of 0.74
(considerable agreement sec. Landis and Koch criteria).
Conclusions. EGFR protein expression could be couple with
gene amplification in most cases of NSCLC, although these results are based on a single institution experience with a relatively
small number of patients and so our data need to be verified in a
larger cohort of patients.
Primary non-Hodgkin’s lymphoma of ovaries:
a case report
1)Barresi E. 2)Schiavo N. 3)Paniccià bonifazi A. 4)Reghellin D.
5)Rucco V. 6)Lestani M.
1)U.o.c. anatomia patologica, Ulss 5 “ovest vicentino”, Arzignano (vi),
Italia 2)U.o.c. anatomia patologica, Ulss 5 “ovest vicentino”, Arzignano
(vi), Italia 3)U.o.c. anatomia patologica, Ulss 5 “ovest vicentino”, Arzignano (vi), Italia 4)U.o.c. anatomia patologica, Ulss 5 “ovest vicentino”,
Arzignano (vi), Italia 5)U.o.c. anatomia patologica, Ulss 5 “ovest vicentino”, Arzignano (vi), Italia 6)U.o.c. anatomia patologica, Ulss 5 “ovest
vicentino”, Arzignano (vi), Italia
Background. Primary ovarian lymphoma is an extremely rare
disease (0.5% of non-Hodgkin’s lymphomas and 1.5% of all
ovarian neoplasm) and limited count reports about it have been
recorded in the literature. Usually it is a secondary localization of
a systemic disease.
Methods. We describe a case of 36-year-old woman with a left
ovary mass incidentally discovered (a CT scan performed for
chronic pelvic pain revealed a tumour). Neoplastic haematic
markers were negative. A left salpingo-ophorectomy, biopsies
of right ovary, endometrium and peritoneum were performed in
laparoscopy, preserving controlateral ovary and uterus.
Results. On gross examination the ovary measured
7,5 × 5 × 4,7 cm, with a smooth and thin surface. Neoplasm
appeared homogeneously solid, white-pink, with areas of necrosis on surface of section. Microscopically ovarian tissue was
replaced by diffuse sheets of mixed medium to large lymphoid
centroblastic-like cells. These elements, frequently in apoptosis
or mitoses, are growing in cords or in alveolar-like structures.
Neither follicular structures nor “starry sky” pattern were observed. Immunohistochemically neoplastic cells expressed CD20,
CD10 and BCL6 (weak); they were negative for CD3, CD5,
BCL2 and MUM1, with a high proliferate rate (> 90%). In addition, neoplasm was negative for primary ovaric neoplastic
markers (calretinin, inhibin, cytokeratin 7, cytokeratin 20 and placental alkaline phosphatase). Ovaric, endometrial and peritoneal
biopsies were negative.
Conclusions. Histological and immunohistochemical findings
revealed a non-Hodgkin diffuse large B-cell lymphoma BCL2and BCL6+. This histotype must be differentiated from B-cell
lymphoma “unclassifiable”, with features intermediate between
diffuse large B-cell lymphoma and Burkitt lymphoma. MYC rearrangement must be studied in order to better defined lymphoma
subtype and appropriate therapy.
Progression of disease in stage I colorectal
carcinoma: are there histo-prognostic markers?
1)V. Barresi, 1)R. Lucianò, 1)E. Vitarelli, 1)A. Ieni, 2)L. Reggiani Bonetti, 4)C. Di Gregorio, 1)C. Crisafulli, 3)M. Ponz de
Leon, 1)G. Barresi
1)Patologia umana, Università di Messina, Messina, Italia; 2)Dip.
Integrato di Laboratorio, Anatomia Patologica, Az. Universitaria
Policlinico,Modena, Italia; 3)Medicina e specialità mediche, Università
di Modena e Reggio-Emilia , Modena, Italia; 4)U.O. di Anatomia patologica, Ospedale di carpi, Carpi, Italia
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Background. TNM stage I CRC is commonly characterized by
a good prognosis, with 5-year survival around 80-90%. According to most recent protocols, affected patients are only submitted
to surgical treatments although a percentage of them experience
disease progression. As a consequence, the identification of prognostic markers able to predict the clinical course of stage I CRC
may be useful in order to select and submit to adjuvant treatments
those patients with higher progression risk. In view of this, in recent studies we tested the eventual prognostic role of the quantity
of neo-angiogenesis, reflected by microvessel density (MVD),
of the immunohistochemical expression of the pro-angiogenic
vascular endothelial growth factor (VEGF) as well as of NGAL,
an iron-binding protein which is involved in colorectal cancer
progression, in stage I CRC.
Methods. MVD as well as VEGF and NGAL immuno-expression were analyzed and compared in two subgroups of surgically
resected stage I CRC: the first included cases obtained from patients deceased because of disease progression, whereby the second comprised cancers from patients still alive with no evidence
of disease progression five years after the initial diagnosis. The
prognostic value of MVD and of VEGF or NGAL expression on
the overall survival to CRC was investigated.
Results. NGAL positive cases as well as high MVD counts and
VEGF expression were significantly more frequent in the CRCs
from patients deceased of the disease in comparison to those
from patients alive after five years from surgery. Furthermore,
NGAL expression as well as high VEGF expression and MVD
appeared as significant negative prognostic markers related to a
shorter overall survival to stage I CRC, with VEGF and NGAL as
independent variables at multivariate analysis. If our preliminary
results will be further validated, assessment of these markers in
CRC specimens might be used in order to select those patients
with a higher progression risk and to submit them to adjuvant
therapies useful to prevent adverse outcome.
Performance of fine needle cytology (fnc)
in Italian breast screening programme
1)Rossi S. 2)Beccati MD. 3)Nenci I.
1) Anatomia, Istologia e Citologia patologica, Azienda Ospedaliero-Universitaria di Ferrara, Italia” e slittare i numeri delle successive di conseguenza. 2)Diagnostica citopatologica, Azienda Ospedaliero-Universitaria
di Ferrara, Ferrara, Italia 3)Anatomia, Istologia e Citologia patologica,
Università di Ferrara, Ferrara, Italia
Introduction. The Italian Breast Screening Programme started in
1996. All data of the target population and the screening-assessment process were annually recorded for quality assurance, since
1997 by GISMa-ONS. Fine needle cytology (FNC) is the most
diffuse technique for assessment of the breast abnormalities after
mammography. Core biopsy (CB) were also used.
Methods. We compared the accuracy for FNC and CB collected
by the SQTM-Project (Computerized Report for Quality of Diagnosis and Therapy for Breast Tumour) by GISMa-ONS, in the
2007 1, according to the parameters defined in the European guidelines for quality assurance, 2006. We have used the five-point
classification system for cytology (C1-5) and core biopsy (B1-5).
We correlated FNC and CB with radiology (R1-5). We calculated
also the Risk of Malignancy (%) on a series of 1688 FNC performed by the Breast-Screening-Programme Ferrara,1997-2006,
to evaluate if cytology may assist in clinical management.
Results. SQTM-Project assembled 3136 screen-detected operated
tumours. The parameters for cytology were (%): AS 66,09; CS
89,97; PPV C5 99,37, C4 92,33, C3 57,61; FN and FP rate 1,68
and 0,42, inadequate rate 9,45. The parameters for histology were
(%): AS 86,41; CS 94,42; PPV B5 99,40, B4 70,96, B3 33,01;
FN and FP 1,72 and 0,51, miss rate from cancer 5,57. Screening
Ferrara. The Risk of Malignancy was (%): C1 14,54; C2 2,18;
C3 32,66; C4 88,84; C5 99,67.
Conclusions. SQTM-Project. The sensitivity and PPV for cancer
were high in both techniques. Since cytology is fast, non invasive
and cost-effective it was the first choice in R4-5 categories. PPV
for B3 were better than C3. Microhistology was preferable in R3.
Screening Ferrara. Based on the Risk of Malignancy we propose
the following clinical management: C1, C2/R4-5, C3/R3 microbiopsy; C2/R1-2, screening; C2/R3, microbiopsy or FU; C3/R1-2,
FU; C3/R4-5 excisional biopsy; C4-5 therapeutic excision and
sentinel lymphnode.
References
1
ONS, Ottavo Rapporto 2009, Ed. M. Zappa.
Ubch10 in cervical intraepithelial neoplasia (CIN)
as a novel marker of cell proliferation
1)Bellevicine C. 2)Desiderio D. 3)Varone V. 4)De luca C. 5)Malapelle U. 6)Troncone G.
1)Scienze biomorfologiche e funzionali, Università di Napoli “Federico
II”, Napoli, Italia 2)Scienze biomorfologiche e funzionali, Università di
Napoli “Federico II”, Napoli, Italia 3)Scienze biomorfologiche e funzionali, Università di Napoli “Federico II”, Napoli, Italia 4)Scienze biomorfologiche e funzionali, Università di Napoli “Federico II”, Napoli, Italia
5)Scienze biomorfologiche e funzionali, Università di Napoli “Federico
II”, Napoli, Italia 6)Scienze biomorfologiche e funzionali, Università di
Napoli “Federico II”, Napoli, Italia
Backgrounds. Morphological diagnosis of CIN has intraobserver
and interobserver variability. Ki67 may sometimes be aspecific. UbcH10 is a novel proliferation marker. Here we analyzed
UbcH10 in CIN by RT-PCR and by immunohistochemistry
(IHC) in relation to Ki-67.
Methods. Cervical biopsies representative of cervicitis (n = 18),
CIN I (n = 14), CIN II (n = 14) and CIN III (n = 6) were UbcH10
and Ki-67 immunostained; a layer(s)-based approach (negative,
1/3+, 2/3+ and 3/3+) was applied. In addition, UbcH10 RT-PCR
was also performed on fresh biopsies.
Results. Most cases of cervicitis were Ki67 (95%) and Ubch10
(78%) negative. In CIN I, UbcH10 and Ki67 yielded the same
staining pattern (negative: 42%; 1/3+: 28%; 2/3+: 14%; 3/3+:
14%). Higher levels of expression were found in CIN II both for
Ubch10 (negative: 21%; 1/3+: 14%; 2/3+: 64%; 3/3+: 7%) and
Ki67 (negative: 14%; 1/3+:21%, 2/3+: 50%; 3/3+: 14%). Similarly, in CIN III UbcH10 (2/3+: 50%; 3/3+: 50%) and Ki-67 (2/3+:
17%; 3/3+: 83%) were highly expressed. Consistently, UbcH10
mRNA levels also increased according to the severity of CIN.
Conclusion. UbcH10 a both qRT-PCR and IHC levels is useful
to increase CIN diagnostic accuracy. Its role in cervical cytology
is currently under investigation.
IMP3 expression in phyllodes tumours of breast
1)Bellezza G. 2)Ferri I. 3)Loreti E. 4)Del Sordo R. 5)Colella R.
6)Sidoni A. 7)Cavaliere A.
1)Institute of Pathological Anatomy, Perugia University, Perugia, Italy
2)Institute of Pathological Anatomy, Perugia University, Perugia, Italy
3)Institute of Pathological Anatomy, Perugia University, Perugia, Italy
4)Institute of Pathological Anatomy, Perugia University, Perugia, Italy
5)Institute of Pathological Anatomy, Perugia University, Perugia, Italy
6)Institute of Pathological Anatomy, Terni, Perugia University, Italy 7)Institute of Pathological Anatomy, Perugia University, Perugia, Italy
Background. Phyllodes tumours (PTs) of the breast are uncommon neoplasms with potential for local recurrence or metastatic
spread. The WHO classification 1 divided PTs into benign, borderline and malignant. However, prognostic assessments based
solely on histological parameters, can be problematic and many
biological markers have been proposed. In this study, we investigated if IMP3, a member of the insulin-like growth factor II
mRNA binding protein, was differently expressed in the three
groups of PTs and could be predictive of behaviour.
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Oral communications and Posters
Methods. Sixty-two PTs were classified by morphological criteria, proposed by WHO, in 40 benign, 13 borderline and 9 malignant. Immunohistochemical expression of IMP3 was evaluated
in stromal neoplastic cells; cases with more than 10% of positive
cells were considered as positive. Some other variables, including surgery, status margin and pathological features, were also
compared among PTs subgroups.
Results. Malignant PTs were more frequent in older patients
(mean: 59 years) and larger in size (mean: 90 mm). Twelve
patients, who experienced local recurrences (7 benign, 3 borderline and 2 malignant), were originally treated mainly by simple
lumpectomy. In these cases surgical margins were positive more
frequently (45% of cases) than in non recurrent tumours (26%).
In 3 malignant cases lymph nodes and lung metastases were also
seen. IMP3 expression was observed in 9 cases (15%). In benign
and borderline PTs IMP3 was present respectively in 5% and 15%
of cases, while in malignant PTs in 56% (p = .001). No differences
were noted between PTs that did and did not recur, while, interestingly, IMP3 expression was higher (50% of cases) in recurrences.
Conclusions. In conclusion, our study showed that IMP3 could
be an helpful diagnostic tool to discriminate benign and borderline from malignant PTs and its expression in recurrences seems
to be related with a more aggressive behaviour.
References
1
Tavassoli et al., WHO, 2003.
Eosinophlic dysplasia of the cervix uteri:
morphology and immunostochemical features
1)Bellisano G. 2)Peer I. 3)Faa G. 4)Ambu R. 5)Tolu G.A.
6)Kasal A. 7)Antoniazzi S. 8)Vittadello F. 9)Egarter-Vigl E.
10)Negri G.
1)U.O. Anatomia Patologica, San Martino, Oristano, Italia / Anatomia
Patologica - Università di Cagliari, San Giovanni di Dio, Cagliari, Italia 2)Anatomia Patologica, Ospedale Centrale, Bolzano, Italia 3)Anatomia Patologica - Università di Cagliari, San Giovanni di Dio, Cagliari,
Italia 4)Anatomia Patologica - Università di Cagliari, San Giovanni di
Dio, Cagliari, Italia 5)U.O. Anatomia Patologica, San Martino, Oristano,
Italia 6)Anatomia Patologica - Università di Cagliari, Ospedale Centrale, Bolzano, Italia 7)Anatomia Patologica, Ospedale Centrale, Bolzano,
Italia 8)Explora, Ricerca ed analisi statistica, Padova, Italia 9)Anatomia
Patologica, Ospedale Centrale, Bolzano, Italia 10)Anatomia Patologica,
Ospedale Centrale, Bolzano, Italia
Background. Eosinophilic dysplasia (ED) of the cervix uteri is a
particular kind of dysplasia that retains metaplastic features. The
aim of this study was to evaluate the biologic potentiality of ED
using p16ink4a (p16) and HPV-L1 (L1) as markers of HPV-induced carcinogenesis 1.
Methods. Histological samples from 82 women with a previous
diagnosis of ED were collected from the archive of the Department of Pathology of the Central Hospital of Bolzano, Italy. All
cases were reviewed using the diagnostic criteria for ED described by Ma et al. 2: 1) lack of normal maturation; 2) relatively
abundant eosinophilic cytoplasm and distinct cell borders compared with conventional HSIL; 3) mildly to moderately increased
nuclear/cytoplasmic ratio; and 4) dysplastic nuclei showing nuclear enlargement, hyperchromasia, variable nuclear membrane
irregularities, and appreciable nucleoli. Immunohistochemical
analysis for p16 and L1 was performed on all ED specimens and
on 31 control specimens with a high-grade Cervical Intraepithelial Neoplasia (CIN 2-3) of usual type.
Results. After revision of the histological samples, features of
ED were confirmed in 66 out of 82 (81%) samples. The original
diagnosis was CIN1 in 6 out of 66 cases, CIN 2 in 37 and CIN3
in 23. In 58 out of 66 (88%) specimens, ED was associated with
CIN of usual type. Diffuse p16 expression was detected in all 66
ED, whereas L1 was expressed in 18 out of 66 (27%) cases. L1+
ED were most often (67%) associated with an original CIN1 di-
agnosis. In conclusion, Eosinophilic dysplasia of the cervix uteri
is frequently associated with CIN of usual type and mostly shows
a high-grade immunohistochemical pattern (p16+/L1-). However,
HPV-L1 may be, expressed in some ED (p16+/L1+), with a pattern similar to that of still productive low-grade lesions, this could
indicate a higher tendency to spontaneous regression.
References
1
Negri G, Bellisano G, Zannoni GF, et al. p16ink4a and HPV L1
Immunohistochemistry is Helpful for Estimating the Behavior of
Low-grade Dysplastic Lesions of the Cervix Uteri. Am J Surg Pathol
2008;32:1715-20.
2
Ma L, Fisk J, Zhang R, et al. Eosinophilic Dysplasia of the Cervix:
A Newly Recognized Variant of Cervical Squamous Intraepithelial
Neoplasia. Am J Surg Pathol 2004;11:1474-84.
Haemaobium eggs detection in human bladder
cancer and sporocysts in snail vectors
1)Benerini Gatta L. 2)Balzarini P. 3)Cadei M. 4)Castelli F.
5)Grigolato P.
1)2nd department of pathology, Spedali civili di brescia, Brescia, Italia
2)2nd department of pathology, Spedali civili di brescia, Brescia, Italia
3)2nd department of pathology, Spedali civili di brescia, Brescia, Italia
4)Institute of infectious and tropical desaese, Spedali civili di brescia,
Brescia, Italia 5)2nd department of pathology, Spedali civili di brescia,
Brescia, Italia
Background. Schistosomiasis or bilharzia is a tropical parasitic disease caused by blood-dwelling fluke worms of the genus
Schistosoma. In Burkina Faso the main schistosomes infecting
human beings are S. haematobium, transmitted by Bulinus snails
and causing urinary schistosomiasis. Schistosoma haematobium
eggs play a central role in the development of bladder cancer.
Investigation of eggs in the urine is the most sensitive and specific method for diagnosing active schistosomiasis. But the eggs
may not be detected in urine during chronic parasitation stages
and cancer. So, the final diagnosis is based on the presence of
granulomas or dysplastic cells and schistosoma eggs in the
submucosa of bladder biopsies. We were interested in set up a
molecular method in which S. heamatobium eggs were detected
by immunohistochemistry and polymerase chain reaction, in
formalin-fixed and paraffin-embedded human bladder cancer or
snail vectors.
Methods. A total of four vesicals carcinoma were obtained from
patients undergoing curative intent surgical resections at the S.
Camille Medical Centre, Nanorò, Burkina Faso. Bulinus snails
were collected from transmission site of Nanorò region of the
Burkina Faso. Immunohistochemistry, polymerase chain reaction
(PCR) and sequencing of S. haematobium eggs was led in formalin-fixed and paraffin-embedded tissues.
Results. We report four cases of vesical cancer schistosomiasis-related. Our data showed that the immunoreaction and amplification
detection led to correct diagnosis of the specific species of Schistosoma in the human cancer. Finally we suggest the use of molecular
methods in the snail vectors for the detection of sporocysts.
L1 AND p16 proteins and HPV DNA in the low-grade
cervical intraepithelial neoplasia (CIN1)
1)Benerini Gatta L. 2)Berenzi A. 3)Balzarini P. 4)Dessy E. 5)Angiero F. 6)Alessandri G. 7)Grigolato P. 8)Benetti A.
1)2nd department of pathology, Spedali civili di brescia, Brescia, Italia
2)2nd department of pathology, Spedali civili di brescia, Brescia, Italia
3)2nd department of pathology, Spedali civili di brescia, Brescia, Italia
4)2nd department of pathology, Spedali civili di brescia, Brescia, Italia
5)Department of pathology, University of milano - bicocca, Milano, Italia 6)Cellular neurobiology laboratory, Fondazione neurological institute
“carlo besta”, Milano, Italia 7)2nd department of pathology, Spedali civili di brescia, Brescia, Italia 8)2nd department of pathology, Spedali civili
di brescia, Brescia, Italia
248
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Background. We evaluated the expression of p16, Ki-67, L1
proteins, and HPV DNA, as molecular markers for diagnosis and
transforming potentiality of low cervical intraepithelial neoplasia
(CIN1).
Methods. Cervical specimens from 72 patients, including 32
cases of CIN1, 10 of CIN2, 10 of CIN3/CIS, 10 of squamous
cell carcinoma (SCC), and 10 cases of chronic cervicitis, were
collected. The expression of p16, Ki-67, L1 antigens was evaluated by immunohistochemical methods. The HPV/nested PCR
method was applied to amplify the HPV/L1 region and high risk
E6/E7 genome 16-18-33-35-52-58. Catalyzed Signal-Amplified
Colorimetric DNA In Situ Hybridization (CSAC/ISH) of high
oncogenic viral risk was also applied.
Results. Ki-67 and p16 increased linearly from control cases to
CIN1, CIN2 and CIN3 cases, with a peak in the SCC cases. In
contrast L1 expression was inversely correlated with malignant
transformation. We divided CIN1 patients into four groups:
L1-p16+, L1+p16-, L1-p16-, and L1+p16+ and we combined
immunohistochemical results with HPV/PCR, L1/PCR and highrisk E6/E7 genome and CSAC/ISH data. We found that only the
L1-p16+ group correlated with malignant transformation (100%
of CIN2, CIN3 and SCC cases) and was present in the 23% of
the CIN1. Moreover, 52% of CIN1 cases showed the presence of
HPV/DNA+. In particular, within L1-p16+ group, in 4 out of 7
cases there was high risk E6/E7 HPV genome and, in one case, it
was integrated into host DNA, as confirmed by CSAC/ISH. We
conclude that in CIN1 patients, the HPV DNA, in particular high
risk E6/E7 genome, has to be investigated in order to distinguish
high from low risk oncogenic patient groups.
Human papillomavirus DNA and p16 protein
expression in squamous cell carcinoma of the lung
1)Benerini Gatta L. 2)Dessy E. 3)Berenzi A. 4)Benetti A. 5)Balzarini P. 6)Tironi A. 7)Angiero F. 8)Grigolato P.
1)2nd department of pathology, P.le spedali civili di brescia, Brescia,
Italia 2)2nd department of pathology, P.le spedali civili di brescia, Brescia, Italia 3)2nd department of pathology, P.le spedali civili di brescia,
Brescia, Italia 4)2nd department of pathology, P.le spedali civili di brescia, Brescia, Italia 5)2nd department of pathology, P.le spedali civili di
brescia, Brescia, Italia 6)2nd department of pathology, P.le spedali civili
di brescia, Brescia, Italia 7)Pathological anatomy, Università di milano
bicocca, Milano, Italia 8)2nd department of pathology, P.le spedali civili
di brescia, Brescia, Italia
Background. HPV is a small DNA virus that usually infects
squamous epithelial cells. In malignant transformation of uterine
cervix, the expression of the E6/E7 viral proteins is associated to
the alterated expression of p16 protein (a key protein in cell cycle
regulation). Data on human papilloma virus (HPV) involvement
in preneoplastic and neoplastic lesions of the lung are limited
and conflicting. To investigate the role of HPV infection in lung
tumorigenesis, we studied the expression of p16 protein and the
relation with the presence of HPV DNA in lung squamous cell
carcinoma (SCC).
Methods. 41 cases of formalin fixed and paraffin-embedded human lung specimens were obtained from the archives of the 2nd
Department of Pathology, Spedali Civili, University of Brescia,
Italy. 31 cases of lung primary SCC and 10 control cases (non
neoplastic non squamous specimens), in the study were included.
Genomic and viral DNA of SCC samples were obtained from the
paraffin block. DNA was extracted and HPV DNA was detected
by nested polymerase chain reaction. The expression of p16 protein was evaluated by immunohistochemistry.
Results. Two cases of SCC were positive for HPV PCR. The
expression of the p16 protein was demonstrated immunohistochemically in the same specimens. The presence of HPV DNA
was correlated to p16 protein expression. The results suggest that
the HPV DNA might play a pivotal role in development and/or
progression of a small group of lung SCC.
Perspective evaluation of proteomic analysis in
prostate cancer and benign prostatic hyperplasia
1)Bergamini S. 2)Bellei E. 3)Monari E. 4)Reggiani Bonetti L.
5)De Gaetani C. 6)Sighinolfi M.C. 7)Micali S. 8)De Stefani S.
9)Bianchi G. 10)Tomasi A.
1) Dipartimento Integrato di Laboratori, Anatomia Patologica e Medicina
Legale, Università di Modena e Reggio Emilia, Modena, Italia 2) Dipartimento Integrato di Laboratori, Anatomia Patologica e Medicina Legale,
Università di Modena e Reggio Emilia, Modena, Italia 3)Dipartimento Integrato di Laboratori, Anatomia Patologica e Medicina Legale, Università
di Modena e Reggio Emilia, Modena, Italia 4) Dipartimento Integrato di
Laboratori, Anatomia Patologica e Medicina Legale, Università di Modena e Reggio Emilia, Modena, Italia 5) Dipartimento Integrato di Laboratori, Anatomia Patologica e Medicina Legale, Università di Modena e
Reggio Emilia, Modena, Italia 6) UO di Urologia, Università di Modena
e Reggio Emilia, Modena, Italia 7)UO di Urologia, Università di Modena
e Reggio Emilia, Modena, Italia 8)UO di Urologia, Università di Modena
e Reggio Emilia, Modena, Italia 9)UO di Urologia, Università di Modena
e Reggio Emilia, Mo
Background. The limited sensitivity and specificity of PSA for
diagnosis of prostate cancer (PCa) highlight the need of more
predictive diagnostic markers: the proteomic analysis might represents a good approach for their discovery and identification. In
this study, we described a proteomic investigation on serum of
82 patients.
Methods. We recruited 28 patients with PCa (Group 1) and 30
subjects with benign prostatic hyperplasia (BPH) and without
PCa histologically confirmed, as control (Group 2). The mean
of the age was 67.0 years (SD ± 6.6) in Group 1 and 67.8 years
(SD ± 7.0) in Group 2, respectively; the mean of PSA value was
9.9 ng/ml in Group 1 and 6.2 ng/ml in Group 2. The serum was
depleted of the 7 high-abundance proteins by Multiple Affinity
Removal System (MARS HuPL7, Agilent) to permit the detection
of the low-abundance proteins. The samples proteomic profile was
obtained using the Surface Enhanced Laser Desorption/Ionization
Time-of-Flight-Mass Spectrometry (SELDI-TOF-MS). Two different types of chromatographic surfaces (ProteinChip) were used:
the IMAC30 (metal affinity) and the H50 (hydrophobic surface).
Results. Proteomic analysis has revealed several cluster of peaks
according to the ProteinChip used. In particolar, the IMAC30
ProteinChip showed three protein peaks differentially expressed
(p < 0.05) in BPH compared to PCa (2210, 2929 and 9082 kDa).
Separating the Group 2 in two different subgroups (BPH with or
without prostatitis) has emerged that these cluster peaks remained
differentially expressed among the BPH/prostatitis patients and
those with PCa. The three protein peaks could therefore selectively characterize the presence of PCa. These data are preliminary
and require additional assessments to confirm the presence of
differentially expressed proteins. However, the SELDI-TOF-MS
technique might represents an innovative and promising approach
for the discovery of potential predictive biomarkers of PCa.
HER2 testing in gastric cancer.
immunohistochemistry (IHC) and fluorescence
in situ hybridization (FISH) comparison
1)Bettelli S. 2)Fontana A. 3)Losi L. 4)Reggiani Bonetti L.
5)Bertolini F. 6)Zironi S. 7)Scarabelli L. 8)Luppi G. 9)Conte PF.
10)Maiorana A.
1)Dip Integr di Lab, Anat Pat e Med Leg, Az. Ospedaliero Universitaria
Policlinico, Modena, Italia 2) Dip. Oncologia, Ematol e Mal Respiratorie,
Az. Ospedaliero Universitaria Policlinico, Modena, Italia 3)Dip Integr
di Lab, Anat Pat e Med Leg, Az. Ospedaliero Universitaria Policlinico,
Modena, Italia 4)Dip Integr di Lab, Anat Pat e Med Leg, Az. Ospedaliero
Universitaria Policlinico, Modena, Italia 5)Dip. Oncologia, Ematol e Mal
Respiratorie, Az. Ospedaliero Universitaria Policlinico, Modena, Italia
6)Dip. Oncologia, Ematol e Mal Respiratorie, Az. Ospedaliero Universitaria Policlinico, Modena, Italia 7)Dip. Oncologia, Ematol e Mal Respiratorie, Az. Ospedaliero Universitaria Policlinico Modena, Italia 8)Dip.
Oral communications and Posters
Oncologia, Ematol e Mal Respiratorie, Az. Ospedaliero Universitaria
Policlinico, Modena, Italia 9)Dip. Oncologia, Ematol e Mal Respiratorie,
Az. Ospedaliero Universitaria Policlinico, Modena, Italia 10)Dip Integr di
Lab, Anat Pat e Med Leg, Az. Ospedaliero Universitaria Policlinico
Background. Overexpression of the HER2 protein in gastric cancer have been reported from 6% to 35% of cases. Tumor localization, histological type, tumor grading and tumor heterogeneity are
mostly related to this wide range.
Methods. From October 2009, we tested HER2 status in 26
gastric cancers newly diagnosed in the Pathologic Anatomy of
Modena. IHC was performed in 20 patients, while FISH analysis
in 16. ICH and FISH were both carried out in 10 patients.
Results. HER2 protein overexpression was observed in 3/20
(15%) patients. HER2 amplification was detected in 5/16 (31%).
Among cases evaluate through both IHC and FISH, 3/10 (30%)
showed IHC score 0, but high amplification by FISH. Differently
from what is generally observed in breast cancer, our data showed
an unexpected discrepancy between FISH and ISH results in the
assessment of HER 2 status in a single institution analysis of
gastric cancer patients.
Muscle spindle and pacinian corpuscle:
conceptions, misconceptions, and the far-fetched
hypothesis of an experienced surgical pathologist
1)M. Bisceglia 2)S. Bisceglia 3)M.L. Bisceglia
1)Department of Pathology, Division of Anatomic Pathology, IRCCS –
Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy; 2)
Nursing School, Faculty of Medicine, University of Foggia,Italy; 3) School
of Pharmacy, University of Parma, Parma, Italy
Background. The muscle or neuromuscular spindle is a proprioceptive microanatomic structure, which together with the Golgi tendon
organ, is responsible for the reflex arc that determines the tonic state
of a muscle. It is penetrated by both sensory and motor nerve fibres,
gamma (mainly) and beta efferents, and therefore has sensory and
motor functions 1. The neuromuscular spindles are found in all skeletal muscles with facial muscles and (perhaps) diaphragm the only
exceptions. The Pacinian corpuscle (or corpuscle of Vater-Pacini) is
a pure mechanoreceptor, responsive to pressure. It is typically found
in the skin, subcutis and superficial soft tissues, but uncommonly
may also be seen in the soft tissue of body cavities and in the serosa
and subserosa of visceral organs.1 Very rarely the Pacinian corpuscle may be found in skeletal muscle, chiefly in relation to fascia
and aponeuroses, and, when found in skeletal muscle, the Pacinian
corpuscle is intimately related to the neuromuscular spindle. Usually both muscle spindle and Pacini corpuscle are too unremarkable
microanatomical findings to focus on during the course of routine
work in surgical pathology.
Objectives. i. To report on the incidental finding of a curious
muscle spindle, the “fibrous” capsule of which mimicked the “lamellar” body of Pacinian corpuscle. ii. To describe the sequence of
events leading to the correct recognition of the muscle spindle. iii.
To emphasize the fundamental anatomical notions ignored by the
pathologist; iv. To list the pathological conditions, partly theoretical, which can affect the two aforesaid microanatomic structures.
Materials. During the microscopic examination of a resection
margin of a skeletal muscle surgical resection specimen harbouring a capillary haemangioma, removed from the thigh of a
42-year old male, a structure which at first glance appeared to
be a neuromuscular spindle was noted by the pathologist (MB),
an unremarkable finding in that context. Two students, one (SB)
who was prepared to take his anatomy exam at the completion
of the first year of nursing school, and the other (MLB), in her
last year of pharmacy school, were asked to identify that microscopic structure. Both students answered that the structure
under the microscope was a Pacinian corpuscle. Their concordant
answers provoked the testing pathologist to scrutinize the slide
more closely and come to suspect that this muscle spindle with a
249
“lamellar” fibrous capsule was likely to be a “hybrid” structure,
in other words a new finding, specifically a composite structure
comprised of a peripherally located Pacinian corpuscle wrapped
around a true central neuromuscular spindle. The suspicion became convincing since nowhere in any of the other sections from
the entire surgical specimen was a similar structure found nor had
the pathologist ever seen something similar, despite having some
experience in musculoskeletal and neuromuscular pathology.
Methods. A true Pacinian corpuscle from an archival skin resection specimen was examined and immunostained, with the
following expected results: the “onion skin-like” lamellar body
of the Pacinian corpuscle was EMA-positive; the sensory nerve
fibre penetrating the lamellar body was neurofilament-positive;
and the Schwann cells enveloping the nerve fibre axon were
S-100 positive. The new finding (thought to be chimeric/composite muscle Pacinian spindle), was also immunostained: the
intrafusal fibres of the spindle were obviously desmin positive;
the “lamellar” sheath, namely what was supposed to be the
wrapping Pacinian corpuscle, was successfully EMA positive
and CD34 negative; neurofilament stained d axons in the center
of the spindle.
Discussion. In essence, in transverse section, the muscle spindle
normally measures 200 µm and is made of skeletal muscle microfibres (variably 5 to 14 in number) surrounded by a “fibrous”
capsule made of 9 to 15 concentric, usually tightly arranged,
layers of flattened epithelial-like cells (also called “capsular
sheet cells”). This “fibrous” capsule represents an extension of
the perineurium enclosing the nerve fibres serving the intrafusal
muscle fibres 1 2. The imaginary hypothesis which was construed
to support fusion of the Pacinian corpuscle and muscle spindle
was based on the misconception that the “fibrous” capsule was
made of EMA negative ordinary fibrous connective tissue.
Actually this “fibrous” capsule of the muscle spindle can be
confidently equated to the terminal perineurium ensheathing the
peripheral nerve twigs, made of EMA positive cells. The VaterPacini corpuscle, normally measuring up 2 mm in length, is made
of a lamellar body and a small central core, the former made of 30
or more concentric loosely arranged lamellae, composed of flat
perineurial cells, the latter containing the terminal non-myelinated sensory nerve fibre. The neuromuscular spindle may normally vary in size and number, but in some circumstances they
also vary in arrangement, appearing in tandem, even sharing a
common capsule, and in groups. The intrafusal fibres are affected
in some neuromuscular diseases (myotonic dystrophy for splitting, poliomyelitis for rarifying), or appear pseudohypertrophic
in others as in Werdnig-Hoffman disease (in comparison with the
extrafusal fibres) 3, and were supposed to be the tissue from which
alveolar soft part sarcoma arises 4. The “fibrous” capsule may
become thickened as in ageing or cellular, fibrosed or edematous
as in Duchenne dystrophy 3. In our case the muscle spindle was
oversized (350 to 400 µm in size after several measurements
on transverse sections), and exhibited a thickened capsule with
separated lamellae encroaching on the periaxial space, which is
normally present between intrafusal fibres and the capsule: we
could not ascertain whether this was only anatomical variation
or due to some unknown cause (?trauma). The Vater-Pacini corpuscles may also normally vary in size and number according to
specific anatomical locations, and may also appear hyperplastic
(“Pacinian corpuscle hyperplasia” 5) or simulate neoplastic conditions (Pacinian neuroma, Pacinian neurofibroma o Pacinian
perineurioma). Finally, at least in theory, one cannot exclude that
perineuriomas might arise from EMA positive perineurial cells
either of the muscle spindle capsule or the lamellar body of the
Vater-Pacini corpuscle.
References
1
Standring S. Gray’s Anatomy. 40th Edition, Churchill Livingstone
2008, pp. 59-60
2
Banks RW, Barker D. The Muscle Spindle. In: Engel AG, Franzini-
250
3
4
5
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Armstrong C (eds). Myology. Third Edition. New York: McGraw-Hill,
2004, pp. 489-509.
Swash M. Pathology of the muscle spindle. In: Mastaglia FL, Walton
J (eds). Skeletal Muscle Pathology. Edinburgh: Churchill Livingstone
1982, pp. 508-36.
Christopherson WM, Foote FW Jr, Stewart FW. Alveolar soft-part sarcomas; structurally characteristic tumors of uncertain histogenesis.
Cancer 1952;5:100-11.
Reznik M, Thiry A, Fridman V. Painful hyperplasia and hypertrophy
of Pacinian corpuscles in the hand: report of two cases with immunohistochemical and ultrastructural studies, and a review of the
literature. Am J Dermatopathol 1998;20:203-7.
Glomus tumor of stomach. Report of 8 cases
and review of the literature.
1) Bisceglia M. 2) Bleiweiss I. 3)Ben Dor D. 4) Magro G. 5)
Sickel J. 6) Carosi I. 7) Miettinen M.
1)Department of Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy 2)Department of Pathology, Mount
Sinai School of Medicine, New York, NY, USA 3)Department of Pathology, Barzilai Medical Center, Ashkelon, Israel 4)Department of Pathology,
University of Catania, Catania, Italy 5)Department of Pathology, El Camino Hospital, Grant Road Mountain View, CA, USA 6) Department of
Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni
Rotondo, Italy 7)Department of Soft Tissue Pathology, Armed Forces Institute of Pathology, Washington, DC, USA. Background. Glomus tumors (GT) are thought to originate from
glomocytes. Glomocytes are round, distinct, epithelioid cells with
ultrastructural and immunohistochemical features of modified
smooth muscle cells, functioning as sphincters of Hoyer-Sucquet
canals of glomera. Glomera represent normal arteriovenous shunts,
abundantly supplied with nerve fibers that act as regulators of temperature in several locations throughout the body. The most common locations of glomera are the skin and peripheral soft tissue of
the distal parts of extremities. However, they are also encountered
in large cavities and visceral organs, including the alimentary tract,
where their function is related to absorption. Paralleling the usual
distribution of glomera, GT are most common in the skin and soft
tissue of acral sites. GT of the stomach is a very rare neoplasm that
was first recognized by Saul Kay et al. in 1951, who reported 3
cases at that time 1. Since then, most published cases have appeared
as single case reports with very few series on record.
Objectives. 1. To present our personal cases of GT of stomach.
2. To comprehensively review the English literature on this
subject and document the total number of gastric GT reported
so far.
Methods. 1 A systematic search of our combined databases was
performed to identify cases of gastric GT. 2. A computerized
literature search of PubMed/Medline was performed between
1951 and April 2010 using [glomus tumor of stomach] as a search
term.
Results. Analysis of cases. 8 original (unpublished) cases were
found in our institutional files: 6 cases were males and 2 cases
were females, all were adults or elderly. The ages of the males
ranged between 38 and 81 (with the others 54, 62, 67, 79 in
between); the two females were 48 and 55, respectively. Gastric bleeding was the presenting symptom in 7 out of 8 cases
(hematemesis in 5, melena 2). The tumors ranged in size from
1.5 to 8 cm. The majority of tumors were located in the antrum
(4); 1 tumor was in the body, 1 was in the fundus, and in 2 the
site was not recorded. Mucosal ulceration was seen in 6 cases.
Either leiomyoma or lipoma was the preoperative diagnosis in
4 cases. All tumors were surgically treated: partial gastrectomy
in 4, wedge resection in 3, lumpectomy in 1. All tumors were
intramural, well circumscribed and confined to the muscularis
propria of the organ, except for one, the largest, which was locally
invasive. All tumors were histologically benign (very low mitotic
index, absence of necrosis, no cytological atypia, no spindling),
except for one, the largest, which exhibited high mitotic rate,
coagulation necrosis, and nuclear atypia, and was diagnosed as
malignant. The morphological pattern was predominantly classic
solid glomus tumor, with associated glomangiomatous areas in 3.
Immunohistochemically: vimentin and alpha-SMA were positive
in all; cytokeratins, CD117, and neuroendocrine markers were
always negative. Follow-up: 6 patients are alive with no evidence
of disease (follow-up ranging from 3 to 18 years; median 8), 1
patient (the eldest) shortly died after surgery, the ma1ignant case
was lost to follow-up. Review of the Literature. To date 104 GT
have been recorded in the English Literature since 1951. Only
4 papers included more than 1 case 1-4. The two largest series
were compiled in 1969 by Appelman and Helwig 3 and in 2002
by Miettinen et al. 4, both from the Armed Forces Institutes of
Pathology (Washington, D.C.), who reported 12 and 31 cases,
respectively. In most cases the tumor was solitary, but in 4 cases
multiple tumors were described. Most GTs are histologically
benign, but 3 malignant cases have been published.
Discussion. GTs most often occur in the gastric antrum of adults,
without any sex predilection. The signs and symptoms can be
variable: bleeding due to surface ulceration is a common finding,
and the bleeding can be quite profuse due to the extensively vascularized nature of the lesion, leading in some instances to anemia or to emergency surgical gastrectomy; other frequent clinical
manifestations are pain, nausea and vomiting. The majority of
the lesions are solitary, although multiple gastric glomus tumors
have been described 3. The main differential diagnosis is with epithelioid GIST and carcinoid. Analogous to their soft tissue counterpart, large size, high mitotic index (≥ 5M:50HPF), cytologic
atypia including spindling, necrosis, and local infiltrative growth
are all possible histologic indicators of malignancy 5 in GT of the
stomach. Additionally we note that, in the experience of one of us
(MM), one gastric GT with limited atypia (spindling present) and
mitotic rate < 5/50 metastasized and killed the patient 4.
Conclusions. 1. GT of stomach is rare (according to Miettinen
et al. 4 < 1% compared to GIST). 2. Most gastric GT are benign.
3. Malignant GT of stomach is extremely rare, but may occur.
4. Smooth muscle differentiation is a constant immunohistochemical finding.
References
1
Kay S, Callahan WP Jr, Murray MR, et al. Glomus tumors of the stomach. Cancer 1951;4:726-36.
2
Allen RA, Dahlin DC. Glomus tumor of the stomach: report of 2 cases.
Proc Staff Meet Mayo Clin 1954;29:429-36.
3
Appelman HD, Helwig EB. Glomus tumors of the stomach. Cancer
1969;23:203-13.
4
Miettinen M, Paal E, Lasota J, et al. Gastrointestinal glomus tumors: a
clinicopathologic, immunohistochemical, and molecular genetic study
of 32 cases. Am J Surg Pathol 2002;26:301-11.
5
Folpe AL, Fanburg-Smith JC, Miettinen M, et al. Atypical and malignant glomus tumors: analysis of 52 cases, with a proposal for the
reclassification of glomus tumors. Am J Surg Pathol 2001;25:1-12.
Lipofuscin-like granules of the juxtaglomerular
apparatus of the kidney. The diagnostic
significance of a quasi-normal subcellular
structure only incidentally encountered in the
course of routine ultrastructural evaluation of
renal biopsies for diagnostic purposes
1) Bisceglia M. 2) D’Errico M. 3) Carosi I. 4) Grasso M.A.
5)Pasquinelli G.
1)Unit of Anatomic Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy 2)Unit of Nephrology, IRCCS Casa
Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy 3) Unit of
Anatomic Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San
Giovanni Rotondo, Italy 4)Hospital Pharmacy Program, School of Pharmacy, “La Sapienza” University, Rome, Italy 5)Department of Clinical
Pathology, University of Bologna, Italy
Oral communications and Posters
Background. In a systematic study of 114 human kidney tissue
specimens, from patients ranging in age from 2 to 70, in 1965 Biava and West reported the light and electron microscopic features
of a particular type of “lipofuscin-like granule” mainly in the
cytoplasm of vascular smooth muscle cells, but also in juxtaglomerular cells, and (more rarely) the lacis cells of human kidneys 1.
The granules were found in all cases, including healthy kidneys,
but their number correlated with age, arterial hypertension and
diabetes mellitus, and were larger in diabetic than in non-diabetic
patients. Neither the awareness of the existence of this finding
nor its clinical significance is widespread among either pathologists or nephrologists. Standard nephropathology textbooks do
not even mention these structures either in the normal anatomy
section nor in any other specific pathology chapter. The afore
mentioned lipofuscin-like granules were incidentally observed
in 4 cases during the routine electron microscope examination of
440 renal biopsies performed at Casa Sollievo della Sofferenza
hospital between 1991 and 2009, for investigation of medical
nephropathies.
Objectives. 1. To review the world literature to find out how
many articles dealing with this particular type of granule in the
context of renal pathology were on record. 2. To report the ultrastructural features of this finding. 3. To see if systemic arterial
hypertension and/or the status of diabetes mellitus were present
also in our cases as originally demonstrated by Biava and West
45 years earlier. 4. To correlate the significance, if any, of these
granules with the specific renal disease for which kidney biopsies
were performed.
Materials and Methods. 1. A comprehensive PubMed-Medline
search was performed between 1962 and May 2010 using widely
several search terms, including lipofuscin, lipofuscin-like granules, and fingerprint in the context of the kidney. 2. The clinical
histories and biochemical data as well as the electron microphotographs of these cases were retrospectively reviewed with regard
to both the key status-symptoms of hypertension and diabetes and
to the specific primary renal disease leading to biopsy.
Results. 1. Only two articles (1 Russian, 1 Japanese), other than
the original one by Biava and West 1, mentioning lipofuscin-like
granules, were found. 2. Our cases included 2 males and 2 females. All patients were adults and their ages were 67, 66, 55, 69,
respectively. All renal biopsies were studied by immunofluorescence, light microscopy and electron microscopy. The pathological diagnoses were the following: minimal change disease, focal
segmental glomerulosclerosis, idiopathic membrano-proliferative
glomerulonephritis type I, and minimal change disease, respectively in the same order. The corresponding suspected clinical
diagnoses were: immunologic glomerulonephritis-NOS vs renal
amlyoidosis, chronic glomerulonephritis-NOS, lupus nephritis,
and membranous glomerulonephritis vs amyloidosis, respectively. Two patients had moderate proteinuria, and two had nephrotic
syndrome. In no case the clinical information of systemic hypertension was given to the pathologist prior to pathological biopsy
examination. Though the retrospective review of the clinical
charts revealed that all patients were affected by this condition,
one in association with diabetes mellitus. All patients had other
associated systemic diseases (rheumatoid arthritis, diabetes mellitus, Sjögren syndrome), except for one (the 69-year old female).
Discussion. In the work by Biava and West the lipofuscin-like
granules were found – in decreasing order of frequency – in
the smooth muscle cells of the afferent glomerular arteriole, in
myoepithelioid juxtaglomerular cells (along with different specific renin-containing granules), and in lacis cells 1. They also
noted that these granules are of 0.5 to 4.0 µ in size and visible at
light microscopy, being argyrophilic, and PAS-positive diastase
resistant 1. We did not directly search for fingerprint profiles
in juxtaglomerular apparatus elements either during electron
microscopical examination, or in examination of standard histological sections. Although these are eye-catching findings, they
251
have always been incidental, and although we were aware of the
existence of these structures as a nonspecific finding in renal arterioles from citation in a neuropathology paper on Kufs’ disease,
due to the interest in this subject matter of one of us (GP), regrettably we admit to never having given them any specific clinical
significance. Instead Biava and West attributed to them a relative
clinical significance, due to their increased number or size in
arterial hypertension and/or diabetes mellitus, respectively 1. In
electron microscopy in our cases, as in the keystone and seminal
paper by Biava and West, these lipofuscin-like granules appear as
dense bodies with a lipid component, a coarsely granular matrix,
and a crystalloidal component which may appear in band or dot
pattern, according to the plane of sectioning. The band pattern of
these crystalloids is homologous to the fingerprint profiles seen
in other diseases such as neuronal ceroid-lipofuscinosis or the
semicircularly organized (fingerprint) linear immune deposits
seen in the above mentioned glomerulopathies. Parenthetically,
but noteworthy, fingerprint profiles have also been observed by
one of us (GP) in the smooth muscle cells of the arterioles in a
rectal mucosal biopsy in a case of neuronal ceroidolipofuscinosis.
Although not in the scope of this report, another open question
concerning these structures is the patho-physiologic mechanism
of their formation. The answer cannot be other than hypothetical,
and is likely due to either oxidative damage to cytosolic structures
or mitochondrial oxidative stress, possibly related to the continual
adrenergic nervous stimulation in connection with blood flow and
to ageing-related impairment of their proteasome processing systems; however for this mechanism we refer to specialized papers
addressing this matter.
Conclusions. 1. Lipofuscin-like granules are subcellular, quasiphysiologic, finding mainly in smooth muscle cells of the walls of
renal arterioles, which increases in number and/or size in subjects
affected by arterial hypertension and diabetes. 2. They do not correlate with a specific primary renal disease. 3. The pathologist has
to be aware of these lipofuscin-like granules in order not to confuse them with other similar findings having a specific diagnostic
significance (such as the fingerprint organized immune deposits
associated with specific glomerulopathies). 4. The nephrologist
should always be alert for either treated or untreated clinical arterial hypertension in their patients and inform the pathologist as
such. 5. Additional systematic observations are needed in order
to further our understanding of these almost completely neglected
subcellular structures.
References
1
Biava C, West M. Lipofuscin-like granules in vascular smooth
muscle and juxtaglomerular cells of human kidneys. Am J Pathol.
1965;47:287-313.
Solitary fibrous tumor of the meninges. Literature
review with a report of 5 additional cases
1)Bisceglia M. 2)Dimitri L. 3)Carotenuto V. 4)Bianco M. 5)Monte V. 6)Giannatempo G. 7)D’Angelo V.
1)Unit of Anatomical Pathology, IRCCS “Casa Sollievo della Sofferenza”
Hospital, San Giovanni Rotondo, Italy 2)Unit of Anatomical Pathology,
IRCCS “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Italy 3)Unit of Neurosurgery, IRCCS “Casa Sollievo della Sofferenza”
Hospital, San Giovanni Rotondo, Italy 4) Unit of Neurosurgery, IRCCS
“Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Italy
5) Unit of Neurosurgery, IRCCS “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Italy 6)Unit of Radiology, IRCCS “Casa
Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Italy 7) Unit
of Neurosurgery, IRCCS “Casa Sollievo della Sofferenza” Hospital, San
Giovanni Rotondo, Italy
Background. Solitary fibrous tumour (SFT) is a spindle cell mesenchymal tumor first described in the pleura by Klemperer and
Rabin in 1931 as a distinct pathologic entity (solitary fibrous mesothelioma, submesothelial fibroma). Suster et al. were the first
252
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
to report the occurrence of these tumors in extrapleural location,
specifically in the somatic soft tissues 1. A number of cases in
many different locations have been added to the literature since 2,
and in 1996 the first seven meningeal cases were reported, five of
which were intracranial and two intraspinal, respectively 3. SFT
is thought to originate from the almost ubiquitous CD34 positive
fibroblast (“dendritic interstitial cell”), which has also been identified in the dural tissue 4.
Objectives. 1. To comprehensively review the world literature
concerning SFT involving the central nervous system (CNS).
3. To report 5 personal additional cases. 2. To ascertain the frequency of this tumor as a proportion of all primary meningothelial and non-meningothelial meningeal-based mesenchymal tumors
in our 17 years experience at the Casa Sollievo della Sofferenza
hospital in S. Giovanni Rotondo. 4. To briefly discuss its distinction from hemangiopericytoma (HPC) of the meninges.
Materials and Methods. 1. A comprehensive PubMed-Medline search was performed between 1996 and May 2010, using
[central nervous system AND solitary fibrous tumor], [meninges
AND solitary fibrous tumor], [brain AND solitary fibrous tumor],
[intraventricular AND solitary fibrous tumor], [medullary cord
AND solitary fibrous tumors], and [intramedullary AND solitary
fibrous tumors] as search terms. 2. A systematic search of our
database was performed to retrieve all meningeal-based primary
mesenchymal tumors from our files. 3. To review the clinical
charts and to follow-up the affected patients.
Results. Literature Review. In 2004 Caroli E et al. found 56 previously reported cases of CNS SFT to which they added 4 cases
of their own 5, and in 2009 Mekni et al. independently reviewed
the same subject and added 8 cases of their own, the number of
cases reported to 2007 thus totalling 96 6. By using all the afore
mentioned search terms, around 150 cases have been found on
record. Most CNS SFT were dural-based, but a significant proportion (≅ 15%) of other CNS locations not directly attached to
the dura (intraventricular, intramedullary, cerebello-pontine, and
subpial-intracerebral, in descending order of frequency) are also
on record A few cases (≅ 10%) exhibited histological malignant
features either at presentation or upon recurrence.
Frequency of SFT in our files. Out of 806 meningeal-based
primary mesenchymal tumors retrieved in total, 786 were meningiomas, 15 hemangiopericytomas, 5 were SFTs, of which 4
intracranial and 1 intraspinal, respectively, and 2 were meningeal
sarcoma unspecified.
Personal SFT Case Reports. All cases were surgically operated,
with or without adjunctive radiation therapy. Case 1: male, aged
47, with an intraspinal (T3-T4) tumor 2 cm in size; the tumor was
totally excised grossly; the patient is alive with no evidence of
disease (ANED) at 11 years. Case 2: male, aged 75, with a tumor
in the posterior cranial fossa (PCF) 3.5 cm in size; the tumor was
grossly incompletely excised and treated with adjunctive radiosurgery (gamma knife); the patient experienced 2 recurrences at 4 and
7 years, which were treated with surgery and radiotherapy, respectively, and eventually – after partial response to radiotherapy – he
died of disease 10 years after diagnosis at the age of 85. Case 3:
male, aged 64, with a PCF tumor 4.5 cm in size; the tumor was
surgically totally excised; the patient is ANED at 5½ years. Case
4: male, aged 76, with a second tumor recurrence 6.5 in size cm in
the PCF (the primary and first recurrence were surgically resected
in an outside institution 15 and 7 years earlier and diagnosed as
fibrous meningioma and SFT, respectively – the slides were not
available for review); the recurrence was grossly totally resected;
eventually the patient died 26 years after the first surgical operation. Case 5: female, aged 59, with a PCF tumor of 4 cm, which
was grossly totally removed; the patient is ANED at 3 years.
Intraoperative findings. All tumors were dural-based. Histological descriptions. All tumors were diagnosed as classical SFT,
except case 4 (myxoid variant). Immunohistochemistry. All cases
showed the classical immunoprofile: Vimentin, CD34, CD99,
BCL2 were all diffusely positive in all cases, except for CD34
in case 4 which was patchily positive; S-100, EMA, alpha SMA,
and desmin all were negative; the mitotic index was very low
(< 1M/10HPF) in all cases, except in case 4 (2M/10HPF); the
MIB-1/Ki67 labeling index was very low (< 2%) in all, except
in case 4 (10%).
Discussion. CNS-SFT is a tumor of adulthood, though occasionally seen in the pediatric population. It is mostly dural-based but
non-dural-based occurrences (intraparenchymal or intraventricular) have also been recorded. The majority of CNS-SFT are intracranial, but one fourth of the cases are intraspinal. Some intraspinal
tumors may also arise from the spinal nerve roots, as in our case
1. Preoperatively they are mostly often diagnosed as meningioma;
intraspinal tumors may also be diagnosed as neurinoma, as in
our case 1. Grossly they are usually well circumscribed, but may
infiltrate into the brain, nerve roots and even skull base. Histologically SFT is comprised of short spindle cells mostly arranged in a
“patternless pattern” (ordinary form), but occasionally organized
in fascicles, with alternating bands of eosinophilic collagen. Similarly to its somatic soft tissue counterpart, morphological variants
(epithelioid, cellular, and myxoid 7) of CNS SFT have also been
observed. Our case 4 was characterized as SFT myxoid variant.
The classic cell morphology is usually bland, but – analogous to
soft tissue – anaplastic variants are also on record, with necrosis,
nuclear atypicalities, high mitotic rate (> 4M:10HPF), and high Ki67/MIB-1 index. In the third tumor recurrence of our case 4 a high
Ki-67/MIB-1 index was seen along with a few mitotic figures, but
necrosis or frank nuclear atypia were absent. SFT is a pathological entity which needs to be distinguished from other dural-based
neoplasms, mostly (fibrous) meningioma, neurinomas, and HPC 8 9.
The correct diagnosis is usually made based on light microscopic
features and the characteristic immunoprofile: CD34+/EMA/S100- (adjunctive markers usually also positive are BCL2 and
CD99), as distinguished from meningioma (usually EMA+/CD34/S100-), and neurinoma (usually S100+/EMA-/CD34-). HPC
exhibits an immunoprofile similar to SFT, but CD34 is expressed
only in a minority of cases and in a weak and patchy pattern 8.
Recently some authors have suggested that CNS HPC should be included in the spectrum of SFT (cellular SFT) 10, but this view is not
shared by others 8 9 11, since CNS HPC is a well-defined CNS entity,
despite controversy regarding its histogenesis. CNS HPC shows a
higher local recurrence rate, and more tumor-related deaths and
extracranial metastases. In a very recent study 12, comparing the
biological behaviour of CNS HPC with that of soft tissue SFT (now
in this setting by definition inclusive of soft tissue HPC) a noticeable clinical difference has been noted, with CNS HPC having a
recurrence rate reaching > 92%. Thus although in soft tissues HPC
has almost disappeared as category separate from SFT, in regards
to CNS the SFT category is still kept separate from it 12. However
difficult cases to discriminate or even transitional (from primary to
recurrent) cases between the two are acknowledged 8 10. From the
literature the biologic behaviour is usually benign, provided total
tumor resection is accomplished, but follow-up data are limited
and they need still be taken cautiously 5. However, in comparison
with its soft tissue counterpart, more aggressive examples (in terms
of recurrence) have been seen in the CNS, but this is due to their
frequent incomplete surgical excision. Extracranial metastases are
extremely rare in CNS-SFT. Our case 2 recurred three times but the
surgical excision of tumor had been grossly incomplete and surgery
was not repeated. Our case 4, who sustained 2 recurrences and
whose tumor eventually developed aggressive histological features
(mitoses, and high Ki-67/MIB1 labeling index), died of tumor after
26 years from primary tumor presentation.
Conclusions. CNS SFT is a tumor distinct from fibrous meningioma and HPC. From the literature the behaviour appears to be
generally benign, but recurrences have been recorded. Surgery is
the treatment of choice, and tumor regrowth is to be anticipated
when removal is not complete. The usefulness of radiotherapy
Oral communications and Posters
is not well documented. Long-term follow-up of the patients is
always mandatory.
References
1
Suster S, Nascimento AG, Miettinen M, et al. Solitary fibrous tumors
of soft tissue. A clinicopathologic and immunohistochemical study of
12 cases. Am J Surg Pathol 1995;19:1257-66.
2
Chan JK. Solitary fibrous tumour-everywhere, and a diagnosis in
vogue. Histopathology 1997;31:568-76. Mod Pathol 1999;12:463-71.
3
Carneiro SS, Scheithauer BW, Nascimento AG, et al. Solitary fibrous
tumor of the meninges: a lesion distinct from fibrous meningioma. A
clinicopathologic and immunohistochemical study. Am J Clin Pathol
1996;106:217-24.
4
Cummings TJ, Burchette JL, McLendon RE. CD34 and dural fibroblasts: the relationship to solitary fibrous tumor and meningioma.
Acta Neuropathol 2001;102:349-54.
5
Caroli E, Salvati M, Orlando ER, et al. Solitary fibrous tumors of the
meninges: report of four cases and literature review. Neurosurg Rev
2004;27:246-51.
6
Mekni A, Kourda J, Hammouda KB, et al. Solitary fibrous tumour
of the central nervous system: pathological study of eight cases and
review of the literature. Pathology 2009;41:649-54.
7
de Saint Aubain Somerhausen N, Rubin BP, Fletcher CD. Myxoid
solitary fibrous tumor: a study of seven cases with emphasis on differential diagnosis. Mod Pathol 1999;12:463-71.
8
Perry A, Scheithauer BW, Nascimento AG. The immunophenotypic
spectrum of meningeal hemangiopericytoma: a comparison with fibrous meningioma and solitary fibrous tumor of meninges. Am J Surg
Pathol 1997;21:1354-60.
9
Tihan T, Viglione M, Rosenblum MK, et al. Solitary Fibrous Tumors
in the Central Nervous System. A Clinicopathologic review of 18 cases
and comparison to meningeal hemangiopericytomas. Arch Pathol Lab
Med 2003;127:432-9.
10
Gengler C, Guillou L. Solitary fibrous tumour and haemangiopericytoma: evolution of a concept. Histopathology 2006;48:63-74.
11 a
Paulus W, Scheithauer BW, Perry A, Hemangiopericytoma, in Louis
DN, Ohgaki H, Wiestler OD, et al. (eds). Mesenchimal, non meningothelial tumors. Lyon: IARC Press 2007, pp. 173-7. bGiannini C, Rushing EJ, Hainfelier. In: Louis DN, Ohgaki H, Wiestler OD, et al. (ed).
Hemangiopericytoma. WHO Classification of tumors of the central
nervous system. Lyon: IARC Press 2007, pp. 178-80.
12
Ambrosini-Spaltro A, Eusebi V. Meningeal hemangiopericytomas
and emangiopericytoma/solitary fibrous tumors of extracranial soft
tissues: a comparison. Virchows Arch 2010;456:343-54.
TTF-1 and WT1 expression in embryonal soft tissue,
visceral, and central nervous system tumors. An
immunohistochemical study of 100 cases
1)M. Bisceglia, 2)C. Galliani, 3)G. Lastilla, 4) J. Rosai
1) Department of Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy; 2)Department of Pathology, Cook Children’s Medical Center, Fort Worth, TX, USA; 3)Department of Pathology,
Polyclinic Hospital of Bari, Bari, Italy; and 4)Centro Diagnostico Italiano
(CDI), Milan, Italy
Background. TTF-1, a member of the NK-2 family of homeodomain transcription factors, is expressed in the early stages of thyroid, lung, and ventral forebrain development, and has been applied as a marker for epithelial-derived neoplasms of the lung and
thyroid, both primary and secondary. In addition, a wide variety
of cell and tissue types have been found to variably express TTF1, including nephroblastoma.1 WT1, encoded by Wilms’ tumor
suppressor gene, controls the expression of growth factors that
regulate glomerular capillary development, activates the bcl-2
gene, and is normally expressed in the kidney (glomerular podocytes) and nephroblastomas. WT1 has also been seen expressed
in other anatomical sites and neoplasms, including ovarian and
endometrial cancer, mesothelioma, desmoplastic small round cell
tumor, melanoma and acute leukemias.
Objectives. To investigate TTF-1 and WT1 immunohistochemical nuclear expression of small round cell tumors of the soft tissues, viscera, and the central nervous system (CNS).
253
Materials and Methods. All cases were retrieved from the pathology files of the participating institutions, 9 were from outside
institutions (6 consultation, 3 courtesy). Formalin-fixed, paraffin
embedded tissues were obtained from 122 patients. Embryonal
soft tissue and bone tumors (64 cases): 26 Ewing’s sarcoma/
primitive neuroectodermal tumors (EWS/pPNET), 13 peripheral (thoracoabdominal) neuroblastomas (pNB), 18 embryonal
rhabdomyosarcomas (ERMS), and 5 desmoplastic small round
cell tumors (DSRCT - 3 intraabdominal, and 2 extra-abdominal:
1 dural-based intracranial, 1 pleural-based thoracic). Embryonal
visceral tumors (12 cases): 5 hepatoblastomas (HB), 4 type I
pleuropulmonary blastomas (PPB-I), 1 retinoblastoma (RB), 1
pancreatoblastoma (1 PTB), 1 paraganglioblastoma (PGB), and
1 embryonal liver sarcoma. Embryonal CNS tumors (24 cases):
14 infratentorial PNET (medulloblastoma – MB), 6 central supratentorial PNET (cPNET), 3 central supratentorial neuroblastoma
(cNB - including 1 olfactory neuroblastoma), and 1 pineoblastoma (PNB). We also studied 9 synovial sarcomas (SVS), a few
differentiated CNS tumors (2 central neurocytomas, 3 pineocytomas, 4 subependymomas), 2 ovarian small cell carcinoma of the
hypercalcemic-type (SCC HC-type), 1 case of Merkel cell tumor,
and one adult case of primitive-looking Merkel-like epithelialderived malignant tumor of the skin for contrast. To compare to
our previous study of nephroblastoma 1, we also tested WT1 reactivity in all these tumors. The medical records were abstracted
for demographic information, specific anatomical sites, and
diagnoses. Heat-induced antigen retrieval was used for detection
of both markers. The following antibodies were used: monoclonal
antibody TTF-1 (1:30 dilution; clone (8G7G3/1) and WT1 (1:50
dilution; clone 6F-H2, directed against the amino terminal domain of WT1 protein). Appropriate positive and negative controls
were used for each antibody. Immunohistochemical staining was
performed using the labeled Envision system according to the
manufacturer’s recommendations.
Results. The series of embryonal tumors included 100 patients in
total, 68 males and 32 females. 62 patients were in the pediatric
age (≤ 21 years), 10 were young adults (> 21 and ≤ 30), and 28
adults. Specifically, the patients’ age ranged between < 1 month
and 42 months (mean 11.4 months), birth and 78 years (mean
24.9 years), 1 and 18 (mean 5.12 years), and 5 and 62 (mean
30.12 years), for embryonal tumors of peripheral nerve tissue
(akin pNB), somatic soft tissue & bone, visceral organs, and
CNS, respectively. The anatomical sites of pNB were: posterior
mediastinum (3), adrenal (7), retroperitoneum (1), and 1 liver
and 1 periaortic lymph node metastasis from adrenal. The rest
of somatic and visceral tissues tumors involved the following
anatomical locations: head & neck (7), genital organs (7), urinary
bladder (4), retroperitoneum (2), liver (1), chest-wall (6), trunk (6
- somatic superficial soft tissue), upper limb (1 - deep soft tissue),
and serosal cavities (DSRCT 5 – intrabdominal 3, intracranial 1,
thoracic 1); 3 tumors affected bone (skull 1, femur 2); 3 were
pPNET metastases (1 each to hilar lymph node from lung, to skin
from kidney, and to liver from unknown primary). There were 13
visceral-based embryonal tumors (5 HB, 4 PPB-I, 1 PGB of the
carotid body, 1 RB, 2 pPNET, 1 each of the kidney and colon).
All CNS tumors were intraaxial (10 supratentorial; 14 infratentorial). Of 9 synovial sarcomas, 6 were female, 3 were male, with
an age range between 10 and 72, and the anatomical sites were
limbs (6 lower limbs, 1 upper limb), trunk (1 chestwall), and lung
(1 metastasis). The 2 females with ovarian SCC HC-type were
17 and 45 years of age, respectively. The immunohistochemical results are as follows: TTF-1 was negative in all the tumors
tested, except for one case of suprasellar cPNET, which showed
widespread immunopositivity in 40% of tumor cell nuclei;
50% of PPB-I had alveolar-epithelial lining with nuclear TTF1 immunostaining, serving as an internal control. No nuclear
positivity for WT1 was found in any case of embryonal tumors.
Of nonembryonal tumors, included in the study, the 2 cases of
254
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
ovarian SCC HC-type showed nuclear moderate immunostaining
in numerous tumor cells (diffuse in 1, and patchy in the other).
However, strong WT1 cytoplasmic positivity was seen in all 18
ERMS; moderate cytoplasmic staining was observed in 53.8%
of pNB, 19.2% EWS/pPNET, 35.7% MB, 50% PPB-I, 60%
DSRCT, 22.2% SVS, 50% cPNET, 40% of HB. WT1 was always
and significantly positive in the endothelium of both normal and
tumor vessels.
Discussion. To the best of our knowledge, TTF-1 has not been
previously investigated in a wide array of embryonal tumors. The
impetus to perform such a study was stimulated by the discovery
of a subset of nephroblastomas expressing nuclear TTF-1 immunopositivity 1. According to our results, negative TTF-1 may
help in the differential diagnosis of primary PNET of the kidney 2
versus nephroblastoma, which can express TTF-1. We could not
confirm nuclear immunostaining in SVS as previously reported in
one case metastatic to the lung 3. The only TTF-1 positive cPNET
is in agreement with reports of other positive peri-diencephalic
neuroepithelial tumors 4. Nuclear WT1 positivity in both ovarian SCC HC-type is in accordance with previous studies and in
support of its müllerian origin 5. Cytoplasmic WT1 positivity has
been reported in ERMS 6, and in the rhabdomyomatous component of nephroblastomas 1. We exploited this property as an
adjunctive marker in a case of spindle cell rhabdomyosarcoma
of the heart 7, supporting cytoplasmic WT1 as a marker for documenting skeletal muscle differentiation. Regarding the absence
of WT1 nuclear immunoreactivity in DSRCT, it is in agreement
with that of other investigators who used a similar monoclonal
antibody 8. The cytoplasmic reactivity for WT1 we observed in
DSRCT might reflect its polyphenotypic nature, but needs to be
elucidated. WT1 cytoplasmic immunopositivity in embryonal
tumors of neural lineage and in the rest of soft tissue tumors is
likely nonspecific, but cytoplasmic positivity in a case of MB is
intriguing, since it was in fact an already known anaplastic MB,
which we had previously diagnosed with immunohistochemical
evidence for early rhabdomyoblastic differentiation (nuclear immunopositivity with myogenin). WT1 was consistently positive
in the cytoplasm of endothelial cells mainly of tumoral vasculature, partly confirming previous experience 9, and leading us
to select WT1 as perhaps one of the most sensitive endothelial
markers currently available (unpublished data of one of us [MB]).
Conclusion. Embryonal tumors of soft tissues or viscera, other
than nephroblastoma, fail to express nuclear reaction with
TTF-1. cPNET of the diencephalic region of the forebrain can
express TTF-1 in tumor cell nuclei. ERMS consistently exhibits
cytoplasmic WT1 immunopositivity. Other embryonal tumors,
mainly of neural lineage and of the somatic soft tissues, may variably express cytoplasmic WT1 in a nonspecific fashion. WT1 is
perhaps one of the most sensitive endothelial markers in surgical
pathology.
References
1
Bisceglia M, Ragazzi M, Galliani CA, et al. TTF-1 expression in
nephroblastoma. Am J Surg Pathol 2009;33:454-61.
2
Parham DM, Roloson GJ, Feely M, et al. Primary malignant neuroepithelial tumors of the kidney: a clinicopathologic analysis of 146 adult
and pediatric cases from the National Wilms’ Tumor Study Group
Pathology Center. Am J Surg Pathol 2001;25:133-46.
3
Lewis JS, Ritter JH, El-Mofty S. Alternative epithelial markers. In:
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the head and neck, lung, and bladderp63, MOC-31, and TTF-1. Mod
Pathol 2005;18:1471-81.
4
Zamecnik J, Chanova M, Kodet R. Expression of thyroid transcription
factor 1 in primary brain tumours. J Clin Pathol 2004;57:1111-3.
5
Carlson JW, Nucci MR, Brodsky J, et al. Biomarker-assisted diagnosis of ovarian, cervical and pulmonary small cell carcinomas: the role
of TTF-1, WT-1 and HPV analysis. Histopathology 2007;51:305-12.
6
Carpentieri DF, Nichols K, Chou PM, et al. The expression of WT1 in
the differentiation of rhabdomyosarcoma from other pediatric small
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round blue cell tumors. Mod Pathol 2002;15:1080-6. Comment in:
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Fraternali Orcioni G, Ravetti JL, Gaggero G, et al. Primary embryonal
spindle cell cardiac rhabdomyosarcoma: case report. Pathol Res Pract
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Barnoud R, Sabourin JC, Pasquier D, et al. Immunohistochemical
expression of WT1 by desmoplastic small round cell tumor: a comparative study with other small round cell tumors. Am J Surg Pathol
2000;24:830-6.
Wagner N, Michiels JF, Schedl A, et al. The Wilms’ tumour suppressor WT1 is involved in endothelial cell proliferation and migration:
expression in tumour vessels in vivo. Oncogene. 2008;27:3662-72.
Primary embryonal rhabdomyosarcoma of prostate
in adults. Report of a case and review
of the literature
1)Bisceglia M. 2)Fiordelisi F. 3)Perrone G. 4)Dicandia L. 5)Cannazza V. 6)Ben Dor D.
1)Unit of Anatomic Pathology, IRCCS “Casa Sollievo della Sofferenza”,
San Giovanni Rotondo, Italy 2) Unit of Anatomic Pathology, IRCCS
“Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy 3) Unit of Anatomic Pathology, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy 4) Unit of Anatomic Pathology, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy 5)Unit of Radiology,
P.O. di Scorrano, ASL - LE 2, Maglie, Italy 6)Department of Pathology,
Barzilai Medical Center, Ashkelon, Israel
Background. Embryonal rhadomyosarcoma (ERMS) is the most
common tumor of the lower genitourinary tract occurring in the
first 2 decades of life, both in males and females, mostly arising
from the bladder, vagina, uterine cervix, prostate, and paratesticular region. ERMS of prostate in adults is extremely rare. In
a systematic review, which was published by Waring et al. in
1992, only 6 cases were found in the literature with adequate
clinicopathological information, to which these authors added 3
cases of their own.1 Occasional case reports of ERMS in adults
were published since.
Objectives. To report one additional personal case of adult
patient with ERMS and to comprehensively review the world
literature on this subject.
Case Report. In 2002, a 49-year-old male, who had undergone
transurethral prostatic resection for benign nodular hyperplasia
and left hydrocelectomy 4 and 2 years previously, respectively,
was admitted for acute urinary retention. Previous pathological
specimens were not available for review. At this time rectal
digital examination revealed an enlarged firm prostate gland.
Abdominal CT scan and US scan showed a prostatic tumor 9 cm
in size with infiltrative margins, bulging into the urinary bladder
and invading the perirectal adipose tissue. Bilateral hydronephrosis due to obstruction of both the ureters and enlarged iliac lymph
nodes were also documented. At cystoscopy a polypoid tumor
obstructing the prostatic urethra was seen and a transurethral
tumor resection was performed. Light microscopic examination
revealed a malignant tumor composed of an admixture of undifferentiated small round cells and scattered groups of spindleshaped cells with bipolar eosinophilic cytoplasmic extensions
showing definite cross striations. Immunohistochemically the
tumor cells were positive for vimentin, muscle specific actin,
desmin, fast myosin, sarcomeric actin, and negative for CD34,
EMA, S100 protein, PSA, PSAP; pan-cytokeratin (MNF116)
was focally positive in a few cells. After several courses of
neoadjuvant VAC-chemotherapy (vincristine, adriamycin, and
cyclophosphomide), which reduced the tumor mass to 5 cm, the
patient underwent radical cystoprostatectomy with bilateral seminal vesiculectomy and pelvic lymphadenectomy. Urinary diversion was accomplished with creation of bilateral ileal conduits.
The original diagnosis was histologically confirmed on examination of the resection specimen. The urethral resection margin
was positive for tumor. Both seminal vesicles, the iliac lymph
Oral communications and Posters
nodes, as well as the resection margins of both ureters, were all
free of tumor. 6 months after surgery a huge pelvic recurrence
of the ERMS, causing intestinal occlusion and bilateral ureteral
obstruction which were relieved with percutaneous nephrostomy
and transverse colostomy, was found and confirmed on needle
biopsy. The patient became cachectic and severely debilitated
and died 1 year after diagnosis. Distant metastases were not
documented. Autopsy was not done.
Literature Review. The literature was reviewed based on a computerized PubMed/Medline search, using [rhabdomyosarcoma
AND prostate] as search terms, and the references lists of all the
available publications on this subject, encompassing the interval
between 1988 (the year when Waring’s et al. 1 review ended) and
May 2010.
Discussion. Sarcoma of prostate is rarely seen in adults, accounting for less than 5% of all malignant prostatic tumors.
ERMS is the rarest type of sarcoma in this age group. Around
40 cases of primary prostatic rhabdomyosarcoma have been reported so far in males ≥ 18 years of age from 1988 to May 2010.
However, in compliance with Waring’s et al. inclusion/exclusion criteria 2, less than 30 cases should be included, which,
in addition to cases recorded in the afore-mentioned review,
amount to a grand total of less than 40. ERMS mostly present
with symptoms of progressive dysuria or urinary obstruction.
Patients often present with locally advanced disease and at times
with metastatic disease. A tumor mass is always discovered and
the diagnosis is made on transrectal needle biopsy or transurethral resection or biopsy specimens. The differential diagnosis
includes both stromal sarcomas arising from specific prostatic
stroma, including STUMP (stromal tumors of uncertain malignant potential), and sarcomas of soft tissue-type, such as inflammatory myofibroblastic tumors, malignant peripheral nerve
sheath tumors, leiomyosarcoma, and other types of rhabdomyosarcoma (alveolar and pleomorphic) 2. Occasionally GIST from
the rectum invading the prostate might also be a consideration.
Immunohistochemistry is of utmost importance in ascertaining
the correct diagnosis, which is based on immunopositivity for
desmin and skeletal muscle markers (MyoD1, myogenin, fast
myosin, sarcomeric actin, myoglobin, …). Predictive prognostic
factors are stage-related. Adults with prostatic rhabdomyosarcomas do not respond to multimodal therapy and have a poor
prognosis. Pediatric patients appear to respond much better than
adults with combined modality treatment for sarcoma in general 3-5, and the rhabdomyosarcomatous group fares better than
the nonrhabdomyosarcomatous one 3. All adult patients with
adequate follow-up died within 20 months after histological
diagnosis with a mean survival of 8 to 10 months vs an overall
5-year survival rate of 70-80% and a median survival of over
10 years, respectively, in children 3. Surgery is the mainstay of
treatment.
Conclusions. ERMS of prostate in adults is a very rare and aggressive disease. The long-term disease specific survival rate is
poor. Stage influences the outcome. Early diagnosis and complete
surgical resection offer the patients the best chance of improved
survival.
References
1
Waring PM, Newland RC. Prostatic embryonal rhabdomyosarcoma in
adults. A clinicopathologic review. Cancer 1992;69:755-62.
2
Hansel DE, Herawi M, Montgomery E, et al. Spindle cell lesions of the
adult prostate. Mod Pathol 2007;20:148-58.
3
Janet NL, May AW, Akins RS. Sarcoma of the prostate: a single
institutional review. Am J Clin Oncol 2009;32:27-9.
4
Sexton WJ, Lance RE, Reyes AO, et al. Adult prostate sarcoma: the
M.D. Anderson Cancer Center Experience. J Urol 2001;166:521-5.
5
Mondaini N, Palli D, Saieva C, et al. Clinical characteristics and overall survival in genitourinary sarcomas treated with curative intent: a
multicenter study. Eur Urol 2005;47:468-73.
255
Aggressive angiomyxoma: a tumour with a wide
morphological spectrum. A clinicopathological
study of 27 cases including recurrent lesions
1)Gurrera A. 1)Amico p. 2)Bisceglia m. 1)Longo f. 3)Kazakov
d. 3)Kacerovskà d. 3)Michal m. 1)Magro g.
1)Dipartimento G.F. Ingrassia, Anatomia Patologica, Università di Catania, Azienda Ospedaliero-Universitaria Policlinico-Vittorio Emanuele,
Catania, Italia; 2)Servizio Anatomia Patologica, IRCCS Ospedale Casa
Sollievo della Sofferenza, San Giovanni Rotondo, Italia; 3)Sikl’s Department of Pathology, Charles University, Medical Faculty Hospital, Pilsen,
Czech Republic
Background. Aggressive angiomyxoma (AAM) is an uncommon
fibro-myofibroblastic tumour, usually occurring in soft tissues of
the vulvovaginal, pelvic and perineal regions of young females.
Similar lesions have occasionally been reported in retroperitoneum and in perineum, para-anal and inguino-scrotal region. AAM
is a locally infiltrative neoplasm with a significant risk of multiple
local recurrences, with a low metastatic potential.
Methods. The clinicopathological features of 27 cases of AAM
are presented with emphasis on morphological heterogeneity of
both primitive (22 cases) and recurrent tumours (5 cases).
Results. Tumours usually presented as painless masses located in
the vagina, vulva, and pelvi-perineum region of women ranging
in age from 43 to 65 years. Grossly, most of tumours presented
with ill-defined margins, ranging in size from 1.5 to 20 cm in
greatest diameter and with a gelatinous to fibrous appearance at
cut surface. Histologically, 59% of tumours were fibro-myxoid
in appearance, while 26% and 15% were purely fibro-sclerotic or
myxoid, respectively. Neoplastic cells were round to spindle or
stellate in shape, with scanty cytoplasm and hyperchromatic nuclei. Cellularity was low in all but in 3 cases that were highly cellular. Mitoses were only rarely observed. Notably, smooth muscle
cells, isolated or arranged in short fascicles, were found scattered
throughout the stroma in 33% of cases. Vascular component was
represented by small capillary-like to large blood vessels with
perivascular hyalinization (48% of cases) and medial hypertrophy
(37% of cases). Recurrent tumours were predominantly hypocellular fibro-sclerotic lesions (3 out 5 cases) that showed keloid-like
collagen bands and a neurofibromatous-like pattern. In two of
these cases, a complete sclerotic obliteration of blood vessels was
seen resulting in confluent nodular structures closely reminiscent
of corpora albicantia.
The present study emphasizes that AAM is a tumour with a
wide morphological spectrum ranging from a purely myxoid
to hypocellular fibro-sclerotic lesion with a neurofibromatouslike appearance. This latter morphological feature, seen both
in primitive and recurrent lesions, should be kept in mind by
pathologist to avoid confusion with benign fibromatous lesions
Extracutaneous involvement of sporadic Kaposi’s
sarcoma. A clinicopathologic study of a case series
1)Bisceglia M. 2)Magro G.3) Panniello G. 4)G. Sanguedolce F.
5)Ben Dor D.
1)Department of Pathology, IRCCS Casa Sollievo della Sofferenza, San
Giovanni Rotondo, Italy 2)Department of Pathology, University of Catania, Catania, Italy 3)Unit of Clinical Dermatology, Ospedali Riuniti, Foggia, Italy 4)Unit of Anatomic Pathology, Ospedali Riuniti, Foggia, Italy 5)
Department of Pathology, Barzilai Medical Center, Ashkelon, Israel
Background. Kaposi’s sarcoma (KS) is a peculiar tumor of vascular derivation and viral etiology (gammaherpesvirus HHV-8),
occurring primarily in the skin 1. KS is rare, comprising 0.1%
of all malignancies worldwide. A variety of clinical forms have
been identified: the sporadic, the endemic, the iatrogenic, and
the epidemic 2. The sporadic (or classical, European, Mediterranean) form primarily affects elderly Caucasian males with a
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
predisposition for Eastern and Southern Europeans, and Jews of
European (mainly Russian and Polish) and North African origin.
KS mostly affects the skin of the acral sites, having a chronic
and indolent clinical course and persisting for many years, with
little propensity to spread to other organs 1 2. In most cases the
course is benign, but a fatal outcome after many years has also
been observed. In sporadic KS soft tissue, bone, lymph nodes,
and visceral organs (mainly the gastrointestinal tract) are rarely
involved 1 2. On occasion these unusual locations represent the
only site of involvement.
Objectives. To report on as well as to present a pictorial review
of a series of extracutaneous KS involving different organs, either
in isolation or in association with cutaneous manifestations.
Materials and Methods. A systematic search of our combined
databases based on institutional and personal consultation files
was performed between 1985 and 2009 to identify extracutaneous cases of KS. All cases have been re-examined and immunohistochemically investigated with anti-LNA-1 (Latent Nuclear
Antigen-1) HHV8 antibody (clone 13B10, dilution 1:20, Novocastra Laboratories, England, UK), if this had not already been
performed at the time of the original examination.
Results. 25 cases with extracutaneous involvement have been
identified from about 750 cases of sporadic KS in our combined
files. Patients’ ages ranged from 10 to 85 yrs. The male to female
sex ratio was 11:1. Of these cases, 5 occurred in the soft tissues (4
in the somatic soft tissue; 1 in the retroperitoneum, involving the
right adrenal), 10 in the gastrointestinal tract (7 in the stomach,
1 in the pharynx, 2 in the rectum), 2 in the bones (1 in the calcaneum and 1 in the lateral malleolus), 8 in the lymph node (3 of the
neck, 4 inguinal, 1 axillary), and 1 in the parotid gland. All cases
exhibited the classic predominantly spindle cell morphology,
alternating with focal angiomatous-like foci. All cases exhibited
diffuse and strong nuclear immunohistochemical reactivity with
anti-LNA-1 HHV 8 antibody.
Discussion. In the Mediterranean basin the frequency of sporadic
KS is that of 1.5:100,000 people. The frequency of extracutaneous involvement for classical KS has been reported at 10%,
which we think is an overestimate. Our rate is much lower,
which may be because of the following: 1. KS patients are not
systematically followed-up for visceral and mucosal involvement
(most frequently in the gastrointestinal tract), which are usually
asymptomatic; 2. we counted lesions per cases diagnosed and
many patients had several skin lesions excised. All our cases
of extracutaneous KS occurred in non-immunocompromised
patients (non-AIDS associated KS, non-iatrogenic KS). However
we included in this case series even those cases in which other
conditions (usually multicentric Castleman’s disease, or nonHodgkin’s lymphoma) were simultaneously found in association
in the same organ (usually lymph node). In 10 cases we were
aware of previous or concurrent skin lesions in the same patient.
Although most cutaneous KS lesions are easily identified by an
experienced pathologist, some lesions are not easy to diagnose if
seen out of the usual anatomic context in which is KS expected
to occur. Soft tissue KS may be misinterpreted as a different type
of spindle cell sarcoma with an inflammatory component (mostly
leiomyosarcoma), but HHV-8 immunohistochemcial testing is
extremely useful for ruling this out 3. On small biopsies KS in
the gastrointestinal tract may be confused with granulation tissue
or other reactive vasoproliferative lesions. KS in lymph nodes
may be overlooked, since at times it is represented by small foci
in association with other reactive or neoplastic lymphoproliferative processes, and may also be confused with foci of nodular
spindle-cell vascular transformation. We did not follow-up these
patients since this was not the scope of this report, however we
are aware that in 2 of these cases the outcome was fatal following
spread to visceral organs (lung, and brain), one of the two cases
having in addition bone involvement, and the other had a huge
local recurrence with primary retroperitoneal involvement (in the
absence of skin changes), after a long disease course of 30 years
and 13 years, respectively. Notably involvement of the adrenal in
a non-HIV KS patient has been previously reported only once 4.
Also of interest is our youngest patient, currently in good health,
who was diagnosed with KS of lymph node, the second pediatric
case in the literature, and who was the subject of a separate report
in 1988 5. Parenthetically the first pediatric patient with classical
KS involving the lymph node was also Italian.
Conclusion. Extracutaneous KS does occur, but is rare. The pathologist should be aware of this occurrence. HHV-8 immunohistochemical testing is critical for KS diagnosis in these cases.
References
1
Bisceglia M, Bosman C, Carlesimo OA, et al. Kaposi’s sarcoma: a
clinico-pathologic overview. Tumori 1991;77:291-310.
2
Geraminejad P, Memar O, Aronson I, et al. Kaposi’s sarcoma and
other manifestations of human herpesvirus 8. J Am Acad Dermatol
2002;47:641-55.
3
Cheuk W, Wong KO, Wong CS, et al. Immunostaining for human herpesvirus 8 latent nuclear antigen-1 helps distinguish Kaposi sarcoma
from its mimickers. Am J Clin Pathol 2004;121:335-42.
4
Lazure T, Plantier F, Alsamad IA, et al. Bilateral adrenal Kaposi’s
sarcoma in an HIV seronegative patient. J Urol 2001;166:1822-3.
5
Bisceglia M, Amini M, Bosman C. Primary Kaposi’s sarcoma of the
lymph node in children. Cancer. 1988;61:1715-8.
Phosphaturic mesenchymal tumor and oncogenic
osteomalacia. A clinicopathologic study of 14
cases with emphasis on unusual features, and
review of the literature
1)Bisceglia M. 2)Parafioriti A. 3)Robbins P. 4)Elmberger G.
5)Fusconi M. 6)Rendina D. 7)Alberghini M. 8)Viti R. 9)Armiraglio E. 10)Spagnolo D. 11)Pasquinelli G. 12)Varenna M.
13)Guglielmi G. 14)Perrone E. 15)Scillitani A. 16)Mossetti G.
1)Unit of Anatomic Pathology, IRCCS Casa Sollievo della Sofferenza
Hospital, San Giovanni Rotondo, Italy 2)Unit of Anatomic Pathology,
Gaetano Pini Institute, Milan, Italy 3)Department of Anatomic Pathology,
PathWest Laboratory Medicine, Perth, Western Australia 4)Department of
Pathology and Cytology, Karolinska University Hospital, Stockholm, Sweden 5)Unit of Rheumatology, University of Bologna, Bologna, Italy 6)Department of Clinical and Experimental Medicine, Federico II University,
Naples, Italy 7)Unit of Anatomic Pathology, Rizzoli Institute, Bologna,
Italy 8)Unit of Endocrinology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy 9)Unit of Anatomic Pathology, Gaetano Pini Institute, Milan, Italy 10)Department of Anatomic Pathology,
PathWest Laboratory Medicine, Perth, Western Australia 11)Department
of Clinical Pathology, University of Bologna, Bologna, Italy 12) Unit of
Metabolic Bone Diseases, Gaetano Pini Institute, Milan, Italy 13) Unit of
Radiology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni
Rotondo, Italy 14) Unit of Nuclear Medicine, IRCCS Casa Sollievo della
Sofferenza Hospital, San Giovanni Rotondo, Italy 15) Unit of Endocrinology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni
Rotondo, Italy 16)Department of Clinical and Experimental Medicine,
Federico II University, Naples, Italy
Background. Most cases of oncogenic osteomalacia (OO) are
caused by mesenchymal tumors (phosphaturic mesenchymal
tumors - PMT), which overexpress fibroblast growth factor-23
(FGF-23), a protein of the “phosphatonin” family, capable of inhibiting renal tubular phosphate transport. The typical laboratory
findings secondary to phosphate loss are hypophosphatemia and
hyperphosphaturia, which finally result in an inadequate mineralization of osteoid in mature bone, the metabolic disorder known
as osteomalacia 1. OO is vitamin-D resistant and dramatically
cured by tumor excision. The 1st case of PMT was described in
1947, but the term PMT was coined in 1987 2. PMT is rare and
consistently located in soft tissue and bone of limbs and trunk;
sinonasal cavities and acral parts are traditionally considered uncommon sites. In a review of the literature up to 2002, Folpe et al.
found 109 cases on record, to which they added 29 new original
cases of their own of 32 they studied in total.3 Histologically PMT
Oral communications and Posters
corresponds to a polymorphous group of neoplastic entities, the
vast majority of cases, particularly in soft tissue, are associated
with a specific histopathologic entity, the mixed connective tissue variant (PMT-MCT) 3, which is characterized by a distinctive
admixture of bland spindled cells, osteoclast-like giant cells, microcysts, prominent and variously sized vasculature, smudgy to
calcified cartilage-like matrix and metaplastic bone. Some cases
have histological features of malignancy. PMT of craniofacial
sinuses usually differs from PMT-MCT and closely resembles
a sinonasal HPC-like tumor variant. Tumor discovery and histological recognition are frequently delayed in OO. Sometimes
(< 10%) OO is not documented, but the diagnosis of PMT can
be proposed reasonably on the basis of the histological features
(aphosphaturic PMT) 3.
Objectives. 1. To describe the clinicopathologic features of our
cases of OO with emphasis on unusual findings. 2. To comprehensively review the world literature between January 2002 and
March 2010.
Methods. 1. A systematic search of our combined databases
was performed to identify cases of possible PMT. All clinicopathologic features, including serum biochemical determinations
and imaging studies were reviewed. Electron microscopy was
performed in one sinonasal case. 2. A computerized PubMed/
Medline search was performed, using 4oncogenic osteomalacia7,
[tumor-induced osteomalacia], and 4phosphaturic mesenchymal
tumor7 as search terms.
Results. 21 cases were initially retrieved from the institutional
files and personal consultation archives, of which 7 PMT with
OO were excluded since they had been previously reported. Of
these latter 7 cases, 4 had been surgically excised and histologically examined (1 intraosseous osteoblastoma of sacrum, 1
sinonasal HPC-like tumor, 2 soft tissue PMT-MCT), and 3 were
not operated on (1 vertebral hemangiomatosis, 2 tumors unidentified). The present study concerns 14 patients (age range 21-70,
median 51) 2 of whom were included previously in Folpe’s et al.
series, though without detailed clinical history or illustrations.
5 were males, 9 were females: 13 with OO, and 1 asymptomatic. Imaging (standard X-ray, and/or CT, and/or MRI, and/or
PET-CT, and/or bone scintigraphy) and appropriate biochemical
studies were performed in all. Octreotide scan for somatostatin
receptor imaging was positive in 3 of 6 cases so studied. FGF-23
serum levels were elevated in all 6 cases assayed (FGF-23 failed
to decrease in 2 cases with incompletely removed tumor, but
normalised on re-excision). Longstanding symptoms and delayed
diagnosis were frequent (6/14); there was failure to recognise the
causal tumor in 2/14. PMT occurred as a soft tissue lesion of the
foot in 3 cases, was intraosseous in 7 (2 in the femur, 1 each in the
humerus, rib, ileum, C1-vertebra) and sinonasal in 2. Histologically PMT was MCT-type in 9 and HPC-like in 3. In sinonasal
cases PMT was HPC-like in 1 and MCT-type in 1. All the excised
tumors were histologically benign (12/12). 1 case examined ultrastructurally displayed suggestive neuroendocrine dense core
granules. 1 case, which was immunostained for FGF-23, was
positive. OO normalized after complete tumor removal in 10/12
surgically treated cases with OO (repeat operations required in
2). The 2 cases of OO with no evidence of PMT were diagnosed
according to ASBMR criteria 4 and medically treated with phosphate and calcifediol supplementation with minimal benefit. The
single case of histologically proven PMT without OO occurred
as a soft tissue tumor of the hand in a 62-year old female. In our
most recent literature review for the years 2002-2010, 107 cases
of PMT were found (mostly adult patients; 2 in pediatric age). Of
the 100 for which the site was known, 58 cases occurred in soft
tissue, 24 in bone, 11 in nasal/paranasal cavities, 4 were adjacent
to or involved the central nervous system coverings (2 intracranial; 2 intraspinal) and 3 were visceral (1 each in tongue, liver and
uterus). Acral location (bone and soft tissue) occurred in 13/100
(foot in 11, hand in 2).
257
Conclusions. 1. PMT is a rare, poorly understood pathologic
entity, often with delayed diagnosis. 2. Acral sites (especially
foot) and sinonasal locations are not uncommon. 3. Aphosphaturic PMT is rare, but may occur. 4. PMT-MCT is the commonest
histological variant, and may also occur in nasal/paranasal cavities. 5. OO is cured by surgery, but fails to regress after incomplete tumor removal. 6. FGF-23 serum level is a sensitive tumor
biomarker that allows clinical management. 7. “Orphan” OO is
rarely established despite careful and repeat investigations.
References
1
Jan de Beur SM. Tumor-induced osteomalacia. JAMA. 2005;294:12607.
2
Weidner N, Santa Cruz D. Phosphaturic mesenchymal tumors.
A polymorphous group causing osteomalacia or rickets. Cancer
1987;59:1442-54.
3
Folpe AL, Fanburg-Smith JC, Billings SD, et al. Most osteomalaciaassociated mesenchymal tumors are a single histopathological entity.
An analysis of 32 cases and a review of the literature. Am J Surg
Pathol 2004;28:1-30.
4
Jan de Beur SM. Tumor-induced osteomalacia. In: American Society
for Bone and Mineral Research (ed). Primer on the metabolic bone
diseases and disorders of mineral metabolism. American Society for
Bone and Mineral Research 2006, pp 345-51.
Primary malignant melanoma of the esophagus.
A clinico-pathologic study of a case with literature
review
1)Bisceglia M. 2)Perri F. 3)Tardio M. 4)Vairo M. 5)Pasquinelli
G.
1)Unit of Anatomic Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy 2)Unit of Gastroenterology, IRCCS
Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy
3)Unit of Surgery, IRCCS Casa Sollievo della Sofferenza Hospital, San
Giovanni Rotondo, Italy 4)Unit of Anatomic Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy 5)Department of Clinical Pathology, University of Bologna, Bologna, Italy
Background. Primary malignant melanoma originating from
internal organs is rare, but has been well documented in the
literature 1. Primary melanoma originating in the digestive tract
is particularly rare, with the majority of cases involving the oral
cavity and anorectum 1,2. Primary malignant melanoma of the
esophagus (PMME) has been the source mainly of case reports,
and its frequency is estimated around 0.1-0.2% of all esophageal
malignancies 3. In a large epidemiologic study in USA the median
age was 69 and the age-adjusted rate incidence that of 0.03 per
million population 4. Again in another large epidemiologic study
in USA, from 1973 to 2004, 39 PMME were found on record
of 659 total primary gastrointestinal malignant neoplasms 5. In
3 separate reviews, presented up to 1989, to 1999, and to (June)
2005 a total of only 139, 154, and 262 cases, respectively, could
be identified in the world literature 3 6 7.
Objectives. To describe a personal case of PMME, and to comprehensively review on a computerized search the world literature
between 2005 and December 2009.
Case Report. A 69-year-old man was admitted for complaints
of abdominal distress and melena, who had never undergone
surgery or been diagnosed previously with malignancy. An
echoscan of the stomach and esophagus revealed an area of mural thickening at the level of the lower 3rd of the esophagus and
involving the cardia. On endoscopic examination, the tumor was
exophytic polypoid. An endoscopic biopsy revealed a poor1y
differentiated, malignant neoplasm. The patient underwent partial
esophagectomy with total gastrectomy. The surgical specimen
was sent for pathological examination. The resection specimen
showed a large, ulcerated, partly fungating, tan red mural mass
of 6 cm in greatest diameter. Histological examination revealed
a malignant neoplasm composed of solid sheets or discrete nests
of monotonous, highly malignant, tumor cells with large nuclei
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
and prominent nucleoli. Areas of hemorrhage and necrosis were
evident throughout all sections. Mitotic figures were numerous
(> 50 per 10 HPF). The tumor was deeply infiltrating into the
muscularis propria, and, in one area, a small focus of junctional
melanocytic activity was found in the basal layer of the squamous
mucosa at the level of the gastroesophageal junction. Histochemical reactions for mucin (PAS-D and mucicarmine) were negative
in the tumor cells as negative was Fontana stain for argentaffin granules. Immunohistochemically the tumor was: strongly
positive for vimentin, S-100 protein, HMB45, and Melan-A, and
negative for cytokeratin AEl/AE3, CEA, MOC31, actin, desmin,
EMA, CD10, CD20, CD45, CD99, CD117 and AFP. Electron
microscopic examination was performed on tissue retrieved from
the paraffin block, which in some of the cells displayed stage I
premelanosomes and stage II melanosomes. All 15 perigastric
and esophageal lymph nodes examined in total were free of tumor. (pT3, pN0, pMx). The final established diagnosis was that
of amelanotic PMME. Follow-up: Careful physical examination
in our patient following the postoperative diagnosis did not reveal
any cutaneous lesion suspicious for melanoma or any tumor elsewhere in the body. Clinical follow-up demonstrated a recurrent
lesion at the site of anastomosis 10 months after surgery. The
patient died of disease 24 months after primary diagnosis.
Discussion. Less than 60 additional PMMEs have been found
up from July 2005 to May 2010, amounting to a grand total of
320 since ever. PMME is thought to originate from foci of basal
melanocytes present in the squamous epithelium 8 9. The clinical
features of PMME similar to those of carcinoma of the esophagus. Most patients are in their sixth and seventh decades, with a
slight male predilection. Dysphagia, substernal pain, heartburn,
and weight loss are the most common symptoms. Grossly the
tumors are most often polypoid and ulcerated, and can vary in
size from small lesions to large, bulky masses. The mucosa at the
edges of the lesion can be pigmented, a phenomenon referred to
as “melanosis” of the esophagus 8 9. The majority of the tumors
are grossly pigmented; however, rarely completely amelanotic
lesions can occur, as in our case. The histologic differential diagnosis for this tumor, particular1y the amelanotic variant, is quite
broad, since it may present either as a large epithelioid cell or
spindle cell or small cell malignant neoplasm: poorly differentiated carcinoma, gastrointestinal stromal tumor (GIST) or other
malignant mesenchymal neoplasms, such as leiomyosarcoma and
malignant peripheral nerve sheath tumor, or non-Hodgkin malignant lymphoma. Immunohistochemistry should be able to clear1y
define the melanocytic nature of the tumor cells. However, the
main differential diagnosis of PMME is with a metastasis to
this organ from melanoma of another site. The most important
histological feature suggestive of PMME is the identification of
junctional activity by atypical melanocytes within the basal layer
of the squamous epithelium. In the present case, the combination
of the absence of a tumor in any other location on thorough clinical examination, absence of development of other lesions in other
organs after 10 months of follow-up, local recurrence at the site of
surgery, and focal Pagetoid involvement in the squamous mucosa
from the resected specimen all supported a diagnosis of primary
melanoma of the esophagus in our patient. PMME is quite aggressive, likely more aggressive that its cutaneous counterparts,
but this may be due to their larger size and depth of invasion at
the time of diagnosis. Common sites of metastases are regional
lymph nodes, liver, mediastinum, lung and brain. The average
survival time following esophagectomy for primary melanoma
is less than 1 year, with a 5 year survival of about 2%. Complete
surgical excision is the standard treatment, followed by adjuvant
radiation and chemotherapy. Local endoscopic laser treatment
may play a role in palliation in locally advanced tumors that are
unresectable.
Conclusions. This thoroughly documented case is presented for
its rarity and the differential diagnosis, especially for amelanotic
PMME is emphasized. The world literature has been reviewed
up to date.
References
1
Batsakis JG, Suarez P. Mucosal melanomas: a review. Adv Anat
Pathol 2000;7:167-80.
2
Mills SE, Cooper PH. Malignant melanoma of the digestive system.
Pathol Annu 1983;18:1-26.
3
Sabanathan S, Eng J, Pradhan GN. Primary malignant melanoma of
the esophagus. Am J Gastroenterol 1989;84:1475-81.
4
Coté TR, Sobin LH. Primary melanomas of the esophagus and anorectum: epidemiologic comparison with melanoma of the skin. Melanoma Res 2009;19:58-60.
5
Cheung MC, Perez EA, Molina MA, et al. Defining the role of surgery for primary gastrointestinal tract melanoma. J Gastrointest Surg
2008;12:731-8.
6
Lam KY, Law S, Wong J. Malignant melanoma of the oesophagus:
clinicopathological features, lack of p53 expression and steroid receptors and a review of the literature. Eur J Surg Oncol 1999;25:168-72.
7
Vandewoude M, Cornelis A, Wyndaele D, et al. Acta Gastroenterol
Belg 2006;69:12-4. (18) FDG-PET-scan in staging of primary malignant
melanoma of the oesophagus: a case report.
8
Sharma SS, Venkateswaran S, Chacko A, et al. Melanosis of the
esophagus. An endoscopic, histochemical, and ultrastructural study.
Gastroenterology 1991;100:13-6.
9
Chang F, Deere H. Esophageal melanocytosis morphologic features
and review of the literature. Arch Pathol Lab Med 2006;130:552-7.
Immunosuppression-associated Kaposi’s sarcoma
complicating chronic inflammatory bowel disease.
A clinico-pathologic study of 2 cases with review
of the literature
1)Bisceglia M. 2)Piscitelli D. 3)Panniello G. 4)Sanguedolce F. 5)
Serviddio G. 6) Bisceglia M.L. 7)Ben Dor D.
1)Department of Pathology, IRCCS Casa Sollievo della Sofferenza, San
Giovanni Rotondo, Italy 2)Department of Pathology, Polyclinic Hospital,
Bari, Italy 3)Division of Clinical Dermatology, Ospedali Riuniti, Foggia,
Italy 4)Division of Anatomic Pathology, Ospedali Riuniti, Foggia, Italy
5) Institute of Internal Medicine, University of Foggia, Foggia, Italy 6)
School of Pharmacy, University of Parma, Parma, Italy 7)Department of
Pathology, Barzilai Medical Center, Ashkelon, Israel
Background. Kaposi’s sarcoma (KS) is a peculiar tumor of vascular histogenesis and viral etiology (gammaherpesvirus HHV-8),
occurring primarily in the skin 1. A variety of clinical forms have
been identified: the sporadic, the endemic, the iatrogenic, and the
epidemic 2. The iatrogenic form supervenes in immunocompromised
hosts, i.e. transplanted patients with immunosuppression, patients
receiving immunosuppressive therapies for haematological malignancies (mainly chronic lymphocytic leukemia, and non-Hodgkin’s
and Hodgkin’s lymphomas), solid tumors (mainly carcinomas of the
breast, followed by lung, colon, larynx, liver, pancreas, and kidney,
in decreasing order of frequency), or inflammatory/autoimmune
diseases, after long-term steroid treatment 1 2. Also included in this
rubric of KS are those rare cases of patients receiving blood transfusions, factor VIII containing plasma fractions, or platelets apheresis.
Excluded from this iatrogenic category are those KS patients who
either subsequently present with a second malignancy 3 or are simultaneously diagnosed with a second malignancy, in the absence
of prior systemic anticancer therapy and/or any clinically detectable
immunosuppression, though both conditions may have developed
independently and coincidentally on the background of an altered
immune system. Non-neoplastic medical conditions treated with
immunosuppressive drugs (mainly corticosteroids), which are on
record as (rarely) associated with, KS are: rheumatoid arthritis, giant
cell arteritis (Horton arteritis), relapsing polychondritis, systemic
lupus erythematosus (SLE), pemphigus vulgaris and inflammatory
chronic intestinal diseases.
Objectives. 1. To report on two cases of iatrogenic KS in patients
receiving immunosuppressive therapy for chronic inflammatory
bowel disease, one with chronic ulcerative colitis and the other
Oral communications and Posters
with Crohn’s disease, neither of whom had HIV infection, or had
been receiving immunosuppressive treatment following transplantation. 2. To review the world literature with regards to iatrogenic KS complicating chronic inflammatory intestinal diseases
recorded between January 1980 and December 2009.
Case reports. Case 1. A 50-year old man diagnosed six months
previously with biopsy-proven severe ulcerative colitis and
treated with corticosteroids and azathioprine, underwent emergency subtotal colectomy due to massive intestinal bleeding.
Histological examination of the surgical specimen revealed
widespread involvement of the colon by KS, in addition to
ulcerative colitis. Following histological diagnosis, the patient
underwent proctosigmoidectomy, and KS with ulcerative colitis
was also seen in the rectum. The postoperative course was uneventful, the immunosuppressive treatment was withdrawn and
the patient recovered. Case 2. A 65-year old man, diagnosed
with biopsy-proven Crohn’s disease involving the duodenum
and receiving near-continuous immunosuppressive treatment for
5 years, underwent surgical resection of a 60 cm long segment of
jejunum, due to repeat episodes of bowel occlusion. Histological
examination of the specimen revealed KS in association with
Crohn’s disease. 3 weeks after surgery the patient was severely
debilitated, and died due to Candida Albicans sepsis. Autopsy
was not performed.
Discussion. Patients who receive immunosuppressive therapy
are at increased risk for KS (immunosuppression-associated
KS), the majority of whom are renal transplant patients. In addition to the role of immunologic impairment, other etiologic
factors also play a role in this form of KS, and one of the recognized risk factors for this type of KS is ethnicity (Eastern and
Mediterranean people as well as Jews of European and North
African origin are at higher risk). The immunosuppression-associated form of KS occurs between a few months and a few
years after starting therapy. Immunosuppression-associated KS
may also affect the skin, but may be limited to the gastrointestinal tract. 9 cases of non-transplant associated iatrogenic KS
afflicting patients with long-lasting inflammatory chronic bowel
disease (chronic ulcerative colitis in 6 and Crohn’s disease in
2) have been described so far in HIV-negative patients 4 5. All
cases were on long-standing immunsuppressive treatment. Two
of these previously reported cases involved Italian patients, both
affected by ulcerative colitis. One of our two patients (both Italian) was on immunosuppressive therapy for 5 months, while
the other one was on it for 5 years. In both cases the initial
diagnosis was morphological, but was subsequently confirmed
with the anti-LAN-1 HHV-8 monoclonal antibody, when it
became commercially available, with both cases showing strong
and diffuse positivity. The differential diagnosis of KS of the
intestine includes other types of spindle cell sarcomas such as
angiosarcoma, GIST, leiomyosarcoma, and inflammatory myofibroblastic tumor (previously known as inflammatory fibrosarcomas): immunohistochemical testing with anti-HHV-8 antibody is
critical, given its high sensitivity (almost 100%) and specificity
(100%) 6. After the skin the gastrointestinal tract is the most frequent anatomic site of involvement by KS, the stomach, being
most frequently affected, followed by the colon. Colon may be
affected by classic KS and may be the only site of involvement
(exclusive of skin). Conversely, KS involvement limited to the
skin may also occur secondarily to medical treatment for colonic
inflammatory diseases. Nonetheless, immunosuppression-associated KS complicating chronic inflammatory diseases of the
bowel is rare. Parenthetically we mention here, that AIDS related
KS presenting as ulcerative colitis-like illness has also been
observed, but this should not be confused with the subject in
question. Genetic susceptibility is definitely part of the complex
interplay in KS between the mechanism of cell proliferation, the
apoptotic controlling machinery and an individual’s immune
regulatory systems.
259
Conclusion. Immunosuppression-associated KS complicating
ulcerative colitis and Crohn’s disease is rare, with 9 cases on
record. Intestinal resection (especially proctocolectomy for ulcerative colitis) and the withdrawal of immunosuppressive drugs
result in improvement of the patient’s general health.
References
1
Geraminejad P, Memar O, Aronson I, et al. Kaposi’s sarcoma and
other manifestations of human herpesvirus 8. J Am Acad Dermatol
2002;47:641-55.
2
Bisceglia M, Bosman C, Carlesimo OA, et al. Kaposi’s sarcoma: a
clinico-pathologic overview. Tumori 1991;77:291-310.
3
Bisceglia M, Zenarola P, Melillo L, et al. Cutaneous presentation of
acute myeloid leukemia in a “classical” Kaposi’s sarcoma patient.
Tumori 1990;76:400-2.
4
Girelli CM, Serio G, Rocca E, et al. Refractory ulcerative colitis and
iatrogenic colorectal Kaposi’s sarcoma. Dig Liver Dis 2009;41:1704.
5
Tedesco M, Benevolo M, Frezza F, et al. Colorectal Kaposi’s sarcoma
in an HIV-negative male in association with ulcerative rectocolitis: a
case report. Anticancer Res 1999;19:3045-8.
6
Cheuk W, Wong KO, Wong CS, et al. Immunostaining for human herpesvirus 8 latent nuclear antigen-1 helps distinguish Kaposi sarcoma
from its mimickers. Am J Clin Pathol 2004;121:335-42.
Unclassified non-pleomorphic sarcoma versus
de novo malignant solitary fibrous tumor versus
monophasic fibrous synovial sarcoma – primary
of the kidney. Pathologic and molecular study
of a case with a long-term survivor
1)Bisceglia M. 2)Trabucco S. 3)Albrizio M. 4)Palmiotti G. 5)Alberghini M. 6)Pasquinelli G. 7)Serio G.
1)Department of Pathology, IRCCS Casa Sollievo della Sofferenza, San
Giovanni Rotondo, Italy 2)Department of Pathology, University of Bari,
Bari, Italy 3)Department of Pathology, Di Venere Hospital, CarbonaraBari, Italy 4)Department of Clinical Oncology, Di Venere Hospital, Carbonara-Bari, Italy 5)Unit of Anatomic Pathology, Rizzoli Institute, Bologna, Italy 6)Department of Clinical Pathology, University of Bologna,
Bologna, Italy 7) Department of Pathology, University of Bari, Bari, Italy
Background. Solitary fibrous tumor of the kidney (SFTK)
was first described in 1996 and can originate either in the renal
capsule or parenchyma 1 2. To date 38 cases of SFTK have been
reported, most of them described by standard criteria as histologically benign, and carrying a favourable clinical prognosis
(follow-up ranging 2 to 89 months) 1-3. Only 2 cases of SFTK
exhibiting malignant histological changes were reported in 2006 1
and 2008 2, respectively. The tumor in the first case was > 10cm
in size and diffusely malignant, with the conventional (benign)
features found only focally: the patient developed lung metastases
4 months after surgery (malignant [secondary] SFTK arising in a
preexisting tumor which had been followed clinically as a stable
lesion for 4 years).1 The tumor in the second case was 9 cm in
diameter with a 3 cm nodular malignant area abruptly emerging
from the surrounding typically bland SFT tissue (dedifferentiated
SFTK or SFTK with sarcomatous overgrowth); this patient was
free of disease 21 months after surgery.3 Malignant SFTK in the
absence of residual histologically benign SFT may be difficult if
not impossible to assess as well as to differentiate from primary
menophasic fibrous synovial sarcoma of the kidney (SSK). SSK,
a rare neoplasm usually carrying a poor prognosis, was first described in 2000 4 5. Primary SSK can exist in either a monophasic
or a biphasic form, and may be misdiagnosed as another type of
sarcomatous or sarcomatoid renal tumor, primary or metastatic.
As its soft tissue counterpart, the diagnosis of primary SSK can
be confirmed by molecular analysis, showing the characteristic
t(X;18) (p11;q11) translocation. To date 54 cases of primary
SSK have been reported, including 3 with rhabdoid features 6
(rhabdoid variant of SSK).
Objectives. To report a case of a primary nonpleomorphic renal
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
sarcoma in a young lady which was difficult to categorize, in
whom lung metastases developed 10 years after nephrectomy,
and which on review was eventually classified as “unspecified
nonpleomorphic sarcoma”.
Methods - Case Report. In 2009 this patient was admitted for
a huge left pulmonary pneumonia-like infiltrate, accompanied
by an abundant pleural effusion. Total body 18FGF-PET scan
revealed high uptake in the corresponding left lung and the patient underwent minimally invasive open lung biopsy. A minute,
2 mm in size, intrapulmonary, mesenchymal malignant tumor
nodule was excised. Given a known history of left nephrectomy
of 10 years earlier, the patient’s previous clinical chart and the
original glass slides of that tumor were retrospectively reviewed
and newly cut sections were either immunohistochemically analyzed or submitted for molecular investigation. In addition small
fragments of paraffin-embedded tissue were deparaffinised and
processed for ultrastructural investigation.
Results. The past renal tumor was hypercellular and mitotically
active, composed of atypical, monomorphic, round to oval closely
apposed medium-sized tumor cells, and showed focal HPC-like
growth pattern, hemorrhagic foci, necrosis and pseudocystic areas.
The greatest tumor diameter was 12.5 cm. The tumor margins were
infiltrative, entrapping the surrounding normal renal parenchyma.
The renal pelvis was infiltrated, but hilar or perirenal adipose tissues as well as 5 paracaval/periaortic lymph nodes submitted for
histological examination were not involved. Examination of the
newly excised lung nodule showed that it was morphologically
consistent with a metastasis from the renal tumor. The immunoprofile of both the renal primary and lung metastasis was: vimentin
diffusely +ve; BCL-2 diffusely +ve; EMA focally +ve; CD34
patchy +ve (strongly diffuse on restaining); CD99 focally +ve; CK
(pankeratin, CK7, CK19) all negative; alpha-SMA, desmin, myogenin all negative; CD117 negative; Fli-1 negative; WT1 negative;
TTF1 negative; S-100 negative; CD10, ER, and PGR all negative.
Electron microscopy showed closely apposed oval-shaped cells
lacking epithelial differentiation (no tonofibrils, no desmosomes),
with absence of actin-like microfilaments. Basal lamina was not
seen. Molecular analyses (RT-PCR and FISH analyses) did not
demonstrate the t(X;18) (p11;q11) translocation of either SYTSSX1 or SYT-SSX2 gene fusion. Taking all these findings into
account the final diagnosis was “unclassified nonpleomorphic renal
sarcoma” – probably de novo malignant SFTK.
Discussion. The diagnosis of malignant SFTK was neither
straightforward nor was it eventually completely accepted since
no foci of benign SFT were found (areas of usual SFT had been
seen so far in both the 2 afore-mentioned cases of malignant
SFTK as well as in all cases of the recently recognized dedifferentiated SFT of soft tissue 7. Furthermore, CD34 may also be
positive in sarcomas other than malignant SFTK and is most often
lost in the malignant and dedifferentiated areas of SFT in both
renal and soft tissue cases 1 8. Notwithstanding, de novo malignant
SFT is a real possibility. SSK, which had not yet been described
at the time of nephrectomy, was the main consideration on review, but its exclusion is based on both the absence of the specific
translocation and presence of CD34 positivity (parenthetically
CD34 positivity was also seen in 3 cases of intrathoracic SS 7);
other renal primaries, excluded for more obvious reasons, were
sarcomatoid renal cell carcinoma, primary renal fibrosarcoma,
malignant nerve sheath tumor, monomorphic angiomyolipoma,
extragonadal endometrial stromal sarcoma, inflammatory myofibroblastic tumor, congenital mesoblastic nephroma, malignant
mixed epithelial stromal tumor, leiomyosarcoma, anaplastic
sarcoma of the kidney with polyphenotypic features 9, of recent
identification, and (atypical) congenital mesoblastic nephroma.
Follow-up: The patient was given several courses of chemotherapy, using Ifosfamide, Epirubicin and MESNA, and temporarily
improved. Currently, 10 months following discovery of the lung
metastases, she is alive with slight disease progression.
References
1
Fine SW, McCarthy DM, Chan TY, et al. Malignant solitary fibrous
tumor of the kidney: report of a case and comprehensive review of the
literature. Arch Pathol Lab Med 2006;130:857-61.
2
Magro G, Emmanuele C, Lopes M, et al. Solitary fibrous tumour of the
kidney with sarcomatous overgrowth. Case report and review of the
literature. APMIS 2008;116:1020-5.
3
Hirano D, Mashiko A, Murata Y, et al. A case of solitary fibrous tumor
of the kidney: an immunohistochemical and ultrastructural study with
a review of the literature. Med Mol Morphol 2009;42:239-44.
4
Argani P, Faria PA, Epstein JI, et al. Primary renal synovial sarcoma:
molecular and morphologic delineation of an entity previously included among embryonal sarcomas of the kidney. Am J Surg Pathol
2000;24:1087-96.
5
Kim DH, Sohn JH, Lee MC, et al. Primary synovial sarcoma of the
kidney. Am J Surg Pathol 2000;24:1097-104.
6
Jun SY, Choi J, Kang GH, et al. Synovial sarcoma of the kidney
with rhabdoid features: report of three cases. Am J Surg Pathol
2004;28:634-7.
7
Bégueret H, Galateau-Salle F, Guillou L, et al. Primary intrathoracic
synovial sarcoma: a clinicopathologic study of 40 t(X;18)-positive
cases from the French Sarcoma Group and the Mesopath Group. Am
J Surg Pathol 2005;29:339-46.
8
Mosquera JM, Fletcher CD. Expanding the spectrum of malignant progression in solitary fibrous tumors: a study of 8 cases with a discrete
anaplastic component – is this dedifferentiated SFT? Am J Surg Pathol
2009;33:1314-21.
9
Vujanić GM, Kelsey A, Perlman EJ, et al. Anaplastic sarcoma of the
kidney: a clinicopathologic study of 20 cases of a new entity with
polyphenotypic features. Am J Surg Pathol 2007;31:1459-68.
Oncocytic adrenocortical neoplasms – a distinct
entity. Report of 13 additional cases with emphasis
on new diagnostic criteria and clinicopathologic
correlation
1)Bisceglia M. 2)Wong D.D. 3)Havlat M.F. 4)McCallum D
5)Platten M.A. 6)Spagnolo D.V.
1)Department of Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy 2)Department of Anatomic Pathology,
PathWest Laboratory Medicine, Perth, Western Australia 3)Department of
Histopathology, St John of God Pathology, Subiaco, Western Australia 4)
Department of Anatomic Pathology, PathWest Laboratory Medicine, Perth, Western Australia 5) Department of Anatomic Pathology, PathWest Laboratory Medicine, Perth, Western Australia 6) Department of Anatomic
Pathology, PathWest Laboratory Medicine, Perth, Western Australia
Background. The first oncocytic adrenocortical neoplasm (OAN)
was reported in 1986 1. Over the ensuing 25 years OAN has been
established as a distinct entity, and interest is now largely focused
on diagnostic criteria and predicting their behaviour. The original
Weiss system 2 for conventional (nononcocytic) adrenocortical
neoplasms does not apply to OAN. The Lin-Weiss-Bisceglia
(LWB) system (applicable for resectable neoplasms only) was
proposed in 2004-2005 3 4, and is gaining widespread acceptance 5 6. In the LWB system definitional criteria, major criteria,
and minor criteria allow classification of OANs as benign, borderline or malignant.
Objectives. 1. To describe the clinicopathologic features of
13 new OANs; 2. to review retrospectively all OANs from the
world literature in the context of the LWB criteria; 3. to analyse
statistically and correlate outcome data according to LWB tumor
categories; and 4. to assess if there are behavioural differences
between malignant OAN and conventional adrenocortical carcinoma.
Methods. A systematic search of our combined databases was
performed to identify cases of “pure” OANs as previously
defined 3 4, and confirmed as oncocytic immunohistochemically and/or ultrastructurally. A comprehensive PubMed-Medline
search was performed between January 1980 and August 2009.
Follow-up data were collected for all reported cases and median
and 5 year survivals were estimated using the Kaplan-Meier
Oral communications and Posters
method. Differences in survival curves between cases classified
histologically as benign, borderline and malignant were analysed
using the Log-Rank test.
Results. We found 13 new OANs in 7 females and 6 males with
a median age of 41 years (range 22-69). 6 patients showed either
clinical or biochemical hormone hypersecretion. All tumors
were encapsulated: median size 80mm (range 7-285), median
weight 155g (range 15-5720). According to LWB criteria 3
were benign, 2 borderline and 8 malignant. Of the latter, local
recurrence occurred in 3, distant metastases in 1 and death in 3.
1 case was associated with an ipsilateral adrenal myelolipoma
and 1 (gigantic) malignant OAN is the largest on record. The
occurrence of “small oncocytes” was a frequent focal finding.
All tumors were strongly immunopositive with mES-13 and
9 were immunoreactive for calretinin, a novel finding in this
context. All 4 cases examined ultrastructurally showed typical
oncocytic features with an abundance of mitochondria. KaplanMeier curves for recurrence/metastases and death (p < 0.001 for
both, using Log-Rank test), were applied to 84 of 109 cases (our
13 OAN and 96 from the literature) with sufficient data to allow
analysis. This revealed the ability of the LWB system to reliably predict future risk in OAN. The overall median survival for
malignant OAN was 58 months (95%CI 27.5 to 88.5), notably
better than the reported 14-32 months for conventional adrenocortical carcinoma 7.
Conclusions. 1. We report 13 new cases of OANs; 2. report the
value of mES-13 immunostaining in establishing oncocytic differentiation; 3. show the value of the LWB criteria in categorising OAN as benign, borderline or malignant; and 4. provide
preliminary evidence of a better prognosis for malignant OANs
compared with conventional adrenocortical carcinomas.
References
1
Kakimoto S, Yushita Y, Sanefuji T, et al. Non-hormonal adrenocortical adenoma with oncocytoma-like appearances. Hinyokika Kiyo
1986;32:757-63.
2
Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol 1984;8:1639.
3
Bisceglia M, Ludovico O, Di Mattia A, et al. Adrenocortical oncocytic
tumors: report of 10 cases and review of the literature. Int J Surg
Pathol 2004;12:231-43.
4
Bisceglia M, Ben-Dor D, Pasquinelli G. Oncocytic Adrenocortical
Tumors. Pathol Case Rev 2005;10:228-42.
5
Lack E. Adrenal Cortical Carcinoma. Tumours of the Adrenal Gland
and Paraganglia. 4th ed. Washington DC: Armed Forces Institute of
Pathology 2008.
6
Lau SK, Weiss LM. The Weiss system for evaluating adrenocortical
neoplasms: 25 years later. Hum Pathol 2009;40:757-68.
7
Bilimoria KY, Shen WT, Elaraj D, et al. Adrenocortical carcinoma in
the United States: treatment utilization and prognostic factors. Cancer.
2008;113:3130-6.
Na+/H+ exchanger regulatory factor 1 (NHERF1)
expression in colorectal cancerogenesis
1)”Mangia A. 1)Bisceglie D. 2)Malfettone A. 3)Asselti M.
4)Bellizzi A. 5)Daprile R. 6)Paradiso A. 7)Simone G.
1)Clinical experimental oncology laboratory, Ncc “Giovanni Paolo II”,
Bari, Italy 2)Clinical experimental oncology laboratory, Ncc “Giovanni
Paolo II”, Bari, Italy 3)Department of pathology, Ncc “Giovanni Paolo
II”, Bari, Italy 4)Clinical experimental oncology laboratory, Ncc “Giovanni Paolo II”, Bari, Italy 5)Department of pathology, Ncc “Giovanni
Paolo II”, Bari, Italy 6)Clinical experimental oncology laboratory, Ncc
“Giovanni Paolo II”, Bari, Italy 7)Department of pathology, Ncc “Giovanni Paolo II”, Bari, Italy
Background. Na+/H+ exchanger regulatory factor 1 (NHERF1)
is a candidate tumor suppressor gene. NHERF1 protein expression has been demonstrated to be altered in several cancers. An
increased cytoplasmic NHERF1 expression suggests a key role of
261
its localization/compartmentalization in defining cancerogenesis
and progression, but its role in colorectal carcinoma remains still
undefined.
Methods. We examined immunohistochemically the expression
pattern and sub-cellular localization of NHERF1 in 51 patients
with advanced colorectal cancers matched with surrounding
nontumoral epithelium, in metastatic lymph nodes and hepatic
metastases from each patient.
Results. NHERF1 showed a different localization and expression in the different compartments of colorectal cancer samples.
In nontumoral epithelium tissues, NHERF1 immunoreactivity
was present as cytoplasmic, membranous and nuclear staining,
while in tumor and metastatic tissues NHERF1 was present as
diffuse cytoplasmic and nuclear staining. The median of cytoplasmic-NHERF1 positive cells was significantly higher in primary tumors (70%), metastatic lymph nodes (60%) and hepatic
metastases (70%) than normal tissues (10%) (p < 0.0001). In
contrast, we had observed a low membranous protein expression
in all tumoral tissues examined respect to normal tissues (0% vs
5% respectively; p < 0.0001). Nuclear-NHERF1 expression was
higher in tumor (18%) and metastatic tissues (15%) than normal
tissues (11%) (p = 0.006; p < 0.01 respectively).
Colorectal cancerogenesis is characterized by increased cytoplasmic expression of NHERF1 as the tumour progresses, suggesting
its role in this process. The switch from membranous to cytoplasmic expression is compatible with a dual role for NHERF1 as a tumour suppressor or tumour promoter dependent on its sub-cellular
localization. Indeed, the increasing nuclear NHERF1 expression
suggest that this protein can move to the nucleus and may induce
expression of genes determining the malignant phenotype.
References
Cardone RA, et al. The NHERF1 PDZ2 domain regulates PKA-RhoAp38-mediated NHE1 activation and invasion in breast tumor cells.
Mol Biol Cell 2007;18:1768-80.
Mangia A, et al. Biological role of NHERF1 protein expression in breast
cancer. Histopathology 2009;55:600-8.
Song J, et al. Expression and clinicopathological significance of oestrogenresponsive ezrin-radixin-moesin-binding phosphoprotein 50 in
breast cancer. Histopathology 2007;51:40-53.
Expression of p-AKT and p-mTOR in a large series
of BP-NETs
1)Boldrini L. 2)Capodanno A. 3)Servadio A. 4)Rotondo M I.
5)Pelliccioni S. 6)Fontanini G.
1)Surgery, Santa Chiara Hospital pisana, Pisa, Italy 2)Surgery, Santa
Chiara Hospital, Pisa, Italy 3)Surgery, Santa Chiara Hospital, Pisa, Italy
4)Surgery, Santa Chiara Hospital, Pisa, Italy 5)Molecular Diagnostic,
Santa Chiara Hospital, Pisa, Italy 6)Surgery, Santa Chiara Hospital,
Pisa, Italy
Background. Bronchopulmonary neuroendocrine tumors (BPNETs) comprise about 20% of all lung cancers. They are separated into 4 subgroups: typical carcinoid tumor (TC), atypical carcinoid tumor (AC), large-cell neuroendocrine carcinoma (LCNEC),
and small-cell lung carcinoma (SCLC), which exhibit different
biological characteristics that have been extensively investigated
to identify features for diagnosis, prognosis and therapy for this
special lung tumor category.
The signalling pathway involving AKT/mTOR (the mammalian target of rapamycin) is one of the main regulators of
cell growth and proliferation and is located at the crossroad of
several major signal transduction molecules (PTEN/Pi3-kinase,
AMKP, Ras/Raf). The only available literature data on AKT/
mTOR in NE lung tumours are represented by experimental
models in SCLC cells. The purpose of this study was to evaluate the expression of phosphorylated AKT/mTOR in a large
series of BP-NETs and to investigate the correlations with
clinicopathological parameters.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Methods. p-AKT (Ser473) and p-mTOR (Ser2448) were
determined by immunohistochemistry in a series of 210 BPNETs, including 85 SCLC, 17 LCNEC, 26 AC, 75 TC, and 7
tumorlets.
Results. High p-AKT expression was found in the majority of
tumorlets and carcinoids, whereas a lower expression was found
in SCLC and LCLNC p = 0.0001). The expression of p-mTOR
was also statistically different in tumorlets and carcinoids, that
showed higher p-mTOR expression, comparing with SCLC and
LCNEC (p = 0.0002). Furthermore, p-mTOR expression was
higher in T1-T2 tumor stages compared to higher stages in all
BP-NETs (p = 0.0008).
Our results suggest a role for Akt/mTOR pathway in BP-NETs,
particularly in carcinoids. Moreover, mTOR could represent a
useful marker in this type of tumors with important applications
in the clinico-therapeutic management of patients.
vessels. Although hemangioma was predominantly exophytic,
there was infiltration of superficial myocardium. No evidence of
atypia, cellular pleiomorphism, high mitotic count, or necrosis
were found. Immunohistochemical profile of tumor consistent
with with strong staining for CD31 and factor VIII. The diagnosis
of cardiac hemangioma, capillary type, was made.
Conclusions. Cardiac hemangiomas are rare tumors therefore
it is difficult to make a definitive preoperative diagnosis. Other
cardiac tumors that may have strong gadolinium enhancement
include pheochomocytoma, angiosarcoma, myxoma, and rhabdomyosarcoma. Cardiac angiosarcomas are exceptionally aggressive, are usually large, centrally necrotic, and frequently extend
into the pericardium.
Granulomatous reaction in gastric carcinomas: an
immunohistochemical and ultrastructural study
1)Endocrinologia, Ospedae S. Maria delle croci, Ravenna, Italia 2)Oncologia, Ospedale Umberto I, Lugo, Italia
1)R. Caruso 1)Bonanno A. 2)Quattrocchi E. 3)Napoli P. 4)Fedele
F.
Background. For pathologist, an essential step in the mastery of
aspiration cytology is the ability to translate the cytologic patterns
into histologic tissue patterns of diagnostic value.
The fine needle aspiration cytology (FNAC) in nodular lesions
has a limited diagnostic use for the impossibility to obtain multiple sections for an immunohistochemical analysis.
Methods. Often from standard FNA is possible to obtain thin
cores or multiple tissue fragments, especially in tissue rich of cell
as lymph node and solid tumours. The FNA samples, previously
centrifugation, are assembled with a drop of tromboplastina to
produce a clot. The clot is fixed in 10% solution of buffered isotonic formalin and processed as for routine histology. Cell blocks
may give some idea of tissue architecture and allow multiple section for immunohistochemistry.
Results. We always prepare the cell blocks and a cytologic
smearing from fresh material in FNA of neoplastic lesions from
different organs and tissues. This gives us tissue fragments for
value histologic pattern of the lesions and on which perform
immunohistochemistry and/or the molecular pathology (FISH,
EGFR, K-ras ecc). In the review of our series we have observed
that the cell blocks is useful to differentiate tumoral histotypes
(in particular of the parotid gland and of the lung), primary from
metastatic tumours, lymphomas, undifferentiated carcinomas
from sarcomas and melanomas, neuroendocrine tumours and it
was essential to diagnose: parotid gland melanoma metastasis,
lymph node alveolar rhabdomyosarcoma metastasis, lymph
node gastric leiomyosarcoma (GIST) metastasis, thyroid gland
colic ADK metastasis, adrenal gland leiomyosarcoma, giant cells
MFH, pulmonary angiosarcoma.
1)Patologia umana, Policlinico universitario, Messina, Italia 2)Patologia
umana, Policlinico universitario, Messina, Italia 3)Servizio anatomia patologica, Ospedale papardo, Messina, Italia 4)Patologia umana, Policlinico universitario, Messina, Italia
Granuloma is a focal, compact collection of inflammatory cells
in which mononuclear phagocytes predominate. The authors
report 9 cases of papillary-tubular gastric adenocarcinomas
characterized by mature granulomas associated with recent microhemorrhages. Mature granulomas were composed of foamy,
CD68-positive histiocytes with occasional giant cells. Hemosiderin-containing macrophages were present in the tumor stroma,
suggesting phagocytosis of erythrocytes. Under electron microscopy, mature (nonepithelioid) granulomas and clusters containing 1 macrophage and 1-3 eosinophils were found. This study
provides morphological examples of skewed type II macrophage
infiltration in gastric adenocarcinomas that is involved in scavenging activity, particularly erythrophagocytosis, formation of
mature (nonepithelioid granulomas), and heterotypic aggregation
with eosinophils.
Left ventricular hemangioma
1) Bondi F. 2)Del Giglio
1)Endocrinologia, Ospedale S. Maria delle croci, Ravenna, Italia 2)Department of cardiovascular surgery, Villa maria cecilia hospital, Cotignola, lugo (ra), Italia
Background. Primary cardiac tumors are rare. The large majority
of cardiac tumors are benign; hemangiomas account for < 10%
of all primary cardiac tumors in children and they are usually asymptomatic when diagnosed after infancy. Cardiac hemangiomas
are often found incidentally at autopsy or with imaging, usually
hocardiography.
Metohods. A 16-year-old previously healthy boy presented with
a heart murmur and was found by transthoracic echocardiography
to have a single mobile tumor in the left ventricular. A diagnosis
of probable cardiac hemangioma was made on the basis of its
MRI signal intensity characteristics indicating high vascularity.
The polipoid mass appeared to be localized in the left ventricle
and its implant base was in the lateral border of the posterior
papillary muscle. The tumor was surgically excised.
Results. At gross inspection, tumor consisted of exophytic
polypoid mass. The size was 1.7 × 1.5 × 1 cm. On cut section,
tumor had microcytic appearance with areas of hemorrhage.
Histopathological features were consistent with an unusual type
of hemangioma composed of large, endothelial-lined, thin-walled
channels and intervening dense proliferation of capillary-sized
The cell blocks: it could be a real -biopsy
1)Bondi F. 2)Salerno V.
Follow-up of borderline cervical cytology cases
negative for atypia with indication to repeat pap
test in a group of spontaneous screening
1)Bonfadini M.G. 2)Magnani C. 3)Rostan I. 4)Marsico A.
5)Navone R.
1)Servizio di citologia, Clinica san gaudenzio, Novara, Italia 2)Servizio di
citologia, Clinica san gaudenzio, Novara, Italia 3)Sc. biomediche e oncologia umana-sez. anatomia pat., Universita di torino, Torino, Italia 4)U.o.
di anatomia patologica, Osp. koelliker, Torino, Italia 5)Sc. biomediche e
oncologia umana-sez. anatomia pat., Universita di torino, Torino, Italia
Background. The Bethesda 2001 System foresees a diagnosis of
ASC-US, ASC-H and AGC for borderline lesions and a subdivision of the cervical slides into negative or positive, with reactive cell changes (RCC) placed into the negative category. Our
research, using an adequate follow-up, i.e. at least 2 cytological
tests and/or 1 negative histological result, in a case group of cervical cytology with a 3-9 year follow-up) aims to establish a final
diagnosis, both for borderline atypia and RCC.
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Oral communications and Posters
Materials. 146,020 cytological cervical samples showed 1,845
ASC\AGC (1.3%), 604 (0.4%) L-SIL, 432 (0.3%) H-SIL, 56
squamous carcinoma (0.04%) and 33 adenocarcinoma (0.02%).
Amongst the 1,845 borderline cases, 455 of the ASC and 54
of the AGC had a follow-up considered to be sufficient, as did
806/4,577 with a negative diagnosis for atypia with indication for
a further cytology test to assess RCC (mainly infections, above
all vaginosis) and ASC-AGC with a reduced legibility (quality)
of the samples.
Results. 305/ 445 cases of ASC (68.6%) were benign, 97 (21.8%)
SIL were low grade, 37 (8.3%) SIL were high grade and 6 (1.3%)
were carcinoma. 35/54 cases of AGC (64.8%), were benign and
19 (35.2%) were malignant (13 H-SIL, 1 squamous carcinoma
and 5 adenocarcinoma). When the ASC were subdivided into
ASC-US and ASC-H, in the 1st group (393 cases), there were
295 (77.0%) definitive benign diagnosis, 75 (17.8%) L-SIL, 20
(4.7%) H-SIL and 5 carcinoma (0.8%). Whilst in the 2nd group,
(37 cases) there were 11 (37.9%) negative cases, 7 (20.7%) LSIL, 18 (37.9%) H-SIL and 2 (3.5%) squamous carcinoma.
764/806 RCC (94.8%) were benign, 29 (3.6%) were low grade
SIL, 11 were high grade SIL (1.4%), 1 (0.1%) was a squamous
carcinoma and 1 (0.1%) adenocarcinoma.
In conclusion, a high predictive capacity was confirmed for ASCH as was the possibility of false negatives for the RCC, due to
poor quality samples. This is in line with the Bethesda System
management recommendations for ASC-US, where a repeated
Pap test after adequate therapy, in the case of infection, is recommended.
Ki67 and p53 immunohistochemical evaluation in
malignant and potentially malignant oral lesions
based on samples obtained by curette
1)Bonfadini M.G. 2)Magnani C. 3)Rostan I. 4)Pentenero M.
5)Gandolfo S. 6)Navone R.
1)Servizio di citologia, Clinica san gaudenzio, Novara, Italia 2)Servizio di
citologia, Clinica san gaudenzio, Novara, Italia 3)Sc. biomediche e oncologia umana-sez. anatomia pat., Universita di torino, Torino, Italia 4)Sc.
cliniche e biologiche-sez. med. e oncologia orale, Universita di torino,
Torino, Italia 5)Sc. cliniche e biologiche-sez. med. e oncologia orale, Universita di torino, Torino, Italia 6)Sc. biomediche e oncologia umana-sez.
anatomia pat., Universita di torino, Torino, Italia
Background. As oral squamous carcinoma is often diagnosed
in the late stages, its survival rate is low. This may be due to
the diagnostic difficulty and the fact that the lesion may develop
without evident dysplastic morphology. It is well known that
both Ki67 and p53, may be good markers for neoplastic and
preneoplastic oral lesions, as is DNA content. Therefore, we
compared the results of immunohistochemistry (IIC) to those of
the DNA ploidy and the microhistological diagnosis, according
to the method already described by our group [Navone R et al.
Oral Potentially Malignant Lesions: First Level Microhistological Diagnosis from Tissue Fragments Sampled in Liquid-Based
Diagnostic Cytology. J Oral Pathol Med 2008;37:358-363].
Methods. Curette sampling (AcuDispo Curette, Acuderm inc)
was carried out in 111 patients with lesions suspicious for carcinoma or potentially malignant lesions (PMLs) of the oral cavity.
Microhistology was done and the immunohistochemical reactions
assessed (Ki 67 e p53) and the data of IIC were compared to the
ploidy data already published by our group (Pentenero M et al.:
DNA Aneuploidy and dysplasia in oral potentially malignant
disorders. Oral Oncol 2009, 45:887-890).
Results. 11/111 cases were squamous carcinoma, 23 high grade
dysplasia, 22 low grade lesions and 55 keratosis without dysplasia. The Ki67 (p = 0.00006) and the dysplasia grade had a statistically positive correlation; suprabasal p53 had a lower correlation
(p = 0.02) as it was positive also for some keratosis cases without
evidence of dysplasia.
In conclusion, in the light of the strong correlation with preneoplastic and neoplastic lesions, above all the possibility of progression, Ki67 and p53 IIC seems to be useful in oral PMLs. Clinical
follow-up is indicated for p53 positive cases without dysplasia.
Expression of stem cells markers CD133, CD117,
and CD44 in prostatic adenocarcinomas is not
associated with stage and grading
1)Bosisio F.M. 2)Leone B.E.
1)Scienze chirurgiche (università milano-bicocca), Desio, Desio, Italia
2)Scienze chirurgiche (università di milano-bicocca), Desio, Desio, Italia
Background. The stem cells phenotype, when present in tumours,
may be able to explain some features of neoplastic progression,
as invasiveness or metastatization, and is hypotetically related to
a more aggressive behaviour. Stemness in prostatic cancer cells
have been studied so far only in cancer cell lines or in restricted
groups of cases. Aim of this study is to correlate the expression
of stem cells markers CD133, CD44, and CD117 in 113 cases of
acinar adenocarcinoma of the prostate primarily with tumor stage
and grading of the neoplasia, then with other prognostic and differentiation variables, such as immunohistochemical staining for
CXCR4, p53, cyclin D1, e-cadherin, vimentin, Ki-67 proliferation index, and basal, luminal or neuroendocrine phenotype.
Methods. Immunohistochemistry for CD133, CD117, and CD44
was performed on tissue microarrays of 113 prostate adenocarcinomas. Data were correlated to stage and grade, and with other
immunohistochemical markers of prognostic or differentiation
significance by statistical analysis.
Results. CD133 resulted positive in 21 out of 113 cases (18.5%),
CD44 in 86 out of 113 (76.1%), CD117 in 87 out of 113 (76.9%).
A simultaneous expression of the three stem cell markers
(CD133+, CD117+, CD44+) was found in 15 cases (13.3%).
None of the stemness markers, individually or simultaneously
considered, showed any kind of correlation with stage, grading,
and prognostic and differentiation markers, with the exception of
CXCR4, a putative mediator of invasiveness and itself related to
staminal phenotype.
Conclusion. The hypothetic stem cell phenotype of the prostate
cancer cells, defined by CD133, CD44, and CD117, is not able to
distinguish a subset of tumours with specific prognostic or differentiation features. Selection of other tumor-initiating cell markers
or resolution of technical problems related to the choice and use
of monoclonal antibodies is mandatory to better explore this field
of knowledge of tumor biology.
Cyclin d1 overexpression in Ewing’s sarcoma/
PNET: a potential marker helpful in the differential
diagnosis of small round cell tumours
1)F. Brancato, 2)R. Alaggio, 1)A. Gurrera, 1)E. Vasquez, 1)G.
Magro
1)Dipartimento G.F. Ingrassia, Anatomia Patologica - Università di Catania, Azienda Ospedaliero Universitaria Policlinico Vittorio Emanuele,
Catania, Italia; 2)Anatomia Patologica, Università di Padova, Azienda
Ospedaliera, Padova, Italia
Background. Ewing’s sarcoma(ES)/neuro-ectodermal primitive
tumour (PNET) is a small round cell sarcoma showing varying
degrees of neuroectodermal differentiation and the characteristic
t(11;22) (q24q12) chromosomal translocation. Although the diagnosis of ES/PNET is morphologically suspected, immunohistochemical analysis, including CD99, HNK1, FLI1 protein and
caveolin-1, is mandatory in confirming it. Unfortunately, these
markers are not highly specific, being also expressed in a wide
variety of pediatric small round cell tumours. In vitro studies have
shown that tumour cell lines of ES/PNET overexpress cyclin D1,
suggesting its key role in the mechanisms that regulate normal
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
cell cycle during G1-S. Additionally, in one immunohistochemical study the expression of cyclin D1 was found in approximately
42% of ES/PNETs, but without any significant correlation with
prognosis.
Methods. The aim of this paper was to study the comparative
immunohistochemical expression of cyclin D1 in 20 cases of pediatric ES/PNETs, 10 cases of embryonal rhabdomyosarcoma, 10
cases of alveolar rhabdomyosarcoma, including the solid variant,
and 5 cases of desmoplastic round cell tumours to assess its potential usefulness in the differential diagnosis of these tumours.
Results. Notably 100% of ES/PNETs expressed cyclin D1,
with a diffuse extension, ranging from 60 to 100% of neoplastic cells. In contrast, desmoplastic round cell tumours showed
immunoreactivity restricted to 10-20% of cells, whereas both
embryonal and alveolar rhabdomyosarcomas lacked cyclin D1
immunoreactivity. Our preliminary results suggest that immunohistochemical cyclin D1 overexpression may be exploitable
as an additional marker in the differential diagnosis of Ewing’s
sarcoma/PNET, rhabdomyosarcoma and desmoplastic small
round cell tumours. This finding, evaluated appropriately in
the context of a large panel of antibodies, may be helpful especially when dealing with small incisional biopsies or ambiguous immunohistochemical results due to sub-optimal fixation,
non-specific immunoreactivity or polyphenotypic profile by
neoplastic cells.
Abnormalities of chromosome 3 and 3q in
squamous lung carcinoma: genotypic patterns
with potential clinical impact
Brunelli M., Eccher A., Martignoni G., Brunello E., Parolini C.,
Pedron S., Menestrina F., Chilosi M.*
1)Department of pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy; *Department of pathology and diagnostic, Policlinico G.B.
Rossi, Verona, Italy
Background. Amplification of chromosome 3q is the most common genomic aberration in squamous pulmonary carcinoma,
however few reports distinguish chromosomal amplification
due to an increase of the locus specific region 3q or to the entire
chromosome 3. Nowadays, the distinction of the primary event
(amplification) vs the second (polysomy of a chromosome) is
mandatory, due to potential impact at a diagnostic or prognostic
levels. We sought to evaluate the subtypes of genotypic abnormalites of the entire chromosme 3 and the distal locus specific 3q
in a serie of squamous lung adenocarcinoma.
Methods. 18 squamous lung adenocarcinomas were recruited.
Immunophenotyping was performed by using antibodies for CK5,
p63, TTF-1 and CK7. Fluorescence in situ hybridization analysis
(FISH) was used to assess the centromeric region of the chromosome 3 (CEP3) and the locus specific gene (LSI) 3q (Olympus).
Polysomy of chromosome 3 without 3q amplification (more than
three CEP3 fluoresecent signals in 18% of the neoplastic nuclei),
polysomy of chromosme 3 with 3q amplification (ratio LSI 3q/
CEP3 > 2.2 in polysomic cells) and amplification of LSI 3q without polysomy of chromosome 3 were differently scored.
Results. All cases displayed CK5 and p63 positivity. TTF-1 and
CK20 were negative. Focal CK7 was observed in 12/18 cases.
4/18 (22%) squamous lung adenocarcinoma showed amplification of 3q without polysomy of chromosome 3, 9/18 (50%) polysomy of chromosome 3 with 3q amplification and 5/18 (28%)
polysomy of chromosome 3 without 3q amplification. Overall,
squamous pulmonary carcinoma usually show centromeric and
locus specific abnormalites on chromosome 3/3q; however there
are three distinctive patterns that may have potential value at
the prognostic level or when evaluating different therapeutical
strategies. The clinical impact of these multifaceted genotypic
abnormalities need further investigation.
Expanding immunophenotypical and molecular
features of tubulo-cystic renal cell carcinoma
1)Brunelli M. 2)Segala D. 3)Gobbo S. 4)Bersani S. 5)Bragantini
E. 6)Gardiman M. 7)Tardanico R. 8)Chilosi M. 9)Menestrina F.
10)Martignoni G.
1)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
2)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
3)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
4)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
5)Pathology, Ospedale “santa chiara”, Trento, Italy 6)Pathology, University of padua, Padova, Italy 7)Pathology, spedali civili, Brescia, Italy
8)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
9)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
10)Pathology, University of verona, policlinico “G.B. Rossi”, Verona,
Italy
Background. Among new emerging description of renal neoplasms, the tubulo-cystic renal cell carcinoma may be considered
a unique morphologic entity, with its distinctive gross and microscopic features. However, before it is accepted as a distinct renal
cell carcinoma subtype, further studies are needed to document
a characteristic molecular signature associated with this tumor.
It has also been questioned its relationship to papillary renal cell
carcinoma. We sought to evaluate the fluorescent molecular signature expanding the chromosomal in situ analysis.
Methods. Ten tubulo-cystic renal cell carcinoma were recruited,
5 of which from a single patient. Clinico-pathological analysis
were recorded. Immunophenotypical analysis using monoclonal
antibody against Cytokeratin 7 (CK7), S100A1, Parvalbumin
(PV), AMACR, CD10 were performed. Chromosomes 7, 12, 16,
17, 20 and Y and locus specific gene 7q31 (c-met), c-myc, EGFR,
p53, Her-2 were tested.
Results. Patients age ranged from 45 to 67, with a male preponderance (5:1). One patient showed 5 similar nodules. Another
patient presented a synchronous papillary renal cell carcinoma.
Tumours had a diameter ranged from 0,8 to 3,5 cm and all staged
pT1a. Grading sec. Furhman was G1-G2 in 7 cases and G3 in 3
cases. Cases stained immuno-positive for CD10 (10/10, 100%),
S100A1 (10/10, 100%) and AMACR (9/10, 90%); PV was
weakly and focally positive in 3 cases (3/10, 30%), while only
one case immunoexpressed CK7 (1/10, 10%). Entrapped tubules
into the neoplasms were positive for CK7. LSI-7q31 c-met was
gained in all cases. Two out of 5 gained chromosome 7 and 17.
Three out of 5 cases showed gains of p53, c-myc, EGFR. One
case showed loss of Y. Chromosome 20 were wild. The LSI
EGFR set wild.
Tubulo-cystic renal cell carcinoma are low staged and graded
tumours. Findings of c-met gains is similar to those reported in
papillary renal cell carcinoma, however the other chromosomes
do not show overlapping features.
New emerging morphological subtypes of papillary
renal cell carcinoma: gains of the 7q31 (C-MET)
as a molecular signature
1)Brunelli M. 2)Segala D. 3)Gobbo S. 4)Bersani S. 5)Eccher A.
6)Chilosi M. 7)Menestrina F. 8)Martignoni G.
1)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
2)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
3)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
4)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
5)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
6)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
7)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
8)Pathology, University of verona, policlinico “G.B. Rossi”, Verona, Italy
Background. Papillary renal cell carcinomas (RCCs) are morphologically divided into type 1 and type 2. Recently, other three
subtypes of papillary RCCs have been described such as the on-
Oral communications and Posters
cocytic and the spindle cells variants and papillary RCCs showing
clear cell changes. While gains of the entire chromosomes 7 and
17 have been described in all aforementioned subtypes of papillary RCCs, the status of the 7q31 gene was not assessed in the
new emerging subtypes. Gains of the locus specific region 7q31
(C-MET) in considered an hallmark of the papillary subtype of
RCCs.
Methods. 35 papillary RCCs including 12 type 1, 12 type 2, 6
oncocytic, 2 with spindle cells and 3 with clear cell changes were
recruited. Immunohistochemical analysis included AMACRracemase and cytokeratin 7. Chromosome 7 and 17 have been
assessed by fluorescence in situ hybridization analysis by using
centromeric CEP7 and CEP17 (Olympus) probes. The locus specific probes mapping the 7q31 region (C-MET) was also used.
Single, double and gains of fluorescent signals was scored per
neoplastic nuclei per each case.
Results. AMACR stained all cases. Cytokeratin 7 stained all
cases except 4 type 2, 3 oncocytic and 2 spindle cells subtypes
of papillary RCCs. Both chromosomes 7 and 17 gains were
observed in 11/12 type 1, in 8/12 type 2, 4/6 oncocytic, in 2/2
spindle cells and in 3/3 with clear cell changes tumours. Gains
of the locus specific gene 7q31 was observed in all type 1, 9/12
type 2, in 4/6 oncocytic, in 2/2 spindle cells and in 3/3 with clear
cell changes tumours. The mean score for assessing chromosomal
gains was 45% (range 15 to 76%).
In conclusion, the new emerging morphological subtypes of papillary RCCs have the molecular signature, such as gains of the
locus specific gene 7q31 (C-MET), that belongs to the genomic
profile of the papillary neoplasms. These findings may have a
primary potential value at a diagnostic level.
Lack of the tailored use of anthracycline
in the lobular subtype of breast carcinoma:
evidence on the Topoisomerase-IIA Amplicon
1)Brunello E. 2)Brunelli M. 3)Manfrin E. 4)Nottegar A.
5)Bersani S. 6)Vergine M. 7)Menestrina F. 8)Martignoni G.
9)Bonetti F.
1)Pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy 2)Pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy 3)Pathology and
diagnostic, Policlinico G.B. Rossi, Verona, Italy 4)Pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy 5)Pathology an, Policlinico G.B.
Rossi, Verona, Italy 6)Pathology and diagnostic, Policlinico G.B. Rossi,
Verona, Italy 7)Pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy 8)Pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy
9)Pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy
Background. In breast carcinoma, topoisomerase-IIa gene amplification appears to be a marker predictor of response to
anthracyclines therapy, with few contrasting data. Interestingly,
the lobular subtype usually does not respond to chemotherapies
such as those including doxorubicin/anthracycline. Few data area
available when matching topoisomerase-IIa gene and lobular
breast carcinoma, thus we sought to analyze the topoisomeraseIIa status in the lobular subtype.
Methods. 46 infiltrative lobular carcinomas, 13 with matched
loco-regional lymph-nodal metastases were recruited. Tissue microarrays have been built by punching three neoplastic cores per
case. Whole tumorous tissue sections were simultaneously evaluated. Topoisomerase-IIa gene amplification was analyzed by
both chromogenic (Zytolight) (CISH) and fluorescent (Olympus)
(FISH) in situ analyses. We also assessed the Her-2/neu status by
CISH, FISH and SISH (Ventana). Amplification was scored as
recommanded criteria. HER-2 immunoexpression was assessed
by using Hercept test.
Results. 44/46 (95%) of the cases did not reveal topoisomerase-IIa amplification whereas two of the 46 (5%) cases were
amplified. Eleven of the 13 metastatic sites did not reveal amplification neither in the primary nor in matched metastases (85%);
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the two remaining were amplified (15%). All cases revealed an
homogeneous status on all three neoplastic cores. The two cases
showing Her-2/neu and topoisomerase-IIa amplification scored
3+ the remaining not-amplified cases scored 0 or 1+ in 40 and
2+ in 4 cases.
In conclusion, the infiltrative lobular subtype of breast carcinoma
does not usually show topoisomerase-IIa gene amplification
neither in the primary nor lymph-nodal metastases. In the era
of personalised and tailored therapies, patients affected by the
lobular subtype of breast carcinoma lack in most of the cases the
biological rationale for receiving the common chemotherapy that
include anthracycline.
Subcutaneous Ewing sarcoma / PNET as a second
cancer in a previously irradiated young patient.
An uncommon type of post-irradiation soft tissue
sarcoma
1)Bruno M. 2)D‚Antona G.I. 3)Vita G. 4) Dicandia L.
5)Bisceglia M.
1)Division of Anatomic Pathology, Madonna delle Grazie Hospital, Matera, Italy 2)Division of Anatomic Pathology, Madonna delle Grazie Hospital, Matera, Italy 3)Division of Anatomic Pathology, IRCCS Institute
of Cancer, Rionero in Vulture, Italy 4)Department of Pathology, IRCCS
Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy
5)Department of Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy
Background. A second cancer is defined as a histologically
distinct cancer that develops in survivors after the first cancer.
The risk determinants of second cancer are multifactorial, and the
younger age of patients at the time of diagnosis of the first cancer,
the exposure to high-dose radiation therapy, the administration
of certain chemotherapeutic agents, and known genetic predisposition to cancer each play a role. Second cancers account for
6-10% of all malignant tumor diagnoses in USA 1. The increased
relative risk of developing a second cancer has been assessed in
both adults and children, and is higher (> 2-fold) in the latter 1.
Soft tissue sarcomas account for a small proportion of second
cancers, with an estimated frequency of < 10% 2. The most common histologic type of soft tissue sarcomas as second cancers
include mostly high-grade sarcomas, such as rhabdomyosarcoma,
malignant peripheral nerve sheath tumor, fibrosarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma, and Ewing sarcoma / primitive neuroectodermal tumor (PNET).
Objectives. To report a case of superficial soft tissue Ewing
sarcoma / PNET as a second cancer in a young patient previously
treated for Hodgkin’s disease (HD).
Case Report. A 18-year old boy developed a palpable soft tissue
mass at the level of the lateral border of his breast region, which
was surgically removed. This young patient had a known history
of HD, nodular sclerosis type (BNLI-II), stage IIB, involving the
neck and mediastinal lymph nodes, which had been diagnosed
approximately 4 years earlier. The patient had been treated with
chemotherapy (ABVD regimen therapeutic protocol) and conventional three-dimensional radiotherapy (Photon 6-10MW with
a total dose delivered of 25,5 Gy with a daily fraction of 150
cGy). The excised tumor was 3 cm in size, grossly circumscribed,
but unencapsulated, and on cut section appeared as a solid, greyish, and fleshy nodule, surrounded by healthy adipose tissue. Microscopically the tumor was composed of undifferentiated small
round cells, with scanty to moderate glycogen-rich cytoplasm
and vesicular nuclei, and showing scattered mitoses. Focally the
resection margins were very near to tumor. Immunohistochemically, the tumor cells were diffusely positive for vimentin, CD99/
O13, and FLI-1, and negative for skeletal muscle, hematolymphoid, neural and neuroendocrine, and epithelial markers (EMA,
cytokeratins w.s.). The tumor was diagnosed as Ewing sarcoma/
peripheral PNET, and a second local excision performed. Follow-
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
up: the patient was given courses of chemotherapy (IVADO regimen therapeutic protocol). Currently he is alive with no evidence
of disease at 1 year after the second cancer diagnosis.
Discussion. The cancer cure rate in children has greatly improved
with modern multimodal treatment protocols and nowadays approximately 70% of overall pediatric patients previously treated
for their (childhood) malignancies are long-term survivors 3.
Patients with childhood or adolescence cancer are at a 3.6-fold
increased risk for development of a second cancer relative to the
general population, and the estimated cumulative incidence of
a second cancer is 3.5% at 25 years 1. The determinants of the
increased relative risk of developing a second cancer are factors
that are partly host-related and partly therapy-related: the former
including the increased susceptibility of stem cells to mutagenic
effects and the higher rate of cell proliferation during development and differentiation (the substrate for their enhanced radiosensitivity), and for the latter radiation therapy and the usage of
certain chemotherapeutic agents (alkylating drugs and anthracyclines) 3. The most common cancers associated with the development of second cancers are hereditary retinoblastoma, soft-tissue
sarcomas, including Ewing sarcoma 2, and HD 1 3. The increased
relative risk for the development of a second cancer in HD, the
highest rate for a cancer with no known genetic predisposition 3
may be at least partly due to the disease itself as an independent
risk factor, and most likely to the specific chemo- and radiationtherapy required for treatment. The therapy-related factor in HD
are anthracyclines and radiotherapy, mostly the latter 1-5. The case
herein described may be qualified as a post-irradiation sarcoma,
based on the following standard criteria: previous exposure to irradiation, tumor site within the radiation field, latency of several
years, and histologic distinction from the primary tumor. Postirradiation soft tissue sarcomas are a well known but rare entity 1 2 4 5. As to their therapy all low-grade tumors and high-grade
tumors 5 cm or smaller may be treated with a margin-negative
surgical excision, and systemic chemotherapy can be considered
when a negative margin is difficult or impossible to obtain 6.
However it is suggested to treat also with chemotherapy those
known chemosensitive soft tissue sarcomas, as Ewing sarcoma
and rhabdomyosarcoma, which proved to be as chemosensitive
as second neoplasms as they are as primaries 7. Ewing sarcoma
/ PNET has already been documented as second malignant neoplasms appearing outside the irradiated area following treatment
for HD (1 case in the Italian joint AIEOP-INT/Milan series of
cases registered during the period of 1979-2004), angiosarcoma
or fibrosarcoma (1 case each 7), and in the irradiated area in a patient who had been treated for chronic myeloid leukemia (1 case
– same series). Ewing sarcoma / PNET often is mostly a sarcoma
of bone and deep soft tissue, occasionally involving even visceral
organs. Ewing sarcoma occurs very rarely as a primary superficial soft tissue tumor 8 9.
Conclusions. Post-irradiation soft tissue sarcomas are rare. They
occur only in irradiated tissues. HD is the most common childhood first malignancy at risk of developing second cancer later
both in childhood and in adulthood. Our case is presented for its
rarity and the described association.
References
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Bhatia S, Sklar C. Second cancers in survivors of childhood CANCER.
Nat Rew Cancer 2002;2:124-32.
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Nguyen F, Rubino C, Guerin S, et al. Risk of a second malignant neoplasm after cancer in childhood treated with radiotherapy: correlation
with the integral dose restricted to the irradiated fields. Int J Radiation
Oncology Biol Phys 2008;70:908-15.
3
Neglia JP, Friedman DL, Yasui Y, et al. Second malignant neoplasms
in five-year survivors of childhood cancer: childhood ancer survivor
study. J Natl Cancer Inst 2001;93:618-29.
4
Schneider U, Lomax A, Timmermann B. Second cancers in children treated with modern radiotherapy techniques. Radiother Oncol
2008;89:135-40.
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6
7
8
9
Hall EJ, Wuu C-S. Radiation-induced second cancers: the impact of
3D-CRT and IMRT. Int J Radiation Oncology Biol Phys 2003;56:838.
Patel SR. Radiation-induced sarcoma. Curr Treat Options Oncol
2000;1:258-61.
Bisogno G, Sotti G, Nowicki1 Y, et al. Soft tissue sarcoma as a
second malignant neoplasm in the pediatric age group. Cancer
2004;100:1758-65.
Banerjee SS, Agbamu DA, Eyden BP, et al. Clinicopathological characteristics of peripheral primitive neuroectodermal tumour of skin and
subcutaneous tissue. Histopathology 1997;31:355-66.
Bisceglia M, Fisher C, Suster S, et al. Tumoral, quasitumoral and
pseudotumoral lesions of the superficial and somatic soft tissue: new
entities and new variants of old entities recorded during the last 25
years. Part VII: excerpta V. Pathologica 2005;97:92-114.
Epigenetic silencing of wnt-inhibitors: activation
of a constitutive wnt signalling in oral cancer
1)Bufo P. 2)Pannone G. 3)Santoro A. 4)Sanguedolce F. 5)Franco
R. 6)Losito S. 7)Botti G. 8)Lo muzio L.
1)Department of surgical sciences, section of anatom, Riuniti, Foggia,
Italy 2)Department of surgical sciences, section of anatom, Riuniti, Foggia, Italy 3)Department of surgical sciences, section of anatom, Riuniti,
Foggia, Italy 4)Department of surgical sciences, section of anatom, Riuniti, Foggia, Italy 5)Istituto nazionale per lo studio e la cura dei tum, Fondazione “G. Pascale”, Napoli, Italy 6)Istituto nazionale per lo studio e la
cura dei tum, Fondazione “G. Pascale”, Napoli, Italy 7)Istituto nazionale
per lo studio e la cura dei tum, Fondazione “G. Pascale”, Napoli, Italy
8)Department of surgical sciences, Irccs crob - centro di riferimento oncologico di b, Rionero in vulture, Italy
Background. Epigenetic DNA methylations plays an important
role in oral carcinogenesis. The soluble frizzled receptor protein
(SFRP) family together with WIF-1 and DKK-3 encodes antagonists of the WNT pathway. Silencing of these genes leads
to constitutive WNT signalling. Because aberrant expression of
ß-catenin might be associated with the epigenetic inactivation of
WNT inhibitors, we analyzed, in a collection of primary OSCC
with matched normal oral mucosa, the methylation status of a
complete panel of genes, SFRP-1, SFRP-2, SFRP-4, SFRP-5,
WIF-1, DKK-3, that are involved directly and indirectly in WNT
pathway, in order to demonstrate WNT-pathway activation in
the absence of ß-catenin and/or APC/Axin mutations during oral
carcinogenesis.
Methods. Methylation-specific PCR (MSP) was performed to
study inactivation of SFRP-1, SFRP-2, SFRP-4, SFRP-5, WIF-1,
DKK-3 genes in 37 cases of paraffin embedded oral cancer.
Results. This study showed that the methylation is an important
epigenetic alteration in oral cancer. In particular, SFRP-2, SFRP4, SFRP-5, WIF-1, DKK-3 revealed methylation status of their
promoter in OSCC, whereas SFRP-1 showed demethylation in
cancer. Fisher’s exact test revealed statistically significant results
(p < 0.05) for all genes. The Wald test confirmed the statistically
significant association between SFRP2-4-5 gene methylation
and OSCC (p < 0.05). SFRP-1 was also characterized by a different statistically significant epigenetic behaviour, because of it
was demethylated in cancer (p < 0.05). Statistical regression test
showed high levels of sensitivity, specificity and accuracy for
SFRP genes, while WIF-1 and DKK-3 have reportedly high specificity, moderate accuracy but low sensitivity. This study suggests
that a cause of catenin delocalization in oral cancer could be due
to WNT pathway activation, by epigenetic alterations of SFRP,
WIF-1 and DKK-3 genes.
267
Oral communications and Posters
Epigenetic profile in endometrial carcinogenesis
1)Bufo P. 2)Pannone G. 3)Santoro A. 4)Sanguedolce F. 5)Losito
S. 6)Pasquali D. 7)Guida M.
1)1. department of surgical sciences - section of an, Riuniti, Foggia, Italy
2)1. department of surgical sciences - section of an, Riuniti, Foggia, Italy
3)1. department of surgical sciences - section of an, Riuniti, Foggia, Italy
4)1. department of surgical sciences - section of an, Riuniti, Foggia, Italy
5)2. istituto nazionale per lo studio e la cura dei, Fondazione “G. Pascale”, Napoli, Italy 6)3. department of clinical and experimental medicin,
Sun, Napoli, Italy 7)4. department of gynaecology and obstetrics, University of naples “Federico II”, Napoli, Italy
Background. Transcriptional silencing by CpG island hypermethylation plays a critical role in endometrial carcinogenesis. In
a collection of benign, premalignant and malignant endometrial
lesions, a methylation profile of a complete gene panel, such
steroid receptors (ER_, PR), DNA mismatch repair (hMLH1),
tumour-suppressor genes (CDKN2A/P16 and CDH1/E-CADHERIN) and WNT pathway inhibitors (SFRP1, SFRP2, SFRP4,
SFRP5) was investigated in order to demonstrate their pathogenetic role in endometrial lesions.
Methods. Methylation-specific PCR (MSP) was performed to assess gene inactivation. P53 and steroid receptors expression were
evaluated by LSAB/HRP immunohistochemistry.
Results. Our results indicate that gene hypermethylation may be
an early event in endometrial endometrioid tumorigenesis. Particularly, ER_, PR, hMLH1, CDKN2A/P16, SFRP1, SFRP2 and
SFRP5 revealed a promoter methylation status in endometrioid
carcinoma, whereas SFRP4 showed demethylation in cancer. P53
immunostaining showed weak-focal protein expression level both
in hyperplasic lesions and in endometrioid cancer. Non endometrioid cancers showed very low levels of epigenetic methylations,
but strong P53 protein positivity. Fisher exact test revealed a statistically significant association between hMLH1, CDKN2A/P16
and SFRP1 genes methylation and endometrioid carcinomas and
between hMLH1 gene methylation and peritumoral endometrium
(p < 0.05). Our data confirm that the methylation profile of the
peritumoral endometrium is different from the altered molecular background of benign endometrial polyps and hyperplasias.
Therefore, our findings suggest that the methylation of hMLH1,
CDKN2A/P16 and SFRP1 may clearly distinguish between benign and malignant lesions. Finally, this study assessed that the
employment of an epigenetic fingerprint may improve the current
diagnostic tools for a better clinical management of endometrial
lesions.
Vulvar angiomyofibroblastoma: a clinicopathological
study of nine cases, including the lipomatous
variant
1)G. Vecchio, 1)R. Caltabiano, 1)A. Gurrera, 2)D. Kacerovska,
3)M. Bisceglia, 2)M. Michal, 1)G. Magro
1)Dipartimento G.F. Ingrassia, Anatomia Patologica, Università di Catania, Azienda Ospedaliero-Universitaria Policlinico Vittorio Emanuele,
Catania, Italia; 2)Sikl’s Department of Pathology, Medical Faculty Hospital, Pilsen, Czech Republic 3)Dipartimento di Anatomia Patologica,
IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italia
Background. Angiomyofibrobastoma (AMF) is an uncommon,
benign stromal tumour usually arising in the vulva. Other sites,
including vagina, urethra, perineum, fallopian tube, inguino-scrotal and para-rectal region in male, may be involved. Clinically,
most of the tumours present as a slowly-growing painless mass,
often misdiagnosed as a Bartholin’s gland cyst, hydrocele, or aggressive angiomyxoma. Clinical behaviour is benign with a low
tendency for local recurrence.
Methods. The clinicopathological features of 9 cases of AMFs of
the vulva are presented with emphasis on unusual morphological
features.
Results. The lesions usually presented as painless masses located
in the superficial vulvar region of women ranging in age from 46
to 60 years. They were well circumscribed and ranged in size from
2 to 3.5 cm in greatest diameter. Histologically, they were composed predominantly of medium-sized spindle to epithelioid cells
variably arranged in cords or nests, and embedded in a fibrous
to only focally myxoid stroma. In most cases neoplastic cells
exhibited a perivascular arrangement around small to mediumsized hyalinized blood vessels. Mitotic activity ranged from 0 to
2 mitoses per 50 HPF. Atypical mitoses, nuclear atypia and necrosis were not observed. Interestingly 3 cases, labelled “AMFs,
lipomatous variant”, contained an abundant intratumoral fatty
component, ranging from 20% to 70% of the entire tumour. In
one case, adipocytes focally exhibited a lipoblast-like appearance.
Additional unusual findings were the presence of neoplastic cells
with vescicular nuclei and CD68+ giant multinucleated osteoclast-like cells. Immunohistochemically the cells were positive to
vimentin, and variably to α-smooth muscle actin, desmin, CD34,
and estrogen/progesterone receptors. No local recurrence was observed after a follow-up period ranging from 3 to 20 years.
Myofibroblastoma of the lower female genital
tract: expanding the morphologic spectrum,
including the mammary-type variant
1)P. Amico, 1)R. Caltabiano, 2)D. Kazakov, 2)D. Kacerovskà,
2)M. Michal, 1)G. Magro
1)Dipartimento G.F. Ingrassia, Anatomia Patologica, Università di Catania, Azienda Ospedaliero-Universitaria-Policlinico Vittorio Emanuele,
Catania, Italia; 2)Sikl’s Department of Pathology, Charles University Medical Faculty Hospital, Pilsen, Czech Republic
Background. Over the last decade, a benign myofibroblastic
tumour with distinctive clinicopathological features has emerged
from the category of the stromal tumours of the lower female
genital tract, including aggressive angiomyxoma, angiomyofibroblastoma and cellular angiofibroma. The terms “superficial
cervicovaginal myofibroblastoma (MFB)” or “MFB of the lower
female genital tract” have been used interchangeably for this entity which characteristically arises from the sub-epithelial stroma
of the vagina, and less frequently, of the vulva or cervix.
Materials. We herein report the clinicopathological features of
10 cases of MFB of the lower female genital tract to expand the
morphologic spectrum.
Results. Tumours clinically presented as polypoid or nodular
masses of variable size (1-3 cm) located in vagina (7 cases) and
vulva (3 cases). Age at diagnosis ranged from 19 to 69 years ().
Histologically, all tumours were well circumscribed and unencapsulated, with the typical localization in the sub-epithelial connective tissue. Unlike previously reported, a band of native connective tissue, separating tumours from the overlying squamous
epithelium, was missing in 5 cases, with tumour cells extending
up to the epithelium. Neoplastic cells, from stellate to ovoid to
spindle in shape, were embedded in a finely fibrous to focally
myxoid stroma. Five tumours, being predominantly composed of
spindle-shaped cells arranged in short fascicles with intervening
thick collagen bands, were closely reminiscent of mammary MFB.
Mitoses were rare. Only focally mild nuclear pleomorphism was
seen. Interestingly, 6 cases showed hyalinized thick-walled blood
vessels. Immunohistochemically, tumours were variably positive
for desmin, α-smooth muscle actin, CD34, CD10, ER and PR.
The present study first identifies the mammary-type variant of
MFB of the lower female genital tract. Based on morphological
and immunohistochemical findings, we postulate that MFB of
the breast and MFB of the lower female genital tract arise from
a common precursor stem cell which typically resides in the hormonally active stroma of women.
268
5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
The mosaic pattern of INI1/SMARCB1 protein
expression is a reliable marker of sporadic
schwannomatosis: an immunohistochemical study
in a series of 10 cases
1)A. Torrisi, 2)R. Caltabiano, 3)M. Ruggieri, 4)P. Nozza, 5)A.
Ortensi, 5)V. D’Orazi, 2)S. Lanzafame, 2)G. Magro
1)Dipartimento G.F. Ingrassia, Registro Tumori Integrato-Messina-Catania-Siracusa, Catania, Italia; 2)Dipartimento G.F. Ingrassia, Anatomia
Patologica, Università di Catania, Azienda Ospedaliero-Universitaria
Policlinico-Vittorio Emanuele, Catania, Italia; 3)Istituto Scienze Neurologiche CNR, Azienda Ospedaliero-Universitaria Policlinico-Vittorio Emanuele, Catania, Italia; 4)Anatomia Patologica, Ospedale Gaslini,Genova,
Italia; 5)Microchirurgia e Chirurgia della Mano, Università La Sapienza,
Fabia Mater, Roma, Italia
Background. Schwannomatosis is characterized by the development of multiple spinal, peripheral, and cranial nerve schwannomas in the absence of diagnostic bilateral vestibular schwannomas of NF2. The majority of cases of schwannomatosis are
sporadic, but familial cases do exist with an autosomal dominant
pattern of inheritance. The INI1/SMARCB1 is a tumour suppressor gene that maps to chromosome band 22q11.2. It affects
the expression of genes that regulate cell cycle, growth, and differentiation. It is also involved in the development of malignant
rhabdoid tumours.
Methods. The immunohistochemical expression of INI1/
SMARCB1 was assessed in a series of 10 cases of sporadic
schwannomatosis and compared with the immunohistochemical
profile of 5 cases of solitary sporadic schwannomas.
Results. As expected, all sporadic schwannomas showed diffuse
nuclear positivity ranging from 97% to 100% of neoplastic cells.
On the contrary schwannomas from sporadic schwannomatosis
showed a mosaic pattern, namely alternating positive and negative nuclei, consistent with the loss of INI1/SMARCB1 expression in a subset of tumour cells, ranging from 10% to 70% in the
different cases. The little data available in the literature showed
that only 55% of schwannomas from sporadic schwannomatosis
exhibit the mosaic pattern of INI1/SMARCB1 expression, as
commonly observed in familial schwannomatosis.
We first report that 100% of schwannomas from sporadic
schwannomatosis have a mosaic pattern of INI1/SMARCB1
expression. Accordingly, apart from familial schwannomatosis,
loss of immunohistochemical INI1/SMARCB1 expression, albeit
with an interlesional variability, is a reliable marker of sporadic
schwannomatosis.
Analysis protocol of the retroareolar margin in the
nipple sparing mastectomy: our experience
1)Costarelli L. 1)Campagna D. 2)Amini M. 3)Fortunato L. 4)Poccia I.
1)Anatomia ed istologia patologica, Az. osp S. Giovanni-Addolorata,
Roma, Italia 2)Anatomia ed istologia patologica, Az. osp S. GiovanniAddolorata, Roma, Italia 3)I chirurgia, Az. osp S. Giovanni-Addolorata,
Roma, Italia 4)I chirurgia, Az. osp S. Giovanni-Addolorata, Roma, Italia
Introduction. The nipple-sparing mastectomy (NSM) has become an accepted treatment for appropriately selected breast
cancers to improve the aesthetic results and the patient’s satisfaction.
The cosmetic results of the mastectomy followed by immediate
reconstruction or by prosthetic implant have been judged to be
good to excellent in 82% of the cases.
The principal postoperative complication requiring a second surgical treatment is the necrosis of the nipple and the retro-areolar
margin positive. To avoid doing a second surgery may be performed the intraoperative frozen sections and HE histopathologic
examination of the retro-areolar tissue.
Methods. The authors performed 43 nipple-sparing mastectomies
on 37 patients (6 bilateral) during 2009/2010. The retro-areolar
tissue obtained was serially sectioned throw 5-6 parallel slices at
the time of the intraoperative frozen section, after painting surgical margin with India ink.
The ablation of nipple-areola complex (NAC) was performed in
the same time of the subcutaneous mastectomy if the neoplasia
was close to margin (< mm 1).
Results. The average age of the patients was 46 years (46 ± 9;
range 32-67). The indications into account to decide to performed the nipple-sparing mastectomy were multifocality in
36 cases (6 bilateral), locally advanced stage in 5 cases and
familiarity in 2 cases. The rate of positive retroareolar margin
was 11.6 percent (5 cases) at the frozen sections following the
excision of the NAC in the same time. The rate of ablation of
NAC in a second time was 11.6 percent (5 cases) for post-operative necrosis and 2.4 percent (1 case) for false negative at
the intraoperative examination. Finally, the intraoperative protocol to examinate the retroareolar tissue reduce the percentage
of reintervention improving cosmetic results with a high level
of surgeons’ and patients’ satisfaction.
Nut midline carcinoma: report of a case with
unusual immunoprofile
1)A. Canesso, 2)R. Alaggio, 3)E.G.S. D’Amore, 4)E. Gaio, 4)R.
Artico, 1)S. Agabiti, 1)F. Sonego, 1)M. Guido
1)Anatomia Patologica, A.o.ulss15 Alta Padovana, Cittadella, Italia,
2)Anatomia Patologica, Università degli Studi di Padova, Padova, Italia;
3)Anatomia Patologica, Ospedale San Bortolo, Vicenza, Italia; 4)ORL,
A.o.ulss15 Alta Padovana, Cittadella, Italia
Background. NUT midline carcinoma is a rare, highly aggressive neoplasia associated with rearrangement of the NUT gene on
chromosome 15q14 most commonly in a balanced translocation
with the BRD4 gene on chromosome 19p13, originally reported
in head and neck and mediastinum in young females. Subsequently NUT-carcinoma has been identified in all age groups
(from 3 to 78 years). We report a case of NUT midline carcinoma
arising in a 52 year-old woman presenting with a left neck mass
and displaying a challenging immunophenotype.
Methods. A 52 year-old woman with no remarkable medical
history and completely asymptomatic presented with a left neck
mass of three months duration. A TC scan showed enlarged,
centrally necrotic lymph nodes that were surgically removed and
submitted for pathology evaluation. The specimen was formalinfixed, paraffin embedded and routinely stained (e.e), then a wide
panel of immunostains was performed.
Results. The neoplasia was composed of undifferentiated cells of
medium size with scant eosinophilic cytoplasm, irregular nuclei
and prominent nucleoli. Mitoses and areas of coagulative necrosis were common. Immunohistochemistry showed reactivity for
CK7, MNF116, 34βE12, CD34 and p63. An unusual dot-like
reactivity for WT1 and Vimentin was noted, as well as cytoplasmic positivity for κ and γ light chain. All the other immunostains
were negative, thus excluding lymphoma, sarcomas, melanoma
or metastatic carcinoma. NUT immunostaining showed a strong
and diffuse nuclear staining. FISH analysis was positive for NUT
rearrangement, but not for BRD4 rearrangement, consistent with
a NUT-variant carcinoma. The patient died two months later for
disseminated disease. NUT-variant carcinoma is an under-recognized entity and should be considered in the spectrum of differential diagnoses in metastatic carcinomas with unknown primary
tumor. Moreover aberrant positive immunostains, like κ and γ in
the present case may represent a potential diagnostic pitfall.
269
Oral communications and Posters
Diagnostic value of automated Her2 evaluation in
breast cancer. A study on 272 equivocal (score 2+)
Her2 immunoreactive cases using an fda approved
system
1)Cantaloni C. 2)Eccher C. 3)Morelli L. 4)Leonardi E. 5)Bragantini E. 6)Aldovini A. 7)Fasanella S. 8)Ferro A. 9)Dalla palma P.
10)Barbareschi M.
1)Anatomia patologica, S Chiara, Trento, Italia 2)Statistica, Fondazione
Bruno Kessler, Trento, Italia 3)Anatomia Patologica, S Chiara, Trento,
Italia 4)Anatomia Patologica, S Chiara, Trento, Italia 5)Anatomia Patologica, S Chiara, Trento, Italia 6)Anatomia Patologica, S Chiara, Trento, Italia 7)Anatomia Patologica, S Chiara, Trento, Italia 8)Oncologia,
S Chiara, Trento, Italia 9)Anatomia Patologica, S Chiara, Trento, Italia
10)Anatomia Patologica, S Chiara, Trento, Italia
Backgroung. Accurate immunohistochemical Her2 evaluation is
fundamental for treatment of breast cancer (BC). The U.S. Food
and Drug Administration (FDA) approved the Aperio IHC Her2
Breast Tissue Image Analysis application for the detection and
semi-quantitative measurement of Her2.
Methods. To validate computer assisted analysis (CAA) in
clinical practice we analyzed 292 equivocally (score2+) Her2 immunoreactive BC; all cases were stained with Dako Herceptest,
evaluated by an experienced pathologist and analyzed with FISH.
The automatic Aperio categorization and the percentage of immunoreactive cells as evaluated by the computer (CPV) and by the
pathologist (PPV) were recorded. CAA classified 7 (2.4%) cases
as negative (0), 136 (46.6%) as score 1+, 134 (40.5%) as score
2+ and 15 (5.1%) as score 3+. CCA classification is associated
with Her2 amplification (p < 0.0001). The mean CPV is 18.44%
sd ± 19.00 (range 0.01-76.10).
Results. CPV and PPV are significantly associated and correlated (p < 0.001), have similar sensitivity and specificity in
identifying Her2 FISH amplified cases. The difference in CPV in
amplified and non amplified subgroups is statistically significant
(p < 0.001). ROC analysis indicates that CPV is good at separating FISH not-amplified from amplified cases (p < 0.001). The
optimal cut-off value maximizing both sensitivity and specificity
is 17.6% (sensitivity = 73.3%, specificity = 71.6%). Reducing
the cut-off value to 0.67% it is possible to reach the sensitivity
of 100% with 16.2% specificity. CCA Her2 IHC evaluation is
feasible and reliable: automated classification is not satisfactory
as some amplified cases might be erroneously clustered as score
1+. Lower CPV cut-off values should be used. CAA can reduce
the number of cases unnecessarily submitted to FISH.
The role of geminin, a DNA replication factor,
in oral squamous cell carcinoma. Preliminary
report of a tissue micro array based
immunohistochemical study
1)Cantile M. 2)Franco R. 3)Aquino G. 4)Losito S. 5)Botti G.
6)Santoro A. 7)Mattoni M. 8)Bufo P. 9)Pannone G.
1)Istituto Nazionale Per Lo Studio E La Cura Dei Tum, Fondazione ‘G.
Pascale’, Napoli, Italy 2)Istituto Nazionale Per Lo Studio E La Cura Dei
Tum, Fondazione ‘G. Pascale’, Napoli, Italy 3)Istituto Nazionale Per Lo
Studio E La Cura Dei Tum, Fondazione ‘G. Pascale’, Napoli, Italy 4)Istituto Nazionale Per Lo Studio E La Cura Dei Tum, Fondazione ‘G. Pascale’, Napoli, Italy 5)Istituto Nazionale Per Lo Studio E La Cura Dei
Tum, Fondazione ‘G. Pascale’, Napoli, Italy 6)Department Of Surgical
Sciences, Institute Of Path, Riuniti, Foggia, Italy 7)Department Of Surgical Sciences, Institute Of Path, Riuniti, Foggia, Italy 8)Department Of
Surgical Sciences, Institute Of Path, Riuniti, Foggia, Italy 9)Department
Of Surgical Sciences, Institute Of Path, Riuniti, Foggia, Italy
Background. The DNA replication licensing machinery is integral
to the control of proliferation differentiation, and maintenance of
genomic stability in human cells. Geminin is a licensing repressor
and prevents re-initiation of cell replication by blocking re-loading
of MCM proteins at replication origins. The recent literature has
proposed that Geminin could be used as sensitive proliferative and
prognostic marker. The aim of this study is the evaluation of Geminin expression in oral squamous cell carcinomas (OSCCs) by Tissue Micro Array based immunohistochemistry (TMA based IHC).
Methods. We performed TMA based IHC on 10 specimens of
normal oral squamous epithelia and 150 OSCCs. IHC was performed by standard streptavidinin-biotin immunoperoxidase method (LSAB-HRP) using specific monoclonal Ab against Geminin.
Results. Geminin is a 25kDa nuclear protein involved in regulation
of the initiation of DNA replication. DNA replication requires the
association of Cdc-6 and minicromosome maintenance (MCM)
protein with chromatin. Geminin blocks this assembly of the
MCM into the pre-replication complex. Expression of Geminin is
regulated throughout the cell cycle with Geminin levels lowest at
G1. Throughout S, G2 and M phases, Geminin levels are elevated
followed by a decrease during mitosis as the protein is targeted for
degradation by the anaphase-promoting complex (APC). Our study
showed Geminin over-expression in the most of OSCCs as compared to normal oral epithelia. In details, this proteins was strongly
up-regulated in OSCCs characterized by high mitotic index. Our
preliminary results indicate that assessment of Geminin may be
useful as prognostic factor in patients with OSCCs.
Adenoid-cystic carcinoma of the breast with
sebaceous and adenosquamous differentiation.
A clinicopathologic study of an aggressive case
1)M. Carlucci, 1)M. Iacobellis, 1)F. Colonna, 2)M. Marseglia,
3)M. Gambarotti, 4)M. Bisceglia, 5)C. Giardina
1)Anatomia Patologica, Ospedale Umberto I, Altamura, Italia; 2)Chirurgia, Ospedale Umberto I, Altamura, Italia; 3)Anatomia Patologica, Istituto Ortopedico Rizzoli, Bologna, Italia; 4)Anatomia Patologica, IRCCS
Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italia;
5) Anatomia Patologica, Università degli Studi di Bari, Bari, Italia
Background. Adenoid cystic carcinoma (ACC) of the breast represents about 0.1% of breast carcinomas and, in contrast to the aggressive nature of the homonymous tumor arising in the head and
neck region, usually has a favorable prognosis. This tumour has
well-demarcated margins, and can be over 10 cm diameter size and
multifocal. ACC is “a morphologically heterogeneous neoplasm”
with trabecular-tubular, cribriform and solid pattern, occasionally
associated with sebaceous and adenosquamous differentiaton.
Case report. An 84-year old woman was admitted with an ulcerated 12 cm tumor mass in her left breast, without palpable axillary lymph nodes. A simple mastectomy was performed. On histological examination a mixed type of adenoid-cystic carcinoma
of the breast with sebaceous and adenosquamous differentiation
was apparent, with trabecular, cribriform and solid patterns, and
in places sharing features of ordinary invasive ductal carcinoma.
Immunohistochemically the cribriform areas were focally positive for actin and for CK34beta12. The pseudocystic spaces were
partly positive for type IV collagen. EMA strongly decorated
areas of sebaceous differentiation and c-kit immunoreactivity was
also focally documented. Estrogens and progesterone receptors
were totally negative.
After mastectomy a total body CT scan showed pulmonary and
osseous metastases that partially responded to chemotherapy.
About 7 months later an intramuscular mass rapidly growing up
to > 10 cm was also noticed in her right thigh. Following a needle
biopsy-based diagnosis of malignancy, the tumor mass was excised, and the histological diagnosis established was “metastasis
of ductal carcinoma G3 with sebaceous differentiation”.
Conclusion. Adenoid-cystic carcinoma has the potential to
differentiate toward skin adnexal structures giving rise to both
sebaceous and adenosquamous cells. However similar tumors
may also be interpreted as “mixed invasive carcinoma” with both
usual features and features commonly seen in salivary gland type
and adnexal skin type carcinomas.
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
Incidence of O6-methylguanine DNA
methyltransferase expression in pituitary
adenomas: our experience at the “Regina Elena”
National Cancer Institute
Carosi M., 1Baldelli R., Panichi D., 1Barnabei A., 2Telera S., 1Appetecchia M., 2Pompili A., Pescarmona E.
Pathology, 1Endocrinology and 2 Neurosurgery, “Regina Elena” National
Cancer Institute
Clinically significant pituitary tumours occur in approximately
in every 1000 individuals. The majority of pituitary tumours are
benign adenomas; however, between 35% and 55% of adenomas
demonstrate invasion into bone, dura or adjacent structures such
as the cavernous or sphenoid sinuses or brain. Although it is a
rare phenomenon, a subset of invasive adenomas display aggressive behaviour and become resistant to medical therapy, causing
substantial morbidity; these tumours require multiple operations
and radiotherapy in an attempt to control tumour growth. Various
chemotherapeutic regimes have been tried in the management of
pituitary carcinoma. Although occasional temporary responses
are reported, the results are usually disappointing. Recent case
studies have successfully used temozolomide, an alkylating chemotherapeutic drug, in the management of pituitary carcinoma
and aggressive pituitary tumours. Temozolomide is widely used
in the management of glioblastoma multiforme and is effective in
other neuro-oncological tumours as well as other neuroendocrine
tumours. Temozolomide is administered orally, readily crosses
the blood–brain barrier and is not cell-cycle specific, advantageous when treating relatively slow-growing pituitary tumours.
O-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that reverses alkylation at the O position of guanine.
As such, MGMT counteracts the effect of temozolomide, which
alkylates DNA at this position. Low tumour MGMT expression
has been shown in some studies to correlate with temozolomide
response and increased survival in patients with brain tumours. A
commonly proposed mechanism of reduced MGMT expression is
methylation of its promoter, although different tumour types vary
widely in the frequency of methylation.
The aim of this study was to evaluate the possible relationship
between MGMT hypermethylation and clinical response to chemotherapy in pituitary adenomas; the method to determine the
hypermethylation status of MGMT, namely methylation-specific
PCR, allowing the selection of patients most likely to benefit
from temozolomide treatment
Fine needle aspiration cytology of thyroid lesions:
cytohistologic correlation and accuracy. Overwiew
of 15 years experience
1)Casadei G.P. 2)Crucitti P. 3)Lega S. 4)Bondi A.
Anatomia Patologica, Dipartimento di Oncologia, Ospedale Maggiore,
Bologna
Objective: The aim of this study was to evaluate the accuracy of
the fine needle aspiration cytology (FNAC) and its contribution
to tumor diagnosis.
Methods. In the period from 1995 to 2009, a total of 12.989
thyroid FNAC were performed at Maggiore hospital and in
two referring smaller hospitals, and examined in a pathology
laboratory at one site. Cytologic diagnoses were re-classified
according to the Italian Society of Pathology (SIAPEC) 5-tiered
category system, as THY 1 unsatisfactory, THY 2 benign, THY 3
indeterminate, THY 4 suspicious, THY 5 malignant. Patients who
underwent surgical treatment were 3.944 (30%). Sample with histologic discrepancy were rewieved, and clinically re-evaluated.
Results. The distribution of cytologic samples by the 5 diagnostic
categories was 3.088 (24%) inadequate (THY 1), 68% THY 2, 5%
THY 3, 1,2% THY 4, and 1,4% THY 5.
Histological examination was performed in 2% of inadequate
samples, 4,7% of benign lesions, 22% in THY 3, and in 33% and
42% of THY 4 and THY 5 category respectively.
Malignancy was histologically observed in 22% of inadequate
FNAC, 16% of benign lesions, 38% of undeterminate lesions, 82% of suspicious lesions and 93% of malignant ones.
Diagnostic discrepancy rate between cytologic and histologic
diagnosis was about 15%. The overall sensitivity and specificity of thyroid FNAC was 77% and 95% respectively. Wrong
diagnostic results arise from errors of sampling, as for smaller
than 1 cm nodules, inadequate smearing, and cytological
equivocal features.
Conclusions. Although FNAC of thyroid nodules can be performed with high sensitivity and specificity, it needs of application of firm rules and guidelines in performing the procedure in
such a way to reduce the rate of false-negative and false-positive
diagnoses. The study of clinical correlation in single case and the
direct involvement of the pathologist with the clinicians on taking
the sample is advisable.
The human claustrum: a microanatomical and
immunohistochemical study
1)Castagna M. 2)Fattori S. 3)Castelluccio E. 4)Quilici F. 5)Perrini P. 6)Pirone A.
1)Dipartimento di Chirurgia, Ospedale S. Chiara, Pisa, Italia 2)Dipartimento di Chirurgia, Ospedale S. Chiara, Pisa, Italia 3)Dipartimento di
Chirurgia, Ospedale S. Chiara, Pisa, Italia 4)Dipartimento di Chirurgia,
Ospedale S. Chiara, Pisa, Italia 5)Neurochirurgia, Ospedale S. Chiara,
Pisa, Italia 6) Dipartimento di Produzione animale, Università di Pisa,
Pisa, Italia
Background. The claustrum is a thin collection of gray matter located deep with respect to the insula. While numerous investigations focused on the chemo- and cytoarchitecture of the claustrum
specific to the localization of calcium-binding proteins (CBPs)
and neuropeptides (Nps) in a variety of mammalian species, few
studies examined the microanatomy and the immunohistochemistry of human claustrum.
Methods. Two normal human cerebral hemispheres were fixed
in a 10% formalin solution for two months and then frozen at -10
to -15C for two to four weeks. The lateral surface of the brain
was dissected by applying Klinger’s fiber dissection technique
under microspcope. One additional brain of a 65-year old male
who died of a myocardial infarction provided the claustrum for
immunohistochemical characterization. Individual sections were
processed for Nissl, parvalbumin (PV) or neuropeptide-Y (NPY)
staining. Tissue was processed with either monoclonal anti-PV
mouse ascites fluid clone PA-235 (P-317; 1:1000: Sigma) or
rabbit anti-NPY porcine serum (IHC 7172, 1:800, Peninsula).
PV-positive neurons were viewed with a confocal microscope using indirect immunofluorescence (Leica TCS-NT, krypton-argon
laser), and NPY-positive neurons were viewed using standard
light microscopy (Leitz Diaplan).
Results. The claustrum presents a ventral (fragmented) and a
dorsal (compact) part which are, respectively, anteroinferior and
posterosuperior. The ventral part of the external capsule forms
the uncinate and occipito-frontal fascicles. The dorsal part of the
external capsule forms the claustrocortical fibers.
The immunoistochemical investigation disclosed evidence of PVand PNY-immunoreactivity in the dorsal claustrum. PV-positive
neurons were generally round, fusiform or pyramidal in shape, often multipolar, with well-filled axonal arborizations. They ranged
in diameter from 10 to 20 µm. NPY-positive neurons were generally round or fusiform in shape, ranging in diameter from 15 to
30 µm. Like previous studies in other mammals, we characterized
claustral PV- and NPY-positive neurons at the light-microscopic
level. In addition, we provided the anatomical bases for a topographical organization of the human claustrum. Further studies
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are necessary to investigate the immunospecific and topographic
subdivision within the claustrum, as well as colocalization and
coexpression patterns with various other CBPs and NPs.
Multifaceted Her-2 and topoisomerase-IIa status in
gastric carcinoma with potential clinical impact
1) Cataldo I. 2) Brunelli M. 3) Barbi S. 4) Pecori S. 5) Beghelli
S. 6) Tomezzoli A. 7) Bersani S. 8) Brunello E. 9) Martignoni G.
10) Scarpa A.
1) Department of pathology and diagnostic, Policlinico G.B. Rossi, Verona,
Italy 2) Department of pathology and diagnostic, Policlinico G.B. Rossi,
Verona, Italy 3) Department of pathology and diagnostic, Policlinico G.B.
Rossi, Verona, Italy 4) Department of pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy 5) Department of pathology and diagnostic,
Policlinico G.B. Rossi, Verona, Italy 6) Anatomic pathology, Ospedale civile maggiore, Verona, Italy 7) Department of pathology and diagnostic,
Policlinico G.B. Rossi, Verona, Italy 8) Department of pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy 9) Department of pathology
and diagnostic, Policlinico G.B. Rossi, Verona, Italy 10) Department of
pathology and diagnostic, Policlinico G.B. Rossi, Verona, Italy
Background. In gastric carcinoma, Her-2/neu gene amplification
is predictive of responsiveness to Trastuzumab whereas Topoisomerase-IIa (Topo-IIa) to anthracycline. A subset of patients
does not respond to these drugs. The heterogeneity may in part
justify the lack of clinical efficacy.
Methods. 172 gastric carcinomas (60 diffuse-type, 98 intestinaltype and 14 mixed) were recruited and 7 tissue micro-array were
built by punching three neoplastic cores per case. Hercept Test
(HER-2) was performed and cases scored by using the TOGA
system (3+, 2+, 1+, 0). Her-2/neu and Topo-IIa gene amplification was assessed by FISH analysis. In all cases, HER-2 heterogeneity was evaluated among each cores per single case; a cases
was registered as heterogeneous when at least one core did differ
from the others. We also evaluated the discrepancy between the
overall score (summing up the values from the three cores) vs the
single observed on whole tissue sections.
Results. Amplification of Her-2/neu and Topo-IIa was respectively observed in 10% and 21% of the cases. Strong 3+ Her-2
immunoexpression was found in 8% of cases and both Her-2/neu
and Topo-IIa resulted amplified in 43% into this group. Among
the 2+, 1+ and 0 groups, Her-2/neu was amplified in respectively
12%, 14% and 1% and Topo-IIa in 25%, 30% and 17%. Polysomy of chromosome 17 (with no amplification) was observed in
7% (Her-2) and 11% (Topo-IIa) of cases.
We found a prevalence of HER-2 immunoexpression and TopoIIa amplification among the intestinal subtype. Heterogeneity
among cores was found in 40% of cases for Her-2; in 46% of this
subset we observed discrepancy among the values in between
sum of the cores vs whole sections.
In conclusion, heterogeneity of HER-2 gene exists in a subset of
gastric carcinomas with potential clinical relevance; the status
of Topo-IIa does not strictly match with that of Her-2/neu. The
impact of these patterns on treatment outcome in gastric cancer
need further investigation.
Atypical leiomyoma of the scrotum is a rare benign entity arising from the muscular dartos tunica. To date fourteen cases of
atypical leiomyoma of the scrotum have been reported in literature. They have been named as atypical, bizarre or symplastic
leiomyoma alternatively, remarking the atypical leiomyomatous
characteristic of the lesion with scanty or no mitosis nor necrosis.
We describe a new case of atypical leiomyoma of the scrotum
with polypoid appearance in a 52 years old man. At microscopic
examination the lesion was constituted by fascicles of leiomyomatous spindle cells, with cellular atypia, without mitosis nor
necrosis. Immunohistochemically, the spindle cells were positive
for desmin and α-smooth muscle actin and negative for CK8-18,
CD34, S100, androgen, progesteron and estrogens receptors. The
immunohistochemical assays confirmed the leiomuscular differentiation of the lesion and rule out any suspicion of malignancy.
Clear cell adenocarcinoma of the colon:
report of a case with 2 years follow-up
1)Cusatelli P. 2)Fiscon V. 3)Pizzi S. 4)Becherini F. 5)Canova E.
1)Anatomia Patologica, ULSS 15 Alta Padovana, Camposampiero (PD),
Italia 2)Chirurgia, ULSS 15 Alta Padovana, Cittadella (PD), Italia 3)Anatomia Patologica, ULSS 15 Alta Padovana, Camposampiero (PD), Italia
4)Anatomia Patologica, ULSS 15 Alta Padovana, Campoasampiero (PD),
Italia 5)Anatomia Patologica, ULSS 15 Alta Padovana, Camposampiero
(PD), Italia
Bakground. Clear cell adenocarcinoma is a very rare entity in
the colon and its prognosis is not clear, since follow-up data are
not available. Clear cell adenocarcinoma usually occurs in the
left colon as a part of a large conventional adenoma. So far, only
one case occurring in the right colon and not associated with
adenoma has been reported. We describe a case of pure clear cell
adenocarcinoma occurring in the left colon without any evidence
of associated adenoma and followed-up for more than 2 years.
Methods. A 63 years old man presented in September 2007 with
melena. Endoscopy showed 3 polyps and an ulcerated, 3 cm diameter, lesion. Polyps were removed endoscopically and all were
conventional adenomas. A biopsy obtained from the ulcerated
lesion showed a small fragment of adenocarcinoma. Radiological
examination did not show evidence of tumour elsewhere in the
body and a left colon resection was performed.
Results. At histology, the entire lesion was composed of clear
cell adenocarcinoma infiltrating the muscularis propria (pt2).
Lymph nodes were not metastatic (15 lymph nodes assessed).
Immunohistochemistry showed the following profile: CK 7-,
CK20+, CDX2+, CD10-, p53+ (strong and diffuse positivity),
hMLH1+/hMSH2+ (consistent with microsatellite stability).
Ki67 was positive in nearly all neoplastic cells.
Despite the high proliferative activity of the tumour, the patient
did not show recurrence or distant metastasis at the last control
in March 2010.
Conclusion. This case confirm that clear cell is a variant of colic
adenocarcinoma and does not necessarily occur in association
with conventional adenoma. Its course does not seem particularly
aggressive.
Cutaneous polypoid atypical leiomyoma of the
scrotum: a case report and a review of literature
Intraparenchymal leiomyoma of the breast:
report of a case with emphasis on needle core
biopsy-based diagnosis
1) Cataldo I. 2) Brunelli M. 3) Grosso G. 4) Pedica F. 5) Magro
G. 6) Menestrina F. 7) Martignoni G.
G.M. Vecchio, 1)A. Cavaliere, 1)F. Cartaginese, 1)A. Lucaccioni, 2)T. Lombardi, 3)A. Bosco, 3)A. Sabino, 3)G. Magro
1) Department of Pathology and Diagnostic, Policlinico G.B. Rossi, Verona, Italia 2) Department of Pathology and Diagnostic, Policlinico G.B.
Rossi, Verona, Italia 3) Urologia, clinica pederzoli, peschiera del garda,
italia 4) Department of Pathology and Diagnostic, Policlinico G.B. Rossi,
Verona, Italia 5) Anatomic pathology, G.F. Ingrassia, Policlinico-Vittorio
Emanuele, Catania, Italia 6) Department of Pathology and Diagnostic,
Policlinico G.B. Rossi, Verona, Italia 7)Department of Pathology and Diagnostic, Policlinico G.B. Rossi, Verona, Italia
1)Dipartimento G.F. Ingrassia, Anatomia Patologica, Università di Catania, Azienda Ospedaliero-Universitaria Policlinico-Vittorio Emanuele,
Catania, Italia; 2)Istituto di Anatomia Patologica, Azienda Ospedaliera di
Perugia, Perugia, Italia; 3)U.O. di Senologia, Ospedale Città di Castello,
Città di Castello, Italia
Background. Leiomyomas are benign smooth muscle tumours
that can potentially occur anywhere, including breast. In this site
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
leiomyomas are usually found both in the skin and periareolar
region, whereas only rarely they involve breast parenchyma. Only
23 cases of intraparenchymal leiomyomas have been reported,
exclusively in women, to date. Histogenesis of these tumours
is still controversial and an origin from vascular smooth muscle
cells or stromal stem cells, or from embryologically displaced
smooth muscle cells, has been postulated.
Materials. We herein report the first case of an intraparenchymal leiomyoma of the breast diagnosed by needle core biopsy.
Tumour was incidentally discovered, on routine ultrasonography,
in the right breast of a 36 year-old woman. Sonographically,
tumour presented as a solid, hypoechoic, 2cm-mass with well
circumscribed margins.
Results. Needle core biopsy showed interlacing bundles of blandlooking eosinophilic spindle cells, closely reminiscent of leiomyoma. A lumpectomy was performed. Cut section showed a firm
and white nodule with smooth external surface. Histologically,
an unencapsulated tumour with the typical features of a classic
leiomyoma was observed. Mitoses, nuclear pleomorphism or
necrosis were absent. Immunohistochemical analyses, revealing a
diffuse staining for desmin, α-smooth muscle actin, h-caldesmon
and ER/PR, confirmed the diagnosis.
The present case emphasizes that the diagnosis of intraparenchymal leiomyoma may be confidentially rendered on needle core
biopsy. In this regard, it should be stressed that making a correct
diagnosis is primarily dependent on awareness that this tumour
may occur in the breast parenchyma.
Diaphragmatic myositis causing unexplained
neonatal sudden death
1)Cesari S. 2)Dal bello B. 3)Perotti G. 4)Silini EM.
1)Anatomia Patologica, Fondazione IRCCS San Matteo, Pavia, Italia
2)Anatomia Patologica, Fondazione IRCCS San Matteo, Pavia, Italia 3)Patologia neonatale, Fondazione IRCCS San Matteo, Pavia, Italia 4)Anatomia patologica, Azienda Ospedaliero-Universitaria, Parma, Italia
Background. The definition of sudden infant death syndrome
(SIDS) requires a full post-mortem investigation to exclude
identifiable causes of death according to detailed protocols. We
describe the pathologic findings of a clinically unexplained sudden death in the perinatal/neonatal period.
Methods. We performed autopsy on a 2 months old female newborn who suddenly died in her cot by an unexplained breathing
arrest. She was born at 34 weeks of pregnancy by vaginal delivery
and was apparently healthy until the acute event. Heart rhythm
was restored after 30’ of cardiopulmonary resuscitation, but brain
death by anoxia occurred after 6 days of mechanical ventilatory
support. All tissues were sampled for histology, including the
diaphragm, and samples from heart and spleen were frozen for
long QT syndrome genetic analysis.
Results. The newborn showed growth retardation (weight 3200
g, crown-rump length 35 cm; total length cm 46). Macroscopic
examination was negative except from pleural and peritoneal
effusions. Histology showed: 1) brain ischemic necrosis and
focal inflammatory meningeal infiltrates; 2) bilateral diffuse
bronchopneumonia with acute alveolar damage, abscesses and
focal organizing areas; 3) lympho-histiocytic myositis with
extensive necrosis of fibres and dystrophic calcification of skeletal muscles, in particular the diaphragm. No bacterial or viral
infections were identified. Long QT syndrome was excluded by
genetic analysis.
We concluded that the main cause of death was necrotizing
myositis specifically involving diaphragm; bronchopneumonia
was likely caused by abnormal respiratory movements and brain
necrosis was due to anoxia during prolonged cardiac arrest.
In conclusion, sudden neonatal death can be caused by diaphragmatic inflammatory pathology, as previously described in 5 newborns (Sundararajan, Med Sci Law 2005, 45 110-114). Sampling
of diaphragm should be included in the post-mortem evaluation
of these events.
Recurrent giant keloid of the sacral region treated
with post-excisional radiotherapy
1)Chiaramonte A. 2) Scaramuzzi G. 3)Tancredi A. 4)Troiano A.
5)Bisceglia M
1)Unit of Surgery, IRCCS Casa Sollievo della Sofferenza Hospital, San
Giovanni Rotondo, Italy 2)Unit of Surgery, IRCCS Casa Sollievo della
Sofferenza Hospital, San Giovanni Rotondo, Italy 3) Unit of Surgery, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy 4)Unit of Radiotherapy, IRCCS Casa Sollievo della Sofferenza Hospital,
San Giovanni Rotondo, Italy 5)Unit of Anatomic Pathology, IRCCS Casa
Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy
Background. Keloid is an abnormal pattern of dermal reaction to
injury, resulting in excess collagen deposition. Various types of
injuries are on record, such as surgery, trauma, burns, inflammatory skin diseases (folliculits, acne), viral dermatological diseases
(chicken pox), vaccinations (Calmette-Guerin/BCG, small pox,
and hepatitis B), fish stings (catfish), foreign bodies. Occasionally the injury may be clinically inapparent. The pathogenesis is unknown, but genetic, hormonal, or local factors may be involved.
Black people are more frequently affected. It is usually sporadic,
but familial occurrences have also been described 1. There’s no
sex prevalence among affected individuals. They can be either
solitary or numerous, and may vary in size from small papules to
large masses. Symptoms may vary from mild local distress (pain,
pruritus) to cosmetic discomfort or anatomic disabilities, even to
disfiguring deformities, associated with dramatic psychological
and social side-effects. Keloids may occur at any age, but they are
more common in the young. No universally accepted treatment
protocol has been standardized, but several choices are available
according to several factors (site, size, clinical history, prior treatments). Keloids are often resistant to treatment and have a high
rate of recurrence 2.
Objectives. To report on a case of a recurrent giant (monstrous)
keloid affecting a young patient, which eventually was treated
with post-surgical radiotherapy.
Case Report. A 22-year old Caucasian short man, 155 cm high
(weight 65 kilos) was hospitalized, complaining of medical,
anatomical, and psychosocial problems relating to a history of
10 year duration of a recurrent keloid. The patient’s standing
and walking were impaired and he needed assistance in coping
with stairs. At physical examination a huge, bulging, oval mass
with a knobby surface 45 cm in length (20 cm wide; 10 cm
thick) was apparent on his lower back. The mass was firm in
consistency and covered by skin which was focally eroded or
moist with evil-smelling secretions, and occluding the anus.
Physical examination also revealed a nodular exophytic mass
5 cm in size, protruding from the umbilical scar, which appeared
two years earlier. No other physical deformities were seen. His
hands and fingers as well as his feet and toes were normal. Past
medical history revealed excision of an intergluteal and perianal,
subcutaneous fibrolipoma of 2 cm in size at the age of 11, which
was histologically examined. At the age of 12 and at the age of
14, he was hospitalized in specialized centers for surgical plastic
reconstruction and underwent second and third surgical excisions
due to keloid formations of 4 cm and 10 cm in size, respectively.
A new keloid became evident shortly afterwards, which was at
times treated with steroid injections without success. The tumor
mass progressively enlarged reaching the above dimensions. The
preoperative clinical suspicion was that of a sarcomatous growth,
which was confirmed by means of PET-CT investigation, but
needle biopsy did show a reactive proliferation of fibroblasts and
myofibroblasts alternating with abundant acidophilic bands of
collagen, typical of keloid. Simultaneoulsy the patient received
genetic counseling and endocrinological evaluation, which
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showed a normal male karyotype, and excluded keloid-associated genetic syndromes, familial history of keloidal formation,
and diabetes mellitus. The patient also underwent neurological
examination since he had been diagnosed with primary epilepsy,
at the age of 5, which was confirmed, and was taking oral anticonvulsivant drugs (Depakine, Gardenal) for prophylaxis and
maintenance since. The tumor mass was surgically extirpated
en bloc (weight 3.400 gr) and sent for pathological examination.
The surgical wound was repaired with a plastic reconstruction
operation. Histological examination confirmed the diagnosis of
keloid, which was only focally present at the lateral excision
margins. 60 days after surgery radiotherapy was undertaken
using photon 8MW (total dose delivered 22Gy in 11 days, with
a daily fraction of 2 Gy). The umbilical keloid was not treated
since surgery was not necessary and due to the patient’s predisposition to keloid formation. Follow-up: no recurrence has been
noticed so far 20 months after this combined treatment (surgery
plus post-surgical radiotherapy).
Discussion. Hypertrophic scar and keloids are common occurrences, estimated to affect 5 to 15% of general population. Giant keloids are extremely rare with only 6 cases recorded in the
literature, the largest reaching the size of 20 cm in its greatest
diameter, all of which with a known history of injury, including unusual etiologies (chicken pox 3, cat-fish sting, vaccination
with BCG 1 case each), except 1 case with no attributed inciting
cause 4: the latter case was also the only arising in a familial context. Three cases showed multiple lesions of various dimensions.
Although unique as to the size and deformity caused, our case
can be categorized as one of the common sporadic cases: non-endocrine, since no endocrinological abnormalities was recognized,
non-familial, since no keloidal inheritance pattern in his pedigree
was ascertained, non-syndromic, since no stigma of keloid-associated syndromes (e.g., Rubinstein-Taybi syndrome) or of the
disfiguring (infantile) hyaline fibromatosis were seen, and nondruggable, since the absence of any plausible role in keloidal proliferations. Instead the inciting factor was well identified as the
first surgical trauma which triggered a likely individual genetic
predisposition to keloid formation. The histological differential
diagnosis include keloidal dermatofibroma, desmoplastic fibroblastoma (collagenous fibroma), hyaline fibromatosis. The case
presented herein is the largest one ever observed, and one with
most dramatic psychological impact (the patient lived almost
in isolation due to shame of the disease) 5. Keloids have been
shown to respond to radiotherapy, pressure therapy, cryotherapy,
intralesional and topical injections of corticosteroids, interferon
and bleomicin or fluorouracil, topical silicone or other dressings,
and laser treatment used alone or in various combinations, with
variable but largely transient success 6 7. Surgery has been used in
case of necessity. Primary radiation therapy has been used for unresectable keloids 8. The combination of surgery and postsurgical
radiotherapy has already been proposed for cases where surgery
is required, and has already been effectively used (follow-up > 2
years) in another case of giant keloid 4.
Conclusion. Giant keloids are extremely rare, and they may
respond to a combined approach (surgery plus postsurgical radiotherapy).
References
1
Marneros AG, Norris JE, Olsen BR, et al. Clinical genetics of familial
keloids. Arch Dermatol. 2001;137:1429-34.
2
Alster TS, Tanzi EL. Hypertrophic scars and keloids: etiology and
management. Am J Clin Dermatol 2003;4:235-43.
3
Gathse A, Ibara JR, Obengui Moyen G. Gigantic keloïds after chickenpox. A case report. Bull Soc Pathol Exot 2003;96:401-2.
4
Jones K, Fuller CD, Luh JY, et al. Case report and summary of literature: giant perineal keloids treated with post-excisional radiotherapy.
BMC Dermatol 2006;6:7.
5
Furtado F, Hochman B, Ferrara SF, et al. What factors affect
the quality of life of patients with keloids? Rev Assoc Med Bras
2009;55:700-4.
6
7
8
Juckett G, Hartman-Adams H. Management of keloids and hypertrophic scars. Am Fam Physician 2009;80:253-60.
Mutalik S. Treatment of keloids and hypertrophic scars. Indian J Dermatol Venereol Leprol 2005;71:3-8.
Malaker K, Vijayraghavan K, Hodson I, et al. Retrospective analysis
of treatment of nresectable keloids with primary radiation over 25
years. Clinical Oncology 2004;16:290-8.
Fibro-myofibroblastic proliferation in the
gallbladder wall. Report of two cases
1)S. Russo, 1)A. Cimmino, 1)A. Napoli, 1)M. Palumbo, 1)M.
Colagrande, 1)M. Silecchia, 1)M. Stolfa, 1)R. Ricco, 2)V. Ninfo
1)Dipartimento di Anatomia Patologica, Azienda Policlinico-Università
di Bari, Bari, Italia; 2)Dipartimento di Scienze Oncologiche e Chirurgiche Azienda Ospedaliera-Università di Padova, Italia
Background. Fibro-myofibroblastic proliferation is a lesion
described in various organs over the last two decades. It is also
called inflammatory pseudosarcomatous fibro-myxoid tumor and
it can mimic sarcoma. Only three cases have been previously
described in gallbladder.
Methods. We report two cases: a 76-year-old female and a
32-year-old male. Both presented with symptoms of chronic
gallstone cholecystitis with recurrent episodes of fever, vomiting,
nausea and epigastric pain.
Ultrasound examination of the abdomen showed diffuse wall
thickening, some calculi, without expansion of intra and extrahepatic bile ducts.
Then both have undergone cholecystectomy.
Results. Macroscopic examination of the gallbladder showed
increased size, and the presence of many calculi.; the gallbladder
wall was thickened.
Histological examination showed in muscle layer and in perimuscular connective tissue fibroblastic and myofibroblastic proliferation with very mild nuclear atypia, associated with diffuse
chronic inflammatory process composed of lymphocytes, plasma
cells, macrophages, and neutrophil granulocytes.
The distinction from solitary fibrous tumor, malignant fibrous
hystiocitoma, fibrosarcoma and other similar entities was based
upon the presence of mild nuclear atypia and the immunohistochemistry.
In both cases the spindle cells stain positive for HHF35 and
vimentin, rarely presented nuclear staining for Ki67 and were
negative for S100, desmin and CD68.
Our final diagnosis in both cases was fibro-myofibroblastic proliferation.
Conclusions. The heterogeneicity in clinical behavior of the
fibro-myofibroblastic proliferations previously described in various sites is probably related to different etiological factors: surgical trauma, infections and autoimmune disorders.
The fibro-myofibroblastic proliferation of gallbladder is a very
rare entity, always related with cholecystitis. Its uncertain malignant potential requires careful follow-up.
A rare case of histiocytoid cardiomiopathy
1)Cocca MP. 2)Nozza P. 3)Marzullo A. 4)Caruso G.
1)Anatomia patologica, Università di bari, Bari, Italia 2)Anatomia patologica, Istituto giannina gaslini, Genova, Italia 3)Anatomia patologica,
Policlinico, Bari, Italia 4)Anatomia patologica, Policlinico, Bari, Italia
Background. Histiocytoid cardiomiopathy (HC) is a rare (about
100 cases reported in literature), genetic cardiac disorder of
infancy or childhood, predominantly affecting girls (M:F = 3:1)
below the age of 2 years, which manifests clinically as severe
cardiac arrhythmias or dilated cardiomiopathy and edema with
heart failure and sudden death. Autosomal recessive, X–linked,
and maternal inheritance has been described. Meanwhile, several
reports indicate that HC is a cardiac manifestation of a mitochon-
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5th triennial congress of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology
drial disorder which involves the Purkinje cells of the conduction
tissue.
Methods. We have seen in consultation a right atrial subendocardial mass of a child aged nine, Arabic and presenting Blackfan–Diamond anemia, which underwent allogeneic bone marrow
transplantation. He then showed an acute GVHD, which became
chronic and was complicated by repeated infections.
Results. We observed nests of enlarged, polygonal, histiocyte–
like cells with foamy granular or vacuolar, weakly eosinophilic
cytoplasm, separated by fibrous branches, with calcium and hemosiderin deposition, but not mitoses. Immunohistochemistry
showed expression of desmin and mithocondrial protein; S100
protein and CD68–PGM1 resulted negative.
Differential diagnosis must be effected between HC and cardiac
rhabdomyoma (CR), the most common pediatric heart tumor. In
CR clinical features includes arrhytmias, outflow tract obstruction, heart failure and hydrops fetalis and is often associated with
tuberous sclerosis complex. It appears as a well demarcated mass,
usually in the ventricles that consists in nodules of enlarged cardiomyocites with cleared cytoplasm PAS + for glycogen content,
with vacuolization and myofilaments. CR has a natural history of
spontaneous regression, without surgery.
Our case deserves to be reported since our patient was male,
older than typical cases for this condition and, finally, because
the diagnosis was made on biopsy material rather than autopsy,
as is usual.
Jugular paraganglioma: report of a case
1)Cocca MP. 2)Palumbo M. 3)Resta L. 4)Cimmino A. 5)Ninfo
V.
1)Anatomia Patologica, Policlinico Di Bari, Bari, Italia 2)Dipartimento
Di Anatomia Patologica, Policlinico Di Bari, Bari, Italia 3)Dipartimento
Di Anatomia Patologica, Policlinico Di Bari, Bari, Italia 4)Dipartimento
Di Anatomia Patologica, Policlinico Di Bari, Bari, Italia 5)Dipartimento
Di Anatomia Patologica, Policlinico Di Padova, Padova, Italia
Background. Paraganglioma of the head and neck (HNP) represent rare tumors that arise from extraadrenal chromaffin cells
of neural crest origin. They represent 10-18% of all chromaffin
tissue-related tumors which are reported at a rate of 2-8 cases/
million·yr They are highly vascular neoplasms that are benign
in the majority of the case. Common sites of origin are carotid
bifurcation, jugular bulb, timpanic plexus and vagal nerve. It is a
common cancer in women between 50 and 70 years. 10% of cases
ar