ISSN 0031-2983
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
ORIGINAL ARTICLE
93 Frequency of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative,
and HER2-negative invasive breast cancer, the so-called triple-negative phenotype:
a population-based study from Trentino, North East Italy
S. Giuliani, E. Leonardi, D. Aldovini, D. Bernardi, M. Pellegrini, F. Soli, A. Ferro,
P. Dalla Palma, N. Decarli, M. Barbareschi
CASE REPORTS
98 Oncocytic papillary renal cell carcinoma: potential pitfall in small enucleation
E. Munari, A. Eccher, D. Segala, A. Iannucci, S. Gobbo, M. Chilosi, M. Brunelli,
G. Martignoni
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
101 Clear cell papillary renal cell carcinoma with characteristic morphology
and immunohistochemical staining pattern
S.M. Gilani, R. Tashjian, H. Qu
105 Idiopathic granulomatous mastitis mimicking breast cancer: report of two cases
F. Limaiem, S. Korbi, T. Tlili, I. Haddad, A. Lahmar, S. Bouraoui, F. Gara, S. Mzabi
ATTI DI CONGRESSO
109 I Congresso Nazionale di Citopatologia SIAPEC-IAP
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Società Italiana di Anatomia Patologica e Citopatologia Diagnostica,
Divisione Italiana della International Academy of Pathology
Vol. 104 June 2012
Ogni anno il carcinoma polmonare colpisce 1.3 milioni di persone
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Cited in Index Medicus/MEDLINE, BIOSIS Previews, SCOPUS
Journal of the italian Society of anatomic Pathology
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italian Division of the international academy of Pathology
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CONTENTS
Original article
Frequency of estrogen receptor (ER)-negative, progesterone
receptor (PR)-negative, and HER2-negative invasive breast
cancer, the so-called triple-negative phenotype:
a population-based study from Trentino, North East Italy
S. Giuliani, E. Leonardi, D. Aldovini, D. Bernardi,
M. Pellegrini, F. Soli, A. Ferro, P. Dalla Palma, N. Decarli,
M. Barbareschi
Objective. Triple negative breast carcinomas (TNT) are infiltrating
breast carcinomas (BC) with negative oestrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER-2) expression, and are associated with frequent BRCA1/
BRCA2 mutations. The aim of the present study is to analyze the
frequency and distribution of TNT in our population where a breast
cancer screening program for women aged between 50 and 69 years
is effective since 2001 with 85% accrual.
Methods. We investigated the records of 2112 consecutive BC and
153 interval BC (i.e. BC detected in the screened negative women in
the interval between screening rounds). Tumours with complete negative expression of ER, PgR and Her2 were considered TNT; tumours
with negative ER and PgR status and faint Her2 expression (score 1)
were considered as possible TNT (pTNT).
Results. We identified 82 (3.8%) TNT and 20 (0.9%) pTNT in the
series of 2112 consecutive BC and 7 TNT and 1 pTNT (5.2%) in the
series of 153 interval BC. In the consecutive series, TNT/pTNT were
observed in 6.5% patients below 50 years and in 4.3% of patients
above 50 years. A high proliferation rate (Ki-67 labelling > 36%) was
observed in 87.8% of TNT (median labelling 56.3%) and in 60% of
pTNT (median labelling 48.4%).
Conclusions. Since TNT/pTNT occurring in women < 50 years is a
criterion for selecting patients whom genetic counselling and BRCA1
testing should be offered, our study is of help in foreseeing the workload of the Unit of Medical Genetics and the Laboratory of Molecular
Pathology.
Case reports
Oncocytic papillary renal cell carcinoma: potential pitfall
in small enucleation
E. Munari, A. Eccher, D. Segala, A. Iannucci, S. Gobbo,
M. Chilosi, M. Brunelli, G. Martignoni
Objective. We describe an emerging entity, recently recognized
as a pitfall in the diagnostic practice among eosinophilic renal
cell tumours.
Methods. A 60-year-old male underwent enucleation of a 1.2
cm nodule. Immunohistochemistry and FISH analysis were performed.
Results. Histology revealed a neoplasm composed of large cells
with eosinophilic cytoplasm, Fuhrman grade 3, arranged in papillae. At the immunohistochemical level, cells showed positivity for
AMACR and CD10. Fluorescence in situ hybridization (FISH)
demonstrated gains of chromosomes 7 and 17 and loss of Y. A
diagnosis of oncocytic papillary renal cell carcinoma was made.
Conclusions. The distinction between renal oncocytoma and
oncocytic papillary renal cell carcinoma is of substantial importance because of their different behaviour and prognosis, since
the latter has malignant potential. Although the available evidence
supporting tumour enucleation as the surgical treatment for renal
cortical tumours ≤ 4 cm, due to aforementioned clinicopathological features such tumours need to be evaluated using appropriate
immunophenotypical and cytogenetic analyses.
Clear cell papillary renal cell carcinoma with characteristic
morphology and immunohistochemical staining pattern
S.M. Gilani, R. Tashjian, H. Qu
Clear cell papillary renal cell carcinoma is newly-defined entity
initially believed to be associated with end stage renal disease.
We report a rare case of this neoplasm in a 70-year-old female
patient with no known history of end-stage renal disease who
presented with haematuria lasting several days. After initial
workup, a computed tomography (CT) scan was performed and
revealed a cystic mass in the right kidney. Material obtained by
fine needle aspiration (FNA) biopsy of the mass was felt to be
suspicious for renal cell carcinoma. The patient subsequently
underwent right nephrectomy, and the lesional tissue was examined microscopically. A 2.3 cm in greatest dimension cystic
space circumscribed by a fibrous wall was surfaced by a single
layer of bland cuboidal cells with abundant clear cytoplasm. The
solid component of the tumour consisted of branching papillae
with delicate fibrovascular cores and uniformly lined by cells
similar to those of the inner wall of the cyst. Some of the fibrovascular cores were markedly expanded by a myxoid-appearing
substance, but no foamy cells were appreciated. Immunohistochemically, the neoplastic cells were diffusely immunoreactive
with cytokeratin 7 (CK7), epithelial membrane antigen (EMA),
high molecular weight cytokeratin (HMWCK) and vimentin.
Neoplastic cells were only focally immunoreactive with CD10,
and were negative for both p63 and α-methylacyl-CoA racemase
(AMACR) (P504S). The cytomorphological features and immunohistochemical staining pattern of this tumour was consistent
with that of clear cell papillary renal cell carcinoma (CCPRCC),
as described by Gobbo et al.
Idiopathic granulomatous mastitis mimicking breast cancer:
report of two cases
F. Limaiem, S. Korbi, T. Tlili, I. Haddad, A. Lahmar,
S. Bouraoui, F. Gara, S. Mzabi
Idiopathic granulomatous mastitis is a rare inflammatory breast
disease of unknown aetiology that is frequently mistaken for
breast carcinoma both clinically and mammographically. In this
paper, the authors report two cases of idiopathic granulomatous
mastitis that occurred in two parous women aged 38 and 45 years.
Clinically, both patients presented with a tender palpable lump
in the left breast. Mammography showed an poorly-defined mass
in both patients with microcalcification in the first case and skin
retraction in the second case. Breast lumpectomy was performed
in both patients. Histological examination of the surgical specimen revealed non-caseating granulomas confined to breast lobules. Special staining for fungi and tuberculosis were all negative.
Correct diagnosis of idiopathic granulomatous mastitis requires
the exclusion of malignancy, other granulomatous disease and
infectious aetiologies. Histopathologic examination remains the
gold standard for diagnosis. This disease is rare, and therefore the
optimum treatment protocol is still being established.
pathologica 2012;104:93-97
Original article
Frequency of estrogen receptor (ER)-negative,
progesterone receptor (PR)-negative, and HER2-negative
invasive breast cancer, the so-called triple-negative
phenotype: a population-based study from Trentino,
North East Italy
S. Giuliani1 2, E. Leonardi1, D. Aldovini1, D. Bernardi3, M. Pellegrini3, F. Soli4, A. Ferro5, P. Dalla Palma1,
N. Decarli1, M. Barbareschi1 2
1
Unit of Surgical Pathology, S. Chiara Hospital, Trento, Italy; 2 Trentino Biobank, Unit of Surgical Pathology, S. Chiara Hospital,
Trento, Italy; 3 Unit of Senology, Azienda provinciale Servizi Sanitari, Trento, Italy; 4 Unit of Medical Genetics, S. Chiara Hospital,
Trento, Italy; 5 Unit of Medical Oncology, S. Chiara Hospital, Trento, Italy
Key words
Breast cancer • Triple Negative • BRCA1 • BRCA2
Summary
Objective. Triple negative breast carcinomas (TNT) are infiltrating breast carcinomas (BC) with negative oestrogen receptor
(ER), progesterone receptor (PgR) and human epidermal growth
factor receptor 2 (HER-2) expression, and are associated with
frequent BRCA1/BRCA2 mutations. The aim of the present
study is to analyze the frequency and distribution of TNT in our
population where a breast cancer screening program for women
aged between 50 and 69 years is effective since 2001 with 85%
accrual.
Methods. We investigated the records of 2112 consecutive BC
and 153 interval BC (i.e. BC detected in the screened negative
women in the interval between screening rounds). Tumours
with complete negative expression of ER, PgR and Her2 were
considered TNT; tumours with negative ER and PgR status and
faint Her2 expression (score 1) were considered as possible TNT
(pTNT).
Results. We identified 82 (3.8%) TNT and 20 (0.9%) pTNT in
the series of 2112 consecutive BC and 7 TNT and 1 pTNT (5.2%)
in the series of 153 interval BC. In the consecutive series, TNT/
pTNT were observed in 6.5% patients below 50 years and in 4.3%
of patients above 50 years. A high proliferation rate (Ki-67 labelling > 36%) was observed in 87.8% of TNT (median labelling
56.3%) and in 60% of pTNT (median labelling 48.4%).
Conclusions. Since TNT/pTNT occurring in women < 50 years
is a criterion for selecting patients whom genetic counselling and
BRCA1 testing should be offered, our study is of help in foreseeing the workload of the Unit of Medical Genetics and the Laboratory of Molecular Pathology.
Introduction
tern of early relapse within the first two years following
diagnosis, with a peak within three years, followed by a
rapid decline over the next five, and a very low risk of
subsequent recurrence. Women with TNT do not benefit
from hormonal or trastuzumab therapy, and are left with
chemotherapy as their only option 4 5 6.
TNT arise more frequently in patients carrying germline BRCA1 and, to a lesser extent, BRCA2 gene mutations, and several studies have demonstrated that
BRCA1-mutation carriers more likely are affected
by TNT than non-carriers 7 8 9. Due to this strict relationship, TNT can be considered as an index of sus-
Breast carcinomas (BC) with negative oestrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER-2/neu) expression are termed triple negative tumours (TNT), and partially correspond to basal-like carcinomas as detected by
gene expression studies 1 2. TNT more frequently affect
younger patients (< 50 years), and are associated with a
high proliferation rate. TNT usually show high expression of p53, EGFR and high molecular weight cytokeratins 3. TNT are aggressive tumours with a unique pat-
Correspondence
This work was supported by the Provincia Autonoma di Trento
and the Fondazione Cassa di Risparmio di Trento e Rovereto.
Mattia Barbareschi, Unit of Surgical Pathology, Laboratory of
Molecular Pathology, S. Chiara Hospital, largo Medaglie Oro
9, 38122 Trento, Italy - Tel. +39 0461 903092 - Fax +39 0461
903389 - E-mail: [email protected]
94
S. Giuliani et al.
Fig. 1. Immunohistochemistry in TNT showing complete negativity for ER (a), PR (b), and HER2 (c). 20x.
Fig. 2. Immunohistochemistry in pTNT showing complete negativity for ER (a) and PR (b), and faint positivity for HER2 (score 1) (c). 20x.
picious BRCA1 mutational status 7 10. TNT occurring
in patients younger than 50 years or with familiarity
for breast cancer is one of the criteria to select patients
whom genetic counselling and BRCA1 testing should
be offered 7 10. Given the relevant workload of genetic
counselling and testing, it is important to know the
incidence of TNT for appropriate organizational and
Tab. I. Hormone receptor and Her2 status in 2112 infiltrating breast
carcinomas.
No. of patients
%
ER
Negative
Positive
296
1816
14
86
PR
Negative
Positive
517
1595
24.5
75.5
HER2
Her0
Her1
Her2
Her3
888
443
621
160
42
21
29.5
7.5
Abbreviations: ER, oestrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor 2;
economical planning. The aim of the present study is
to describe the frequency of TNT in a large series of
consecutive unselected breast carcinomas in the general population in Trentino, Italy.
Patients and methods
The study population includes 2112 women affected by
infiltrating BC observed at the Santa Chiara Hospital,
Trento, Italy between 2005 and 2010. In our region, Trentino, North Italy, a breast cancer screening program for
women aged between 50 and 69 years has been effective
since 2001 with 85% accrual. Coded data on these markers were available in the database of the Unit of Surgical
Pathology, and served as a basis for this study. We also
investigated 153 interval BC detected between 2001 and
2009, which were defined as BC detected in screened
negative women during the interval between screening
rounds. All invasive BC specimens had been routinely
evaluated for ER, PR, Ki-67 and HER-2/neu status using
immunohistochemistry (IHC) at the time of diagnosis.
Her2 immunoreactivity was evaluated using the HercepTest kit (DakoCytomation, Glostrup, Denmark) and
scored according to the manufacturer’s FDA-approved
95
Frequency of triple negative breast cancer
Tab. II. Age at diagnosis in all breast cancers (n=2112), in TNT (82) and pTNT (20).
Age at diagnosis
< 30
No. of patients
TNT (%*)
% of TNT within
the age group
Possible TNT (%**)
% of Possible TNT
within the age
group
1
1 (1.2%)
100
0
-
30-39
84
8 (9.8%)
9.5
1 (5%)
1.2
40-49
362
18 (22%)
4.5
1 (5%)
0.3
50-59
476
16 (19.5%)
3.3
5 (25%)
1
60-69
519
17 (20.7%)
3.2
8 (40%)
1.5
70-79
390
16 (19.5%)
4.1
2 (10%)
0.5
≥ 80
280
6 (7.3%)
2.1
3 (15%)
1
* = percentage among all TNT
** = percentage among all possible TNT
system. ER, PgR and Ki-67 were evaluated using 6F11
(Leica-Novocastra, Newcastle, UK.), Pgr636 (Dako)
and MM1 (Leica-Novocastra) antibodies, respectively.
ER and PgR were considered negative when no nuclear
staining was seen in spite of positive appropriate internal
and external controls 11. The Ki-67 labelling index was
evaluated manually and with computer assisted image
analysis in the most densely labelled areas, as previously described 12. Tumours with negative expression of all
three markers were considered TNT (Fig. 1). Tumours
with negative ER and PgR status but with faint Her2
expression (score 1) were considered as possible TNT
(pTNT) (Fig. 2).
Results
Immunohistochemical data concerning ER, PgR and Her2
status of the entire series of 2112 cases of BC are shown
in Tab. I. Among these cases, there were 82 (3.8%) TNT
and 20 (0.9%) pTNT. Twenty-seven of 82 TNT (33%)
were detected in women < 50 years at the time of diagnosis, corresponding to 6% of the 447 patients < 50 years old
(detailed data on age distribution are shown in Tab. II and
Fig. 3. Age at diagnosis in TNT and pTNT in our series of 2112
infiltrating breast carcinomas.
Fig. 3). Two of 20 (10%) pTNT were detected in women
< 50 years old at the time of diagnosis, corresponding to
0.4% of the 447 patients < 50 years old (detailed data on
age distribution are shown in Tab. II and Fig. 3). One patient affected by TNT was also affected by ovarian cancer.
The age distribution of TNT and pTNT was slightly different, with TNT occurring at a younger age than pTNT:
globally, their frequency was 6.5% for patients below 50
years and 4.3% for older patients (Fig. 4).
The histopathological characteristics of the TNT and
pTNT are shown in Tab. III. The most frequent histotype
in both TNT and pTNT was infiltrating ductal carcinoma (86.6 % and 95%, respectively). A high proliferation
rate (Ki-67 labelling >30% according to Goldhirsch et
al 2009) 13 was observed in 87.8% of TNT with a median value of 56.3%, and in 60% of pTNT with a median
value of 48.4%.
Fig. 4. Distribution of TNT/pTNT among BC in patients under 50
and over 50 years in our series of 2112 infiltrating breast carcinomas.
96
S. Giuliani et al.
Tab. iii. Characteristics of TNT and pTNT in the consecutive series of
2112 patients.
Variable
TNT (N=82)
n%
Possible TNT
(N=20), n %
Histotype
Ductal
Lobular
Medullary
Apocrine
Squamous
Metaplastic
71(86.6)
1 (1.2)
6 (7.3)
0
1 (1.2)
3 (3.7)
19 (95)
1 (5)
Tumour size
1a (< 5mm)
1b (6-10 mm)
1c (11 – 20 mm)
2 (> 20 mm < 50 mm)
> 50 mm
Unknown
2 (2.4)
14 (17)
34 (41.5)
28 (34.2)
4 (4.9)
-
1 (5)
3 (15)
3(15)
8(40)
2 (10)
3 (15)
Grade
1
2
3
Unknown
10 (12.2)
66 (80.5)
6 (7.3)
3 (15)
15 (75)
2 (10)
MIB1/Ki67
Median value
Low proliferation (≤ 20%)
Medium proliferation (21-35%)
High proliferation (≥ 36%)
Unknown
56.3%
3 (3.7)
3 (3.7)
72 (87.8)
4 (4.8)
48.4%
0
7 (35)
12 (60)
1 (5)
Tab. IV. Frequencies of TNT in population-based studies.
Authors
Nation
Rakha EA et al.
UK
(2007)
Dent R et al.
Canada
(2007)
Tischkowitz et al.
Canada
(2007)
Among 153 interval BC, we identified 7 TNT and 1 pTNT, representing 5.2% of all interval BC. Five of these
were detected in women between 50-59 years of age and
two in women between 60-69 years old.
Discussion
The present study shows that in our region, in a population of Caucasian ethnicity and with an effective BC
screening program for women between 50 and 69 years,
TNT/pTNT represent a small subset (around 4.8%) of all
BC. We show that TNT/pTNT occured in 6.5% of our
patients below 50 years, in 4.3% of patients above 50
years, and in 5.2% of interval BC.
At variance with our study, the average reported frequency of TNT in Caucasian populations is 10-17%
(Tab. IV) 7 14-19. This discrepancy could be related to
several facts, including our strict criteria to define ER,
PR and Her2 negativity, the impact of our screening
program, and the genetic background of our population.
The presence of a screening program, which is known to
increase the detection of low grade, ER and PgR positive
tumours, may indeed reduce the relative frequency of
TNT. However, this does not seem to be the case as in
our series the frequency of TNT/pTNT in young women
not included in the screening program (i.e. younger than
50 years) is well below in the range of literature data for
age-matched groups of patients 14-19. The genetic background of our population may also be relevant20 21 since
our population has been relatively stable in the past, and
further studies could address this topic.
% TNT
Sample size
TNT definition criteria
16.3
1726
ER-PR-HER2-
11.2
1601
ER-PR-HER2-
14
264
ER-PR-HER2-
51.074
ER < 5%, PR <5 %,
HER2 score 0 or 1
Overall 12.5
Bauer KR et al.
(2007)
CA, USA
Lund MJ et al.
(2009 )
GA, USA
Morris GJ et al.
( 2007)
PA, USA
Adamo et al.
(2011 )
Present study
10.8 in non-Hispanic white
24.6 in African-American
17.2 in Hispanic
11.7 Asian/Pacific Islander
Overall 29.5
46.6 in African-American
21.8 in Caucasian
20.8 in African-American
116 African American
ER-PR-HER2360 Caucasian
2230
ER-PR-HER2-
10.4 in Caucasian
Italy
9.8
1894
Italy
4.7
2112
ER-PgR 0-9%
HER2 score ≤ 2 with no gene
amplification
ER- PR- HER score 0
ER- PR- HER2 score 1
97
Frequency of triple negative breast cancer
Interval BC are usually considered rapidly growing tumours with reduced ER expression 22 23, and it could be
hypothesized that they should be more frequently of the
basal cell type. However, the frequency of TNT in our
series of interval BC is low, and does not differ significantly from the one observed in the entire group of our
consecutive patients.
Beside concerns about the reasons for the lower frequency of TNT observed in this series, our data are important to anticipate the workload of the Unit of Medical
Genetics and the Laboratory of Molecular Pathology.
In fact, TNT occurring in young women (< 50 years) or
in women with familiar history of breast cancer represent a criterion for selecting patients to whom genetic
counselling and BRCA1 testing should be offered 7 9 24.
Both genetic counselling and laboratory testing are
time consuming, expensive and not always clear (e.g.
because of variants, polymorphisms, etc.), and it is
important to accurately plan their activity. This is
even more important when psychological and emotional aspects of the genetic investigations are taken
in account: once a patient has been told that she could
be a BRCA1 mutation carrier, it is mandatory to provide the results of the genetic test within a reasonable
time to avoid unnecessary anxiety for herself and her
family.
The role of TNT as an index of suspicious BRCA1 mutational status highlights the importance of proper communication between the Units of Pathology and Medical Genetics. Because of progressive reduction of the
members of modern families, which among Western
countries is especially evident in Italy, it will become
progressively more difficult to identify probands to submit for genetic testing, based on criteria of familiarity.
Therefore, the relevance of identifying TNT, not only
for therapeutic purposes but also for genetic purposes,
highlights the important role of pathologists in early detection and prevention of familial BC.
References
1
Perou CM, Sørlie T, Eisen MB, et al. Molecular portraits of human
breast tumours. Nature 2000;406:747-52.
3
Irvin WJ Jr, Carey LA. What is triple-negative breast cancer? Eur
J Cancer 2008;44:2799-805.
4
Reis-Filho JS, Tutt AN. Triple negative tumours: a critical review.
Histopathology 2008;52:108-18.
5
Dent R, Trudeau M, Pritchard KI, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res
2007;13: 4429-34.
6
7
8
9
sus on the primary therapy of early breast cancer. Ann Oncol
2009;20:1319-29.
Rakha EA, El-Sayed ME, Green AR, et al. Prognostic markers in
triple-negative breast cancer. Cancer 2007;109:25-32.
15
Tischkowitz M, Brunet JS, Bégin LR, et al. Use of immunohistochemical markers can refine prognosis in triple negative breast
cancer. BMC Cancer 2007;24:7-134.
16
Bauer KR, Brown M, Cress RD, et al. Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative,
and HER2-negative invasive breast cancer, the so-called triplenegative phenotype: a population-based study from the California
cancer Registry. Cancer 2007;109:1721-8.
17
Minami CA, Chung DU, Chang HR. Management options in
triple-negative breast cancer. Breast Cancer: Basic and Clinical
Research 2011;5:175-99.
18
Nanda R. “Targeting” triple-negative breast cancer: the lessons
learned from BRCA1-associated breast cancers. Semin Oncol
2011;38:254-62.
19
Gadzicki D, Schubert A, Fischer C, et al. Histophatological criteria and selection algorithms for BRCA1 genetic testing. Cancer
Genetics and Cytogenetics 2009;189:105-11.
Atchley DP, Albarracin CT, Lopez A, et al. Clinical and pathologic characteristics of patients with BRCA-positive and BRCAnegative breast cancer. J Clin Oncol 2008;26:4282-8.
10
11
12
13
14
Gonzalez-Angulo AM, Timms KM, Liu S, et al. Incidence and
outcome of BRCA mutations in unselected patients with triple receptor-negative breast cancer. Clin Cancer Res 2011;17:1082-9.
Kwon JS, Gutierrez-Barrera AM, Young D, et al. Expanding the
criteria for brca mutation testing in breast cancer survivors. J Clin
Oncol 2010;28:4214-20.
Mauri FA, Veronese S, Frigo B, et al. Er1d5 and h222 (er-ica)
antibodies to human estrogen receptor protein in breas carcinomas: a result of a multicentric comparative study. Appl Immunohistochem 1994;2:157-63.
Fasanella S, Leonardi E, Cantaloni C, et al. Proliferative activity in
human breast cancer: Ki-67 automated evaluation and the influence of different Ki-67 equivalent antibodies. Diagn Pathol 2011;6
Suppl 1:S7.
Goldhirsch A, Ingle JN, Gelber RD, et al. Thresholds for therapies: highlights of the St Gallen International Expert Consen-
Lund MJ, Trivers KF, Porter PL, et al. Race and triple negative
threats to breast cancer survival: a population-based study in Atlanta, GA. Breast Cancer Res Treat 2009;113:357-70.
Morris GJ, Naidu S, Topham AK, et al. Differences in breast carcinoma characteristics in newly diagnosed African-American and
Caucasian patients: a single-institution compilation compared
with the National Cancer Institute’s Surveillance, Epidemiology,
and End Results database. Cancer 2007;110:876-84.
Adamo V, Ricciardi GRR, De Placido S, et al. Management and
treatment of triple-negative breast cancer patients from the NEMESI study: an Italian experience. European Journal of Cancer
2011. Epub ahead of print.
20
Saunders KH, Nazareth S, Pressman PI. Case report: BRCA in the
Ashkenazi population: are current testing guidelines too exclusive? Hered Cancer Clin Pract 2011;9:3.
21
Halbert CH, Kessler L, Troxel AB, et al. Effect of genetic counseling and testing for BRCA1 and BRCA2 mutations in African
American women: a randomized trial. Public Health Genomics
2010;13:440-8.
22
Van der Vegt B,Wesseling J, Pijnappel RM, et al. Aggressiveness
of ‘true’ interval invasive ductal carcinomas of the breast in postmenopausal women. Mod Pathol 2010;23:629-36.
23
Raja Ma, Hubbard A, Salman AR. Interval breast cancer: is it a
different type of breast cancer? Breast 2001;10:100-8.
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Foulkes WD, Stefansson IM, Chappuis PO, et al. Germline BRCA1
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Cancer Inst 2003;95:1482-5.
25
pathologica 2012;104:98-100
Case report
Oncocytic papillary renal cell carcinoma:
potential pitfall in small enucleation
E. Munari, A. Eccher, D. Segala, A. Iannucci*, S. Gobbo, M. Chilosi, M. Brunelli, G. Martignoni
Department of Pathology and Diagnostic, University of Verona, Italy; * Anatomic Pathology, Ospedale Maggiore,
Azienda Ospedaliera Universitaria Integrata, Verona, Italy
Key words
Oncocytic papillary RCC • Renal oncocytoma • FISH
Summary
Objective. We describe an emerging entity, recently recognized
as a pitfall in the diagnostic practice among eosinophilic renal
cell tumours.
Methods. A 60-year-old male underwent enucleation of a 1.2 cm
nodule. Immunohistochemistry and FISH analysis were performed.
Results. Histology revealed a neoplasm composed of large cells
with eosinophilic cytoplasm, Fuhrman grade 3, arranged in papillae. At the immunohistochemical level, cells showed positivity for
AMACR and CD10. Fluorescence in situ hybridization (FISH)
demonstrated gains of chromosomes 7 and 17 and loss of Y. A
diagnosis of oncocytic papillary renal cell carcinoma was made.
Conclusions. The distinction between renal oncocytoma and
oncocytic papillary renal cell carcinoma is of substantial importance because of their different behaviour and prognosis, since
the latter has malignant potential. Although the available evidence
supporting tumour enucleation as the surgical treatment for renal
cortical tumours ≤ 4 cm, due to aforementioned clinicopathological features such tumours need to be evaluated using appropriate
immunophenotypical and cytogenetic analyses.
Introduction
oncocytomas represent 3-7% of all renal tumours and
are characterized by compact nests, acini, tubules or
microcysts composed of round to polygonal cells, with
a densely granular eosinophilic cytoplasm and regular
nuclei with centrally placed nucleoli 6.
The distinction between the aforementioned tumours
is of significant importance since renal oncocytomas
are benign neoplasms with an indolent course, whereas
papillary RCCs are malignant tumours characterized by
potential malignant behaviour 1. The morphologic features are usually sufficient to distinguish renal oncocytomas from papillary RCCs among most routine cases.
In certain scenarios, such as small enucleation or renal
biopsies from small renal masses, there are challenging
neoplasms combining both an extensive papillary pattern together with true oncocytic cells 7.
Herein we describe a case of a 60-year-old male who
underwent enucleation after the incidental finding of a
renal nodule 1.2 cm in maximum diameter localized to
the left kidney and finally diagnosed as oncocytic papillary RCC.
Papillary renal cell carcinoma (RCC) is a well-established subtype of RCC with characteristic macroscopic
and histological features, comprising approximately 1015% of RCCs 1.
This tumour can be subdivided into two morphologic
types with different prognostic implications: type 1,
with small cells arranged in a single layer on delicate
papillary cores, and type 2 with large eosinophilic cytoplasm and pseudostratified nuclei with higher Fuhrman
grade arranged on broader papillae 2. Cytogenetically,
papillary RCC is mostly characterized by trisomies
of chromosomes 7 and 17 and deletion of chromosome Y as well as additional gains of chromosomes
12, 16, and 20 3. The existence of additional variants
of papillary RCC may be inferred by the recognition
of a few cases with different morphological features,
such as those composed entirely of oncocytes 4; typical signs of oncocytic papillary RCC in routine clinical
practice has also been highlighted 5. In contrast, renal
Correspondence
Enrico Munari, Department of Pathology and Diagnostic,
University of Verona, piazzale L.A. Scuro, 37100 Verona, Italy Tel. +39 045 8124843 - Fax +39 045 8027136 - E-mail: enrico_
[email protected]
99
Pitfall in oncocytic papillary RCC
Case report
A 60-year-old man was incidentally found to have a
nodule 1.2 cm in diameter on the left kidney and underwent enucleation. On intraoperative consultation,
the lesion appeared macroscopically solid and browncoloured, with a 4 mm adjacent wide rim of normal renal
parenchyma. Histological examination of the tumour tissue with frozen-sections revealed that the neoplasm was
mainly composed of large cells with dense eosinophilic
cytoplasm, Fuhrman grade 3 nuclei, and arranged mainly
in papillary structures with delicate fibrovascular stalks.
Areas of oncocytic cells arranged in a solid pattern were
also present. Foamy macrophages were also observed
within the fibrovascular cores of the papillae. Necrosis
was not present. A diagnosis of oncocytic renal cell neoplasm, not otherwise specified, was made (Fig. 1a).
On definitive examination, the tumour was completely
confined by a thick pseudo-capsule without infiltrating
the renal parenchyma. Morphology revealed a neoplasm
composed of both papillary and solid-papillary arranged
neoplastic cells, with oncocytic features (Fig. 1b). The differential diagnosis was for papillary RCC or renal oncocytoma. At the immunohistochemical level, neoplastic cells
showed strong and diffuse positivity for α-methylacylCoA racemase (AMACR) (Fig. 2a), together with positivity for CD10. Tumour cells showed dim immunostaining
for CK7, and no labelling for parvalbumin.
In situ hybridization (FISH) demonstrated three or more
signals for both chromosomes 7 and 17 in 21% of the
nuclei, while no signal for chromosome Y was detected
in 41% of nuclei (Fig. 2b).
Discussion
Papillary RCC was first recognized as an independent
entity based on its particular morphology characterized
Fig. 1. Small solid neoplasm (a) composed of large cells with dense eosinophilic cytoplasm and Fuhrman grade 3 nuclei, arranged mainly in
papillary structures with delicate fibrovascular stalks (b).
Fig. 2. Strong and diffuse AMACR immunoexpression in neoplastic cells (a); FISH showing three or more signals for both chromosomes 7
and 17 in neoplastic nuclei (b).
100
E. Munari et al.
Tab. I. Case series of oncocytic papillary RCCs treated with nephrectomy or partial nephrectomy/enucleation reported in the literature.
Author
Lefévre et al.
Hes et al. 4
kunju et al.
Masuzawa et al.
Park et al.
Okada et al.
Ürge et al. 5
Total
No. of cases
10
12
7
1
7
1
12
50
Median age
(years)
71
67
57
75
67
81
68
68
Median tumour size
(mm)
28
50
20
150
12
54
35
35
by a papillary architecture 2. It was then divided into two
types based on morphological characteristics and clinical behaviour: type 1 with small cells and low nuclear
grade (grade 1-2), and type 2 composed of large cells
with a higher nuclear grade (grade 3-4) 8. The clinical
utility of dividing papillary RCCs into 2 types according to histological characteristics was demonstrated to
have prognostic significance 8. Here we described a case
of oncocytic papillary RCC, a neoplasm characterized
by oncocytic cells arranged in a papillary pattern with a
specific immunophenotypic and cytogenetic profile.
Oncocytic papillary RCC have been discussed in terms
of morphological characteristics and immunophenotype in different studies with particular attention to the
features that distinguish it from renal oncocytoma. The
distinction between renal oncocytoma and oncocytic
papillary RCC is of primary importance because of their
different clinical behaviour and prognosis, as the latter
has malignant potential 4.
The case described here showed morphology characterized by a population of large cells with eosinophilic
cytoplasm and Fuhrman grade 3 nuclei arranged mainly
in papillary structures with delicate fibrovascular stalks.
Areas of oncocytic cells arranged in a solid pattern were
also present, and a diagnosis of oncocytic renal cell neoplasm was made during intraoperative consultation. In
differential diagnosis, all renal neoplasms with granular
cytoplasm must be considered, i.e., renal oncocytoma,
chromophobe RCC, clear cell RCC with extensive granular areas, oncocytoid RCC after neuroblastoma and oncocytoma-like angiomyolipoma. The tumour in the pres-
2
3
4
5
Eble JN, Sauter G, Epstein JI, et al. WHO: Tumours of the Urinary
System and Male Genital Organs. Lyon: IARC Press 2004.
Delahunt B, Eble JN. Papillary renal cell carcinoma: a clinicopathologic and immunohistochemical study of 105 tumors. Mod
Pathol 1997;10:537-44.
Brunelli M, Eble JN, Zhang S, et al. Gains of chromosomes 7, 17,
12, 16, and 20 and loss of Y occur early in the evolution of papillary renal cell neoplasia: a fluorescent in situ hybridization study.
Mod Pathol 2003;16:1053-9.
Hes O, Brunelli M, Michal M, et al. Oncocytic papillary renal cell
carcinoma: a clinicopathologic, immunohistochemical, ultrastructural, and interphase cytogenetic study of 12 cases. Ann Diagn
Pathol 2006;10:133-9.
Urge T, Hes O, Ferda J, et al. Typical signs of oncocytic papil-
10
12
2
1
2
1
7
35
Partial Nephrectomy/
Enucleation (%)
0 (0)
0 (0)
5 (71)
0 (0)
5 (71)
0 (0)
5 (42)
15 (30)
ent case differs from chromophobe RCC and clear cell
RCC for the presence of papillary areas. The most difficult differential diagnosis is renal oncocytoma because
of the oncocytic features of cells. Moreover, oncocytoma
can show papillary foci; similarly, papillary neoplasms
may show extensive areas of solid components. Thus,
differential diagnosis based solely on morphology can be
extremely difficult. In these cases, the use of an immunohistochemical panel including racemase, CD10 and
parvalbumin can be very useful 9. However, the specificity of some of these antibodies is limited. In particular,
racemase has been described in 15% of renal oncocytoma and parvalbumin, a marker present in 70% of renal
oncocytomas, has been reported to be positive in some
cases 9. On definitive diagnosis, immunohistochemistry
demonstrated that cells stained for AMACR and CD10,
while immunostaining for parvalbumin was negative.
The numerical abnormalities on chromosomes 7 and 17
and loss of chromosome Y are specific for papillary RCC,
but not for oncocytoma; we found the presence of three or
more signals for chromosomes 7, 17 and the loss of chromosome Y. Considering the above-mentioned features, a
diagnosis of oncocytic papillary RCC was made.
Although the available evidence supports tumour enucleation or partial nephrectomy as the standard surgical
treatment for renal cortical tumours ≤ 4 cm 10, including
oncocytic papillary RCC (Tab. I), it should be kept in
mind that these latter tumours rarely develop metastases 4. Due to aforementioned clinicopathological features, these tumours must be diagnosed with appropriate
immunophenotypical and cytogenentic analyses.
References
1
Nephrectomy
6
7
8
9
10
lary renal cell carcinoma in everyday clinical praxis. World J Urol
2010;28:513-7.
Trpkov K, Yilmaz A, Uzer D, et al. Renal oncocytoma revisited: a
clinicopathological study of 109 cases with emphasis on problematic diagnostic features. Histopathology 2010;57:893-906.
Russo P, Goetzl M, Simmons R, et al. Partial nephrectomy: the rationale for expanding the indications. Ann Surg Oncol 2002;9:680-7.
Delahunt B, Eble JN, McCredie MR, et al. Morphologic typing of
papillary renal cell carcinoma: comparison of growth kinetics and
patient survival in 66 cases. Hum Pathol 2001;32:590-5.
Martignoni G, Brunelli M, Gobbo S, et al. Role of molecular markers in diagnosis and prognosis of renal cell carcinoma. Anal Quant
Cytol Histol 2007;29:41-9.
Thompson RH, Kurta JM, Kaag M, et al. Tumor size is associated with malignant potential in renal cell carcinoma cases. J Urol
2009;181:2033-6.
pathologica 2012;104:101-104
Case report
Clear cell papillary renal cell carcinoma
with characteristic morphology
and immunohistochemical staining pattern
S.M. Gilani, R. Tashjian, H. Qu
Department of Pathology, St. John Hospital & Medical Center, Detroit MI, USA
Key words
Renal cell carcinoma • Clear cell papillary type • Classification • Immunohistochemical staining • Cytokeratin 7
Summary
Clear cell papillary renal cell carcinoma is newly-defined entity
initially believed to be associated with end stage renal disease.
We report a rare case of this neoplasm in a 70-year-old female
patient with no known history of end-stage renal disease who presented with haematuria lasting several days. After initial workup,
a computed tomography (CT) scan was performed and revealed a
cystic mass in the right kidney. Material obtained by fine needle
aspiration (FNA) biopsy of the mass was felt to be suspicious for
renal cell carcinoma. The patient subsequently underwent right
nephrectomy, and the lesional tissue was examined microscopically. A 2.3 cm in greatest dimension cystic space circumscribed
by a fibrous wall was surfaced by a single layer of bland cuboidal
cells with abundant clear cytoplasm. The solid component of the
tumour consisted of branching papillae with delicate fibrovascular cores and uniformly lined by cells similar to those of the inner
wall of the cyst. Some of the fibrovascular cores were markedly
expanded by a myxoid-appearing substance, but no foamy cells
were appreciated. Immunohistochemically, the neoplastic cells
were diffusely immunoreactive with cytokeratin 7 (CK7), epithelial membrane antigen (EMA), high molecular weight cytokeratin (HMWCK) and vimentin. Neoplastic cells were only focally
immunoreactive with CD10, and were negative for both p63 and
α-methylacyl-CoA racemase (AMACR) (P504S). The cytomorphological features and immunohistochemical staining pattern of
this tumour was consistent with that of clear cell papillary renal
cell carcinoma (CCPRCC), as described by Gobbo et al.
Introduction
which showed atypical cells suspicious for, but not diagnostic of, renal cell carcinoma. The subsequent laparoscopic total nephrectomy specimen weighed 771 grams.
Serial sectioning of the specimen revealed multiple 1.0 to
8.5 cm in the greatest dimension cystic foci, the majority
of which were lined by a pale-tan to grey, smooth inner
surface. However, a 2.3 cm in greatest dimension cavity in the mid-portion of the kidney contained a red-tan,
solid focus that projected from the fibrous cyst wall. Microscopic examination of this focus revealed a neoplasm
with branching papillae and well-formed fibrovascular
cores. The papillae were uniformly lined by a single layer of bland cuboidal cells with abundant, clear cytoplasm
(Figs. 1-2). Some of the papillae were markedly expanded by a myxoid-appearing substance, but no foamy cells
were identified. The inner lining of the cyst was composed of neoplastic cells with features similar to those
lining the papillae. Immunohistochemically, the neoplastic cells were diffusely immunoreactive with cytokeratin 7 (Fig. 3), epithelial membrane antigen (EMA), high
Clear cell papillary renal cell carcinoma (CCPRCC) is a
rare, newly-defined entity with very few reported cases
in the English language literature. Typically, CCPRCC
is associated with end stage renal disease (ESRD) and
should be distinguished from the other variants of renal
cell carcinoma. We report a unique case of CCPRCC appearing in a patient with no known history of ESRD.
Case report
A 70-year-old female with a past medical history significant for colon cancer, hypertension and coronary artery
disease presented with the history of haematuria lasting
several days. After initial workup, a computed tomography (CT) scan demonstrated a partially-calcified cystic
mass in the right kidney. The patient underwent CTguided fine needle aspiration (FNA) biopsy of the lesion,
Correspondence
Syed M. Gilani, St. John Hospital & Medical Center, Detroit, MI
48236 - Fax +1 313 881 4727 - E-mail: [email protected]
102
S.M. Gilani et al.
Fig. 1. Low-power view of neoplastic cells within the cystic cavity.
(H&E, Original magnification x40).
Fig. 3. Branching papillae with fibrovascular cores. (H&E, Original
magnification x200).
Fig. 2. Histological features of clear cell papillary renal cell carcinoma. The papillae are uniformly lined by a single layer of bland
cuboidal cells with abundant clear cytoplasm. (H&E, Original magnification x100).
Fig. 4. Immunohistochemical features of clear cell papillary renal
cell carcinoma. Neoplastic cells diffusely immunoreactive for cytokeratin 7. (Original magnification x100).
molecular weight cytokeratin (HMWCK) and vimentin.
The same cells were only focally immunoreactive with
CD10 and were negative for both p63 and α-methylacylCoA racemase (AMACR) (P504S). Based upon the
cytomorphological features and immunohistochemical
staining pattern, a diagnosis of clear cell papillary renal
cell carcinoma (CCPRCC) was rendered. Recent studies have demonstrated that conventional clear cell renal
cell carcinoma, papillary renal cell carcinoma, renal cell
carcinoma with Xp11.2 translocation and clear cell papillary renal cell carcinoma are distinct entities, each with
characteristic morphology, immunohistochemical staining profile and cytogenetic characteristics.
renal epithelial neoplasia are based on the histological,
immunohistochemical and cytogenetic characteristics of
the neoplasm in question. In the past, classification of renal cell carcinomas with both papillary architecture and
clear cell features had presented a diagnostic challenge.
However, a new entity designated as clear cell papillary
renal cell carcinoma (CCPRCC) has recently been described by Gobbo et al. 1, allowing for better categorization of such lesions. This variant of renal cell carcinoma
has distinctive histological and immunohistochemical
features and should not be mistaken for either clear cell
renal carcinoma or papillary renal cell carcinoma. Some
investigators have postulated an association between
CCPRCC and end-stage renal disease (ESRD) 2.
Clear cell papillary renal cell carcinoma is a low stage
and low Fuhrman grade neoplasm with distinct histological features. Microscopically, these predominantly
cystic neoplasms are surrounded by a fibrous stroma
with branching papillae containing well-formed fibro-
Discussion
Several variants of renal cell carcinoma have been described in the literature. The criteria for classification of
103
Clear Cell Papillary Renal Cell Carcinoma
vascular cores. These papillae are lined by small- to
intermediate-sized neoplastic epithelial cells with abundant clear cytoplasm. Nuclei tend to be small, round, and
polarized towards the luminal aspect of the cells. The
chromatin pattern corresponds to a low Fuhrman nuclear
grade (Fuhrman grades 1 or 2). Foamy macrophages are
not identified, a key feature that distinguished CCPRCC
from papillary renal cell carcinoma. Necrosis is typically
not present, and there is minimal to no mitotic activity.
The immunohistochemical staining patterns show positive immunoreactivity with cytokeratin 7 (CK7) and carbonic anhydrase 9 (CA IX). CD10, α-methylacyl-CoA
racemase (AMACR) (P504S), translocation factor E3
(TFE3), and translocation factor EB (TFEB) 3 are generally negative. Based on the limited data, it is suggested
that this tumour has low potential for malignancy 3.
Cytogenetic analysis is increasingly being utilized to aid
in the differentiation between variants of renal cell carcinoma. Each of the most common histologic subtypes
harbours specific recurrent genetic abnormalities, such
as deletion of 3p in conventional clear cell renal cell
carcinoma and trisomies 7 and 17 in papillary renal cell
carcinoma 4. Several genetic mutations associated with
3p deletions have been described for clear cell renal cell
carcinomas. Furthermore, Rohan et al. emphasized the
role of hypoxia-inducible factor (HIF-1α) pathway in
the pathogenesis of CCPRCC 5. Renal cell carcinomas
with Xp11.2 translocation have also shown multiple genetic alterations involving the Transcription Factor E3
(TFE3) gene on Xp11.2 and various other fusion partners.
The differential diagnosis of CCPRCC includes conventional clear cell renal cell carcinoma, papillary renal
cell carcinoma, cystic clear cell carcinoma and renal cell
carcinoma with Xp11.2 translocation. Neoplastic cells
found in conventional clear cell renal cell carcinoma
are typically immunoreactive for CD10 and AMACR
(P504S), but not for CK7. A VHL gene mutation has
been detected in conventional clear cell renal cell carcinoma, but not in CCPRCC 6. Papillary renal cell carcinoma can be distinguished from CCPRCC based on
immunohistochemical staining patterns. The former is
immunoreactive for CK7, CD10, and AMACR (P504S)
and not for CA IX 7. The presence of clear cells in papillary renal cell carcinoma is associated with more aggressive lesions and a poorer prognosis 8. Renal cell carcinoma with Xp11.2 translocation is a rare entity and
predominantly reported in children and young adults 9.
They are positive for CD10 and TFE3. Based on recent
studies, it has been postulated that conventional clear
cell renal cell carcinoma, papillary renal cell carcinoma,
CCPRCC and renal cell carcinoma with Xp11.2 translocation are four discrete entities 10, each with distinct
cytomorphological, immunohistochemical (Tab. I) and
cytogenetic characteristics.
In our patient, a few months of postoperative follow-up
showed that the symptoms were markedly diminished and
there were no post-operative complications. CCPRCC is
a unique entity with distinct cytomorphological, immunohistochemical and cytogenetic characteristics. It is critical
to recognize and accurately diagnose CCPRCC because
of its tendency to mimic other subtypes of renal cell carcinoma, especially in borderline neoplasms that possess
both papillary architecture and clear cell histology. Definitive pathological diagnosis depends on the collective
interpretation of histopathological findings, immunohistochemical staining pattern and cytogenetic analysis. Surgical resection is the mainstay of treatment.
Tab. I. Differential diagnosis of clear cell papillary renal cell carcinoma.
Histopathologic findings
Immunostaining
Prognosis
RCC, Conventional Clear Cell
Type
• Multiple architectural
patterns with network of
small, thin-walled vessels.
• Neoplastic cells with
abundant clear cytoplasm
filled with lipid and glycogen
• Positive for CD10, AMACR,
• Usually negative for CK7.
• Tumour stage is the most
important prognostic
feature.
• Nuclear grade is the second
most important prognostic
feature.
Papillary RCC with Clear Cell
Features
Malignant epithelial cells
forming papillae and tubules.
• Papillae contain fibrovascular
cores, aggregates of
foamy macrophages, and
cholesterol crystals.
• Positive for CK7 and AMACR
• Five-year survival depends
on tumour grade, stage and
presence of sarcomatoid
dedifferentiation (Type I
tumours have longer survival
than Type II tumours).
RCC, Xp11.2 Translocation
Malignant epithelial cells with
clear to eosinophilic, granular
cytoplasm forming papillae or
arranged in a nested pattern.
Positive for CD10, AMACR and
TEF3
• Usually present at an
advanced stage. Poor
prognosis in adults.
AMACR = Alpha-Methylacyl-CoA Racemase, TEF3 = Transcription Factor E3.
104
S.M. Gilani et al.
References
1
2
3
Gobbo S, Eble JN, Maclennan GT, et al. Renal cell carcinomas
with papillary architecture and clear cell components: the utility
of immunohistochemical and cytogenetical analyses in differential
diagnosis. Am J Surg Pathol 2008; 32:1780-6.
Tickoo SK, dePeralta-Venturina MN, Harik LR, et al. Spectrum
of epithelial neoplasms in end-stage renal disease: an experience
from 66 tumor-bearing kidneys with emphasis on histologic patterns distinct from those in sporadic adult renal neoplasia. Am J
Surg Pathol 2006;30:141-53.
Adam J, Couturier J, Molinié V, et al. Clear-cell papillary renal
cell carcinoma: 24 cases of a distinct low-grade renal tumor and
a comparative genomic hybridization array study of seven cases.
Histopathology 2011; 58:1064-71.
4
Hagenkord JM, Gatalica Z, Jonasch E, et al. Clinical genomics of
renal epithelial tumors. Cancer Genet 2004;204:285-97.
5
Rohan SM, Xiao Y, Liang Y, et al. Clear-cell papillary renal cell carcinoma: molecular and immunohistochemical
analysis with emphasis on the von Hippel-Lindau gene and
hypoxia-inducible factor pathway-related proteins. Mod Pathol
2011;24:1207-20.
Kuroda N, Shiotsu T, Kawada C, et al. Clear cell papillary renal
cell carcinoma and clear cell renal cell carcinoma arising in acquired cystic disease of the kidney: an immunohistochemical and
genetic study. Ann Diagn Pathol 2011;15:282-5.
6
Molinié V, Balaton A, Rotman S, et al. Alpha-methyl CoA
racemase expression in renal cell carcinomas. Hum Pathol
2006;37:698-703.
7
Klatte T, Said JW, Seligson DB, et al. Pathological, immunohistochemical and cytogenetic features of papillary renal cell carcinoma with clear cell features. J Urol 2011;185:30-35.
8
Armah HB, Parwani AV. Xp11.2 translocation renal cell carcinoma. Arch Pathol Lab Med 2010;134:124-9.
9
Kato H, Kanematsu M, Yokoi S, et al. Renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion: radiological findings mimicking papillary subtype. J Magn Reson Imaging
2011;33:217-20.
10
pathologica 2012;104:105-108
Case report
Idiopathic granulomatous mastitis mimicking breast
cancer: report of two cases
F. LIMAIEM, S. KORBI, T. TLILI, I. HADDAD, A. LAHMAR, S. BOURAOUI, F. GARA*, S. MZABI
Department of Pathology and *Gynecology, Mongi Slim Hospital La Marsa, Tunisia
Key words
Idiopathic granulomatous mastitis • Breast • Inflammatory disease • Mammography
Summary
Idiopathic granulomatous mastitis is a rare inflammatory breast
disease of unknown aetiology that is frequently mistaken for
breast carcinoma both clinically and mammographically. In this
paper, the authors report two cases of idiopathic granulomatous
mastitis that occurred in two parous women aged 38 and 45
years. Clinically, both patients presented with a tender palpable
lump in the left breast. Mammography showed an poorly-defined mass in both patients with microcalcification in the first
case and skin retraction in the second case. Breast lumpectomy
was performed in both patients. Histological examination of the
surgical specimen revealed non-caseating granulomas confined
to breast lobules. Special staining for fungi and tuberculosis
were all negative. Correct diagnosis of idiopathic granulomatous
mastitis requires the exclusion of malignancy, other granulomatous disease and infectious aetiologies. Histopathologic examination remains the gold standard for diagnosis. This disease is
rare, and therefore the optimum treatment protocol is still being
established.
Introduction
amination revealed a well-circumscribed, firm but mobile
1.5 cm lesion in the upper outer quadrant of the patient’s
left breast. The overlying skin was slightly erythematous
with no ulceration. There was no nipple discharge or
lymphadenopathy. Results of standard laboratory analyses were normal. Mammography showed asymmetric
density in the outer upper quadrant of the left breast with
scattered microcalcification (Fig. 1). Ultrasonography
demonstrated a heterogeneously hypoechoic lesion with
posterior acoustic shadowing. The patient underwent local excision of the mass. Histopathological analysis of
the surgical specimen showed non-caseating granulomas
involving the mammary lobules with Langhans-type
multinucleated giant cells, neutrophils, lymphocytes and
plasma cells (Figs. 2, 3). Microabscess formation was
seen focally, but there were no areas of caseation. Special staining for fungi (periodic acid-Schiff) and tuberculosis (Zieh-Neelsen) were negative. The final pathological diagnosis was idiopathic granulomatous mastitis. The
patient developed a chronic wound sinus and was treated
with antibiotics. This problem persisted for 2 months after the initial excision and required curettage before healing with no further problems.
Idiopathic granulomatous mastitis is a rare, chronic,
non-caseating, granulomatous lobulitis of uncertain aetiology. It usually affects women of child-bearing age and
is frequently mistaken for a malignancy, particularly if
regional lymph nodes are enlarged 1 2. Herein, we report
on two cases of idiopathic granulomatous mastitis that
clinically and radiologically mimicked breast cancer.
Our aim was to highlight the clinicopathological features of this entity with special emphasis on differential
diagnosis.
Clinical history
Case 1
A 38-year-old woman with no significant past medical
history presented with a tender palpable lump in her left
breast discovered one month prior to consultation. There
was no history of recent pregnancy, breast-feeding, breast
trauma, use of oral contraceptives, family history of
breast disease, or exposure to tuberculosis. Physical ex-
Correspondence
Faten Limaiem, Department of Pathology, Mongi Slim Hospital,
La Marsa, Tunisia - Tel. +216 96 552057 - E-mail: fatenlimaiem@
yahoo.fr
106
F. Limaiem et al.
Case 2
A 45-year-old woman with no significant past medical
history presented with a two-month history of a very tender and erythematous left breast mass with sinus tracts
and ulceration of the skin. The patient was not pregnant
and did not recently breast-feed her children. Moreover,
there was no history of breast trauma, use of oral contraceptives, family history of breast disease, or exposure
to tuberculosis. Physical examination revealed a painful,
firm immobile 3.5 cm mass in the upper outer quadrant
of the left breast. The overlying skin showed ulceration
as well as signs of inflammation. Axillary lymph nodes
were palpable. Mammography showed asymmetric den-
sity in the outer upper quadrant of the left breast with
skin retraction and thickening (Fig. 4). Ultrasonography
revealed a heterogeneously hypoechoic lobular lesion
with posterior acoustic enhancement. The patient underwent lumpectomy. Histopathological analysis of the
surgical specimen showed non-caseating granulomas
involving the mammary lobules with Langhans-type
multinucleated giant cells, neutrophils, lymphocytes and
plasma cells. There were no areas of caseation within
the granulomas and special stains for bacteria, acid-fast
organisms and fungi were negative. Postoperative recovery was uneventful. At present, the patient is still on
follow-up.
Fig. 1. Case 1: Preoperative mammography of the left breast
showing asymmetric increased density with indistinct margins
and focal microcalcification.
Fig. 2. Case 2: Left upper mammographic image demonstrating
nipple retraction, with skin thickening and a poorly-defined density of the mass.
Fig. 4. Non-caseating granuloma involving mammary lobules
with Langhans-type multinucleated giant cells, neutrophils, lymphocytes and plasma cells. (haematoxylin & eosin; original magnification × 40).
Fig. 3. Destruction of lobular architecture by granulomatous inflammation (haematoxylin & eosin; original magnification × 10).
107
Idiopathic granulomatous mastitis
Discussion
Since its initial description by Kessler and Wolloch in
1972, more than 120 cases of idiopathic granulomatous
mastitis have been reported in the English language literature to date 1. Most patients who develop this form
of mastitis are of reproductive age ranging from 17 to
42 years (mean, 32 years) 2. 3. Idiopathic granulomatous
mastitis occurs commonly in recent pregnancy and lactation 1 2. There was no history of recent pregnancy or
breast-feeding in either of our cases. The most common
clinical presentation of idiopathic granulomatous mastitis is a unilateral firm-to-hard extra-areolar mass ranging from 0.5 to 9 cm in size 4. The lump may involve
the overlying skin or penetrate the underlying pectoralis muscle with nipple retraction, sinus formation and
axillary lymphadenopathy clinically mimicking breast
carcinoma 2 3 5. With its retro-areolar or central location, features of inflammation and its common occurrence in recent pregnancy and lactation, the breast mass
can also be mistaken clinically for a lactating breast
abscess 1 4. Routine radiological examination including
ultrasonography and mammography cannot differentiate idiopathic granulomatous mastitis from carcinoma.
Ultrasonography usually reveals inhomogeneous, irregular hypoechoic lesions with focal posterior shadowing
or multiple relatively circumscribed heterogenous hypoechoic lesions associated with a large mass 6 7. Doppler
examination reveals increased vascularity of the lesions
and surrounding tissues. On MRI, it is still impossible to
differentiate an active inflammatory process from a tumoural process. However, MRI is of substantial value in
demonstrating the extent and reduction of the lesions in
time 6-8. macroscopically, specimens typically consists
of firm-to-hard mammary parenchyma that contains a
palpably distinct mass. Margins are less apparent on visual inspection of the cut surface where the grey-to-tan
tissue appears to have a faintly nodular architecture 9.
The primary pathologic change in idiopathic granulomatous mastitis is a granulomatous inflammatory
reaction centred on lobules, a granulomatous lobulitis.
Granulomas composed of epithelioid histiocytes, Langhans giant cells accompanied by lymphocytes, plasma
cells and occasional eosinophils are found within and
around lobules. With progression of the inflammatory
References
process, confluent granulomas may obscure or obliterate the lobulocentric distribution of the process, particularly toward the central portion of the tumour. Fat
necrosis, abscess formation and fibrosis contribute to
effacement of the lobular distribution in confluent lesions. There are no areas of caseation within granulomas, and special stains for bacteria, acid-fast organisms
and fungi are negative 9. Other causes of granulomatous
lesions in the breast must be excluded. The distinction
between granulomatous mastitis and various other granulomatous inflammatory conditions, such as tuberculosis, sarcoidosis, cat scratch disease and foreign body
reaction, can usually be made by correlating clinical
and pathological findings. It is critically important that
a granulomatous reaction in carcinoma also be considered. Whereas most examples of this unusual complex
have areas composed of easily identified intraductal or
invasive carcinoma, the associated carcinoma is sometimes obscured by this reaction 9. Although the aetiology of idiopathic granulomatous mastitis is unknown,
it has been postulated that this entity could result from
an autoimmune response. It is possible that damage to
the ductal epithelium produced by local trauma, chemical irritation or infection may allow the extravasation of
luminal fat and protein-rich secretion into the lobular
connective tissue, thereby inducing a granulomatous response with lymphocyte and macrophage migration 10-12.
Prior use of oral contraceptives and reaction to childbirth are other proposed aetiological factors of idiopathic granulomatous mastitis 1. The optimal management
of granulomatous mastitis is still controversial. Some
authors have recommended the use of corticosteroids
following a diagnosis of granulomatous mastitis, while
others do not 13-15. Most authors recommended surgical
excision coupled with corticosteroid therapy 12 16. Recurrence rates of 16-50% after excision have been reported
in the literature. Other complications include fistula formation and secondary infection.
In conclusion, idiopathic granulomatous mastitis is a
rare inflammatory disease of the breast that can mimic
breast carcinoma. Radiologically, there is no characteristic appearance and accurate preoperative diagnosis is
often difficult. Histopathologic analysis in correlation
with clinical, biological and radiological findings is
mandatory for definitive diagnosis.
5
Kessler E, Wolloch Y. Granulomatous mastitis: a lesion clinically
simulating carcinoma. Am J Clin Pathol 1972;58:642-6.
6
Tuncbilek N, Karakas HM, Okten OO. Imaging of granulomatous
mastitis: assessment of three cases. Breast 2004;13:510-4.
7
Han B, Choe YH, Park JM, et al. Granulomatous mastitis: mammographic and sonographic appearances. Am J Roentgenol
1999;173:317-20.
1
Kok KYY. Telisinghe PU. Granulomatous mastitis: Presentation,
treatment and outcome in 43 patients. The Surgeon 2010;8:197201.
2
Van Ongeval C, Schraepen T, Van Steen A, et al. Idiopathic granulomatous mastitis. Eur Radiol 1997;7:1010-2.
3
Yilmaz E, Lebe B, Usal C, et al. Mammographic and sonographic
findings in the diagnosis of idiopathic granulomatous mastitis. Eur
Radiol 2001;11:2236-40.
8
Engin G, Acunas G, Acunas B. Granulomatous mastitis: gray
scale and color Doppler sonographic findings. J Clin Ultrasound
1999;27:101-6.
4
Erhan Y, Veral A, Kara E. A clinicopathologic study of a rare entity mimicking breast carcinoma: idiopathic granulomatous mastitis. Breast 2000;9:52-6.
9
Rosen PP. Inflammatory and reactive tumours. In: Rosen’s Breast
pathology. 2nd ed. Philadelphia, PA: Lippincott Williams and
Wilkins 2005, pp. 38-40.
108
F. Limaiem et al.
10
Asoglu O, Ozmen V, Karanik H. Feasibility of surgical management in patients with granulomatous mastitis. Breast J
2005;11:108-14.
14
11
Imoto S, Kitaya T, Kodama T. Idiopathic granulomatous mastitis. Case report and review of the literature. Jpn J Clin Oncol
1997;27:274-7.
Hovanessian Larsen LJ, Peyvandi B, Kilpfel N. Granulomatous
lobular mastitis: imaging, diagnosis and treatment. Am J Roentgenol 2009;193:574-81.
15
12
Schelfout K, Tjalma WA, Cooremans ID. Observations of an idiopathic granulomatous mastitis. Eur J Obstet Gynaecol Reprod Biol
2001;97:260-2.
Baslaim MM, Khayat HA, Al-Amoudi SA. Idiopathic granulomatous mastitis: a heterogeneous disease with a variable clinical presentation. World J Surg 2007;31:1677-81.
16
13
DeHertogh DA, Rossof AH, Harris AA, et al. Prednisone man-
Azlina AF, Ariza Z, Arni T. Chronic granulomatous mastitis: diagnosis and therapeutic considerations. World J Surg 2003;27:515-8.
agement of granulomatous mastitis. N Engl J Med 1980;303:799800.
Pathologica 2012;104:109-173
110
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
I CONGRESSO NAZIONALE
DI CITOPATOLOGIA
SIAPEC-IAP
Trieste, 28-30 giugno 2012
Palazzo dei Congressi – Stazione Marittima
Presidente del Congresso
Luigi Di Bonito
Consiglio Direttivo SIAPEC-IAP
Presidente
Claudio Clemente
Past President
Gian Luigi Taddei
Vice Presidente
Gaetano De Rosa
Segretario
Anna Sapino
Consiglieri
Arrigo Bondi, Paolo Dalla Palma, Ambrogio Fassina, Roberto
Fiocca, Domenico Ientile, Leonardo Resta, Luigi Ruco,
Marco Santucci, Giuseppe Zamboni
Rivista “PATHOLOGICA”
Marco Chilosi
Commissione Informatica
Roberto Mencarelli
Rappresentante POF
Laura Viberti
Rappresentante AITIC
Carmine Lupo
Segreteria Locale
D. Bonifacio
S. Dudine
F. Zanconati
Comitato Scientifico
Tutti i relatori e moderatori per il contributo al programma
scientifico del Congresso.
Segreteria Organizzativa
via San Nicolò, 14
34121 Trieste
Tel. +39 040 368343 – int. 15
Fax +39 040 368808
Sito: www.theoffice.it/SIAPEC2012
E-mail: [email protected]
111
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
M
Giov
28-giu
P
9-13.00
Test di competenza
R. Navone
15.00-18.00
Corso di
Immunocitochimica
M. Chilosi - C. Doglioni
Corso di Statistica
applicata
L.Torelli
Corso al microscopio
Urine
P. Dalla Palma - M. Bonzanini
18.00-19.30
Assemblea soci SIAPEC-IAP
19.30
Inaugurazione e presentazione del Congresso (prof. Vito Ninfo, Padova)
20.00
Cocktail di benvenuto
8.30-10.30
M
P
Versamenti
Linfonodi e mediastino
Ginecologia
Immunocitochimica
Simposi satelliti
Ginecologia
Citologia aspirativa
degli organi profondi
Tiroide
Cena sociale
8.30 - 9.00
DISCUSSIONE POSTER
Mammella
Nuove tecnologie
10.30-11.00
11.00-12.30
12.30
Corso al microscopio
Ginecologia
G. Negri - S. Prandi
Corso al microscopio
Mammella
A. Sapino - F. Zanconati
Coffee break
20.00
9.00-10.30
M
Citotecnici
PAUSA PRANZO
16.00.16.30
16.30-18.30
Corso al microscopio
Ginecologia
G. Negri - S. Prandi
Coffee break
13.00-14.00
14.00-16.00
Sab
30-giu
Statistica applicata
agli screening
Urine
10.30-11.00
11.00 -13.00
Ven
29-giu
Polmone
Comunicazioni libere
Corso al microscopio
Mammella
A. Sapino - F. Zanconati
Corso al microscopio
Tiroide
G. Fadda – F. Nardi
Coffee break
Mammella
Controllo di qualità
e risvolti medico-legali
Comunicazioni libere
CHIUSURA DEL CONGRESSO E PREMIAZIONI
Corso al microscopio
Capo-collo
S. Fiaccavento
112
sessioni scientifiche
Venerdì, 29 giugno 2012
ore 8.30-10.30
Polmone
Chairmen: Gerardo Botti (Napoli), Gian Luigi Taddei (Firenze)
I tumori neuroendocrini del polmone
G. Pelosi
Dipartimento di Patologia Diagnostica e Laboratorio e Facoltà di
Medicina, Università di Milano
I tumori neuroendocrini del polmone (TNE) rappresentano
circa il 15-20% dei carcinomi polmonari e comprendono
quattro entità clinico-patoligiche distinte sulla base di criteri
epidemiologici, genetici, clinici e patologici (WHO, 2004),
vale a dire carcinoide tipico (TC), carcinoide atipico (AC),
carcinoma neuroendocrino a grandi cellule (LCNEC) e
carcinoma a piccole cellule (SCLC). Essi costituiscono uno
spettro classificativo organizzato tradizionalmente in quattro
gradi dal punto di vista patologico e in tre gradi dal punto di
vista clinico in cui il CT corrisponde a tumori di basso grado
con lunga sopravvivenza anche in caso di malattia metastatica, il AC a tumori di grado intermedio con sopravvivenza
intermedia e gli SCLC e LCNEC a tumori scarsamente differenziati con sopravvivenza scadente. Mentre la diagnosi
di SCLC è agevole anche su campioni citologici, che anzi
rappresentano un materiale di prima scelta poiché la maggior parte dei pazienti si presentano con malattia localmente
avanzata o metastatica, la diagnosi delle altre entità richiede
l’esame di campioni chirurgici. In particolare non è possibile
distinguere carcinoidi tipici e atipici, come pure il LCNEC,
su preparati citologici per le uniformità morfologiche o la
possibilità di confusione con altri carcinomi polmonari. La
diagnosi dei TNE polmonari è un processo “stepwise”, in
cui è necessario dapprima identificarne la qualità neuroendocrina utilizzando criteri morfologici ampiamente condivisi
basati sulla valutazione delle caratteristiche citocariologiche
ed architetturali, successivamente confermarne la natura
neuroendocrina con marcatori immunoistochimici pan-endocrini (cromogranina A e sinaptofisina). Questi ultimi
sono mandatori per la diagnosi di LCNEC, che altrimenti
potrebbe essere confuso con altri carcinomi non a piccole
cellule, in particolare adenocarcinomi poco differenziati.
Molto utile e raccomandabile è l’uso sistematico della valutazione semiquantitativa della frazione proliferante con
immunoreazione per Ki-67 per distinguere i TNE di grado
basso-intermedio (CT e AC) dalle forme scarsamente differenziate (SCLC e LCNEC), come pure la proteina del
retinoblastoma e la ciclina D1 per separare il LCNEC dal
SCLC, la cui sensibilità ai medesimi protocolli di terapia è
variabile e non soddisfacente. Numerosi dati di letteratura si
stanno poi accumulando sulla caratterizzazione genetica dei
TNE polmonari, utilizzabili dal punto di vista classificativo,
prognostico e predittivo. Molto attuale è la problematica
classificativa alla luce della prevista nuova classificazione
WHO dei tumori polmonari che uscirà nel 2014-2015, che
potrebbe prendere in considerazione anche terminologie già
in uso nei TNE del tratto gastro-entero-pancreatico, come
pure l’uso sistematico di tecnologie genetiche d’avanguardia
che porteranno ad aumentare la messe delle informazioni a
nostra disposizione per le finalità della patologia terapeutica.
Role of cytological specimen in molecular
evaluation of NSCLC
G. Fontanini*, A. Proietti*, E. Sensi, C. Lupi, A. Servadio, L.
Boldrini*, G. Alì
Dipartimento di Chirurgia, Università di Pisa; Azienda Ospedaliera
Universitaria Pisana (AOUP), Pisa
*
Introduction. Lung cancer is the leading cause of cancer-related deaths worldwide. Non small cell lung cancer (NSCLC)
accounts for approximately 80% of these. The World Health
Organization (WHO) pathological classification of lung cancer is based on the use of standard histochemical stains of
surgical resected neoplasms, but in the clinical practice, the
resection rate for lung cancer ranges from 10-15% of all the
tumours. In particular 60% of patients with NSCLC present
with unresectable stage IIIB or IV disease. Hence the largest
part of patients are treated on the basis of a diagnosis made on
a single small tumour biopsy or a cytological sample. In the
last few years, the histotype has been shown as a critical variable in clinical decision making, as a result of the introduction
of newer biologically targeted chemotherapies for advanced
disease and of development of specifically tailored systemic
treatments. In NSCLC, prospective randomized studies have
shown that new chemotherapeutic and molecular-targeted
agents may lead to improved results, as compared with prior
standard therapeutic options. On the other hand, therapy inefficacy and toxicity are largely reported if the treatment is not
correctly suited to the tumour histotype. Particularly, it was
discovered that epidermal growth factor receptor (EGFR)
and K-RAS mutations, which are most frequently observed
in adenocarcinomas (ADC), are predictive of responsiveness
and resistance, respectively, to EGFR tyrosine kinase inhibitors, erlotinib and gefitinib, and accurate subtyping as ADC
or squamous cell carcinoma (SCC) became important for the
selection of patients for molecular testing. More recently, two
other agents, bevacizumab and pemetrexed, were found to
have differential toxicity and activity, respectively, in patients
with SCC versus non-SCC.
The cytologic sampling of lung cancer allows minimally invasive procedures and immediate on-site assessment of specimen adequacy. Moreover cytology has exceptional accuracy
for distinguish small cell carcinoma (SCLC) from NSCLC.
However, the feasabilty of NSCLC subtyping in cytology is
not well established. In particular immunohistochemistry has
came up as a powerful tool for revealing a line of differentiation as ADC or SCC, hence it is increasingly incorporated in
routine diagnostic practice. However, cell dispersal and low
cellularity are sometimes viewed as a strong limitation for
using cytologic specimens fo r ancillary studies, such as IHC
or molecular testing.
Materials and methods. A review of the cytohistological
database of the Division of Pathology of Pisa University,
Department of Surgery was conducted to identify all small
biopsy and cytology specimens performed for a diagnostic
purpose in patients with a thoracic lesion from 2009 to 2011.
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sessioni scientifiche
Totally, 341 patients with a cytological diagnosis of NSCLC
were studied. No selection based on the final diagnosis subtyping was done. All the cases were retrieved and reviewed
in blinded fashion. To establish the accuracy the cytological
diagnoses were compared with subsequent histological diagnosis, when available. Cytological samples were exfoliative
(bronchial washing and bronchial brushing) and aspirative
(transbronchial needle aspiration cytology, transthoracic needle aspiration cytology and pleural effusion). The cytological
preparation were conducted according to standard specimen
processing protocol in our laboratory. All the smears were
fixed in Cytofix and stained with Papanicolau. Cell blocks
were available for cases with a sufficient needle rinse, which
yielded a visible pellet after centifugation. A panel of immunohistochemical markers was used during the diagnostic
workup, including TTF-1, p63 and CK34βE12. Exact marker
panels for various cases were selected at the discretion of
individual pathologist but a minimum included TTF-1 and/
or p63. Interpretation was based on the following algorithm:
TTF-1-negative/p63-positive or CK34betaE12-positive was
interpreted as supportive of SCC, whereas expression of
TTF-1 as supportive of ADC. On the basis of other studies,
we allowed p63 and CK34betaE12 coexpression with TTF-1
for classification as ADC. If cytomorphology was equivocal
for NSCLC subtype, but cellularity was insufficient for IHC a
case was diagnosed as “NSCLC-NOS”.
On selected 106 positive sample molecular testings were conducted. Only sample judged by the pathologists as adequate
(easily identifiable tumor cells by light microscopy, with
tumor cells representing at least 25% of overall cellularity).
Genomic DNA was isolated from cytological specimens by a
standard method. The paraffin was removed by xylene extraction, and the sample was subsequently lysed by proteinase-K.
DNA extraction was then performed using the spin column
procedure. Mutational profiling of EGFR (exons 18-21) was
performed by direct dideoxy sequencing. Pyrosequencing
assays was performed for sequencing analysis of K-RAS (codons 12 and 13). All statistical analyses were conducted using
a statistica software (JMP® 8.0.2). A Chi-square test was used
to analyze the associations between the different variables.
The a priori level of significance was set at a p-value of less
than 0.05.
Results. Three hundred forty-one patients were studied. 256
(75.1%) were male and 85 (24.9%) were female. The mean
age at diagnosis was 68.3 years.
Among cytological samples 75 were bronchial washing (BW),
211 were bronchial brushing (BB), 214 were transbronchial
needle aspiration (TBNA), 24 were transthoracic needle aspiration (TTNA) and 24 were pleural effusion (PE). NSCLC
samples were recorded as adenocarcinomas in 160 (160/341,
47.2%) cases and as squamous cell carcinomas in 129
(129/341, 37.9%) cases. Fifty-two cases (52/341, 15%) were
diagnosed NSCLC unclassified (NOS) because cytological
features of a specific histotype could not be identified.
Eighty of 341 cases (23.5%) could not be subtyped by morphology but after analysis with IHC, a tumour subtype was
identified. Histologic correlation revealed that 92.8% of IHCaided diagnoses were correct. Six of 341 cases (6/341, 1.7%)
could not be subtyped neither by morphology nor by IHC. All
of these cases remained unclassified (NSCLC-NOS).
Of 69 analyzable specimens, EGFR mutations were identified
in 9 cases (12.5%). The mutations were as follows: 4 in exon
19, 1 in exon 19 and 20, 2 in exon 21, 1 in exon 19 and 21
and 1 in exon 20 and 21. Mutations of exon 19 consisted of
in-frame deletions involving five amino acids from codons
746 through 750 (ELREA), and amino acids around codons
747 to 759. Mutations in exon 21 were missense substitutions
resulting in leucine to arginine at codon 858 (L858R). Other
mutations in exons were single-nucleotide substitutions. Only
2 cases were judged inadequate for the molecular analysis
because of the insufficient cellularity (2/69, 3,4%).
K-RAS mutations were detected in forty samples of the 126
analyzable (40/126, 31.8%); almost all the mutations (39/40)
involved codon 12. Five cases were excluded from the molecular analysis because of the scanty material (5/126, 4%).
In ADCs of EGFR mutations were observed in 7 cases (7/40;
17,5%), in particular mutations of exon 19 in 4 cases (4/40
10,5%) and mutation of exon 21 in 3 cases (3/40, 7,5%).
Whereas K-RAS codon 12 mutation was observed in 41 cases
(41/160; 26%).
Conclusions. This is a large study focus on accuracy of
NSCLC subtyping in pathology practice, where IHC is routinely used for subtyping of morphologically unclassifiable
NSCLC. In addition, we made a large review of utilization of
cytologic specimens for EGFR/K-RAS molecular aanalysis.
We found thaht cytologic subtyping is highly feasible and accurate (92.8% of cyto/histologic concordance). Furthermore,
various cytologic specimens are suitable for EGFR/K-RAS
molecular analysis with mutational rates comparable with
those detected in surgical specimens.
Venerdì, 29 giugno 2012
ore 8.30-10.30
Urine
Chairmen: Paolo Dalla Palma (Trento), Oscar Nappi (Napoli)
The patient with clinical hematuria: what’s the
role of the cytology?
C. Di Loreto, G. Raiti, F. Riosa, E. Pegolo
Istituto di Anatomia Patologica, Università di Udine
Introduction. Urinary cytology is a safe, non invasive and
inexpensive method to investigate gross and microscopic
hematuria. As most of the urothelial carcinomas produce hematuria, even at a non invasive stages, urinary cytology test,
could provide a means of early detection of urothelial lesion,
thus reducing cancer related morbidity and mortality.
Urine cytology can detect cancerous cells shed from any part
of the entire urothelium (from the renal pelvis to the urethra)
in a voided urine specimen. Because urinary cytology has a
114
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
notoriously variable sensitivity and specificity its value is still
debatable.
The aim of this study was to assess the role of urinary cytology in the evaluation of cancer of the urothelial tract.
Materials and methods. The archive diagnosis of 10,524
consecutive fresh voided urine samples were reviewed for
this study. The specimens belonged to 3508 patients without
any known history of urothelial cancer, that attended the outpatient services of our hospital over a period of 5 years from
January 2006 to June 2011. Urine sampling was performed by
the patients from second morning voided urine for 3 consecutive days. All samples were placed and stored in Cytolyt solution and then processed by the ThinPrep 2000 method (Cytyc
Corp., Boxborough, Mass) according to the manufacturer’s
instructions. Final cytologic results were classified into 1 of 4
categories: inadequate, normal-benign, atypical-suspicious or
malignant. The normal-benign category included inflammation and reactive cellular changes. Depending on the clinical
evaluation and on the cytology diagnosis, the patients had a
cystourethroscopy and/or a radiologic workup of the urinary
tract. Any abnormal urothelial lesions were biopsied and frank
tumors were resected. Pathologic samples were sent in 10%
formalin. In patients with a malignant or atypical-suspicious
cytology and no obvious tumor, random bladder biopsies were
obtained.
Biopsy specimens were diagnosed according to WHO 2004
classification of tumours of the urinary system. The 7th edition of the TNM-AJCC staging system was used for clinicalpathological staging.
Diagnostic accuracy was calculated being the biopsy the gold
standard. χ2 square and Fisher test were applied for statistical
significance.
Results. The median age of the population was 70 years
(range: 22-97 years). The male to female ratio was 1.33:1.
Of the 3508 cases, 63 (1.8%) were unsatisfactory for interpretation due to scant number of cells or severe degradation.
3172 (90.4%) cases were diagnosed as normal-benign, 159
(4.5%) as atypical-suspicious and 114 (3.3%) as malignant.
A total of 197 patients (5.5%) had a biopsy of the urinary
tract. The percentage of performed biopsies in each cytology category was as follow: 3.1% in the inadequate category, 2.4% in normal-benign, 27.6% in atypical-suspicious,
66.6% in malignant category. The inadequate category was
excluded from any further statistical analysis. The majority
of the biopsies were performed in the urinary bladder (n =
182, 92.3%), 5 in the ureters, 2 in the renal pelvis, and 8 in
the urethra. Out of the 197 biopsies, 131 (66.5%) were diagnosed as positive for carcinoma and 66 (33.5%) as negative
for carcinoma. 124 (94.6%) of the overall tumors showed
an urothelial differentiation. In the bladder we also found
2 primary squamous carcinoma, 1 adenocarcinoma and 3
metastatic cancer.
Among the urothelial carcinoma, 74 (59.2%) were high grade,
50 (40%) low grade; 28 (22.4%) had a flat growth pattern and
96 (76.8%) a papillary architecture. In 1 tumor (0.8%) neither
the grade nor the architecture was evaluable.
Biopsy results
Positive for cancer
(N = 131)
Negative for cancer
(N = 66)
Regarding the pathological stage, 109 (87.2%) were superficial urothelial carcinomas (44 pTa, 19 pTis and 46 pT1), 13
(10.4%) were muscle invasive carcinomas (6 pT2, 5 pT3, 2
pT4) and 3 (2.4%) were unassessable.
Biopsy proven cancer was evident in 36 (45.6%) patients
with normal cytology, 32 (72.8%) of patients with atypicalsuspicious, and 63 (85.2%) of patients with malignant cytology (table). Considering the high positive predictive value of
the atypical-suspicious category, we counted this category as
positive.
The overall diagnostic accuracy was 70% with a sensitivity and
specificity of 72.5% and 65.1% respectively. The positive and
negative predictive values were 80.5% and 54.4% respectively.
High grade urothelial carcinomas were associated with a higher
sensitivity (85.1%) compared to low grade urothelial carcinoma
(56%) with a statistically significant difference (p = 0.0001).
The severity of cytology report was associated with tumor
grade with 33% of high grade in the normal-benign, 51.6% in
the atypical-suspicious and 78.3% in the malignant category.
There was no statistically significant evidence between sensitivity and growth pattern (flat vs papillary) and between
sensitivity and tumor stage (p = 0.4 and p = 0.17 respectively).
Conclusions. The mainstay for diagnosis of urothelial cancer
is the combination of cystoscopy, biopsy and voided urine cytology. Among these, voided urine cytology is a non invasive
technique suitable for diagnosis and follow up for urothelial
carcinomas.
Because most urinary tract carcinomas produce hematuria,
urine cytology is considered as part of the diagnostic workup
for urothelial malignancy in hematuric patients. However, recent studies have reported the limitations of urinary cytology
in the evaluation of patients with hematuria because of the low
sensitivity and low prevalence of urothelial carcinomas in the
general population and because of the challenging interpretation of the urinary cytology.
Paez and colleagues reported that no tumor could be diagnosed
with cytology alone and that a negative cytology could not
exclude a malignancy 1. Because of its limitations, Nabi et al.
recommended the judicial use of cytology in the proper clinical
context 2. Nakamura et al concluded that the utility of urinary
cytology in the patient with hematuria may be minimal 3.
In our study, we found that 3.24% of hematuric patients were
positive for malignant cells in urine cytology and 4.53%
had an atypical-suspicious cytology results. A biopsy of the
urinary tract was performed in 2.4% of the patients in the
normal-benign category, 27.6 in the atypical-suspicious and
66.6% in the malignant category thus only a total of 5.6% of
the patients required further investigations including biopsy.
The overall sensitivity for cancer (72.5%) is higher compared to the average reported values in the literature but a
lower specificity (65%) has been found 4. This high value of
sensitivity could be related to the evaluation of a series of 3
cytology samples per patient and to a better treatment of the
samples. The sensitivity for detecting high grade urothelial
carcinoma was higher (85.1%) than for low grade carcinoma
(56%). It’s known that well differentiated urothelial tumors
Negative
(N = 79)
Cytologic diagnosis
Atypical/suspicious
(N = 44)
Malignant
(N = 74)
36
32
63
43
12
11
115
sessioni scientifiche
are often missed by cytology but this group of tumors has a
minor clinical importance compared to the more aggressive
high grade tumors.
We found that applying the urinary cytology test as a screening
method in hematuric patients, the rate of biopsies performed is
very low when the cytology results are negative. Considering
the good sensitivity showed by our study, especially for the
clinically relevant high grade tumors, urinary cytology along
with an appropriate clinical evaluation can reduce the number
of patients that undergo a more invasive workup including
biopsy. Thus, urine cytology has an adjunct role in diagnosing
urothelial malignancy.
References
1
Paez A, Coba JM, Murillo N, et al. Reliability of the routine cytological diagnosis in bladder cancer. Eur Urol 1999;35:228-32.
2
Nabi G, Greene DR, O’Donnell M. How important is urinary cytology in
the diagnosis of urological malignancies? Eur Urol Jun 2003;43:632-6.
3
Nakamura K, Kasraeian A, Iczkowski KA, et al. Utility of serial urinary cytology in the initial evaluation of the patient with microscopic
hematuria. BMC Urol 2009;9:12.
4
Turco P, Houssami N, Bulgaresi P, et al. Is conventional urinary
cytology still reliable for diagnosis of primary bladder carcinoma?
Accuracy based on data linkage of a consecutive clinical series and
cancer registry. Acta Cytol 2011;55:193-6.
Consequence of 1973 and 2004 World Health
Organization grading systems on urinary
cytology
P. Dalla Palma
Trento
Translation of cytological diagnosis into corresponding histological diagnosis is a difficult job. The five categories
generally used (inadequate, negative, atypical, suspicious
and positive for malignant cells) are not always qualified for
urinary cytology.
From a clinical (and even cytological) point of view only two
categories (negative and positive) should be useful even if it
is well known that most differentiated tumors can not be accurately diagnosed by cytology due to the substantial lack of
cellular abnormalities.
The intermediate cytological categories (atypical and suspicious) must be limited at few cases while these diagnoses have
little value for the urologist. In these cases it is important to secure a patient’s history and cystoscopic findings before formulating a clinical recommendation. Urothelial tumors leading
to an “atypical” diagnosis predominantly include low-grade
(papillary) tumors. A distinction between histological entities
like papilloma, papillary urothelial neoplasia of low grade of
malignancy (PUNLMP) and papillary carcinoma of low grade)
are impossible with urinary cytology. Suspicious diagnoses in
most cases reflect the uncertainty of the cytologist.
The most important accomplishment of urinary cytology is the
diagnosis of high-grade (in situ or invasive) carcinomas with
very high accuracy. In this occurrence a cytological diagnosis
can be superior to a histological one. The false positive value
is very low if the clinical data are sufficient.
The distinction in two categories (low versus high) of the
2004 classification (formerly 1998 WHO/ISUP) can be more
reproducible than the 1973 classification but a translation
between the two classification can be difficult while older G2
lesions must be splitted in part together with low grade tumors
and in part with high grade ones and we can impossible to
reclassify a three-tiered classification in a two-tiered one.
Even if the 2004 classification seen to be superior in term of
reproducibility and clinical prognosticator, the 1973 classification still has clinical validity and id widely adopted. Nevertheless a good cyto-histological correlation remains difficult
and the identification of most differentiated tumors maintains
a histological indication.
Role of the needle aspiration cytology in the
diagnosis of metastatic urothelial carcinoma
E. Bollito, A. Fornari, P. Dalla Palma*
SCDU Anatomia Patologica Azienda Ospedaliera-Universitaria San
Luigi Gonzaga, Orbassano; * S.C. Anatomia Patologica Ospedale di
Trento
Urothelial carcinoma is usually seen by pathologists in both
histological and cytological specimens from upper and more
frequently lower urinary tract
Cytology is usually performed on voided urine or bladder
washing samples and some discussions are still open about
sensitivity of this test in detecting urothelial cancers. Particular problems are found in examining samples from the upper
urinary tract. In fact cytology from renal pelvis or urether may
be very difficult but even sometimes more informative than
histological methods made on biopsy specimens taken from
the same sites.
Occasionally some difficulties may be found in differentiating
renal collecting duct (CDC) from urothelial carcinoma (UC):
even in this case there are no relevant differences in therapies
this differential diagnosis is important to set different followup, however it’s usually a histological problem on nephrectomy specimen. PAX8+/p63- is a more common immunophenotype of CDC instead vice versa are the immunostains in UC.
A similar problem exist when we are trying to differentiate a
micropapillary variant of the UC from a micropapillary ovarian carcinoma: PAX8 and Ca125 may be helpful in this case
in both histological and cytological samples.
However the hardest challenge in diagnostic cytology regarding urothelial carcinoma is the evaluation of specimens
from needle aspiration from different sites suspected to be
metastatic localizations on an UC. Sometimes lung nodules
occurs in patient smokers who have high risk for both urothelial and primary lung carcinoma: TTF1 usually permit an
easy recognition of primary lung adenocarcinomas but poorly
differentiated squamous cell and UC may share morphological characteristics but also positivity for p63 and some citokeratins and negativity for PAX8 stains; CD141 and uroplakin
may be helpful but they are not sufficient for a conclusive differentiation, so these differential diagnosis should frequently
be resolved morphologically only.
La refertazione nell’esame citologico dell’urina:
criticità e proposte di lavoro
P. Maioli
U.O. Aziendale di Anatomia Patologica Ravenna
In Italia, negli ultimi quattro anni, il cancro alla vescica è
risultato il 4° tra i tumori più frequenti negli uomini. Anche se
viene spesso visto come una “condizione maschile” che colpisce 3-4 volte di più gli uomini, la sua diffusione tra le donne
non deve essere sottovalutata. Tra le donne, il cancro alla
vescica è l’11° tra le forme di tumore maligno più comuni:
ogni anno in Italia si registrano 15.987 nuovi casi di cancro
alla vescica tra gli uomini e 3.326 tra le donne. Ogni anno in
Europa si registrano circa 120.000 nuovi casi di cancro alla
vescica e il numero è in aumento.
116
Il cancro alla vescica colpisce soprattutto in età avanzata,
viene diagnosticato in prevalenza verso 65-70 anni». Tra le
possibili cause riveste un ruolo rilevante il fumo di sigaretta.
Questa associazione epidemiologica è stata notata per la prima
volta nel 1950. Si stima che siano necessari 20 anni di fumo
per sviluppare il cancro alla vescica. Inoltre, la frequenza con
cui si fuma è direttamente correlata alla probabilità che si
sviluppi il tumore. Tuttavia tutti i fattori scatenanti non sono
ancora completamente chiari. Mentre l’esposizione a sostanze
cancerogene, per lavoro o stili di vita, ha un ruolo rilevante.
Non è ancora stata è accertata una connessione con fattori
ereditari.
Gli strumenti per la diagnosi. La cistoscopia e la citologia urinaria sono entrambe utilizzate nella diagnosi e nel
monitoraggio del cancro alla vescica, da diverso tempo. La
cistoscopia consente l’ispezione visiva diretta dell’urotelio
e della mucosa. La biopsia con cistoscopia, ovvero la resezione transuretrale (TUR) di lesioni e aree sospette, rimane la
procedura standard per il rilevamento del cancro vescicale,
compreso il carcinoma in situ (CIS). Tuttavia, tale metodica
presenta alcune limitazioni nell’individuazione delle lesioni
piatte, come il CIS, che possono essere diffuse e non distinguibili rispetto alla mucosa normale o caratterizzata da un
quadro infiammatorio aspecifico. Tale approccio, tuttavia,
è di dubbio vantaggio, in quanto il rilevamento avviene su
base casuale ed è limitato. I tumori transizionali della vescica
presentano, rispetto ad altre neoplasie, caratteristiche peculiari
che ne favoriscono lo studio dal punto di vista biologico e
soprattutto diagnostico. I tumori, infatti, vengono a trovarsi
in continuo contatto con l’urina, nella quale possono essere
rilasciate notevoli quantità di cellule. In linea teorica risulta
pertanto possibile ottenere materiale biologico senza ricorrere
ad approcci invasivi.
Diagnosi. Nei tumori superficiali della vescica la caratterizzazione biologica può trovare applicazione in tre diversi scenari:
1) per migliorare l’accuratezza diagnostica nei soggetti in cui
si sospetta, per la prima volta, una neoplasia;
2) nello screening dei soggetti a rischio, ad esempio quelli
esposti a noti carcinogeni quali le amine aromatiche, la ciclofosfamide, l’irradiazione pelvica;
3) nel controllo dei soggetti che hanno già subito un trattamento endoscopico di tumori transizionali, allo scopo di
migliorare l’accuratezza diagnostica e diminuire la frequenza
di esami invasivi costosi quali la cistoscopia.
Sino ad oggi, la citologia urinaria ha rappresentato lo
standard di riferimento per tutti gli altri test diagnostici.
L’esame citologico comunque risulta fortemente dipendente
dall’esperienza dell’osservatore e pertanto è difficilmente
standardizzabile e fornisce scarse informazioni nel caso di
tumori a basso grado. L’esame cistoscopico presenta una più
elevata specificità, la quale però non è mai assoluta anche in
mani esperte in quanto l’accuratezza diagnostica dipende da
numerosi fattori quali il tempo di osservazione, la tolleranza
del paziente, l’eventuale sanguinamento indotto e la presenza
di alterazioni infiammatorie della mucosa vescicale. La cistoscopia presenta, inoltre, gli svantaggi di essere un esame
relativamente invasivo, anche nella versione con strumenti
flessibili, costoso e non scevro di complicanze, quali infezioni
urinarie (10% circa) e stenosi uretrali nel maschio.
L’uso della cistoscopia a fluorescenza appare incrementare ulteriormente l’accuratezza diagnostica, particolarmente
per quanto riguarda il carcinoma in situ. Nel corso degli
ultimi anni sono stati messi a punto numerosi test per migliorare l’accuratezza diagnostica delle neoplasie vescicali e
l’industria biomedica continua a proporne di nuovi.
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Tali test possono essere suddivisi in 2 fondamentali categorie:
1) quelli ad interpretazione rapida, che sono anche di semplice esecuzione tecnica e che vengono effettuati e valutati
dall’operatore sanitario al momento della visita del paziente,
con produzione di un campione di urina;
2) quelli ad interpretazione differita, che richiedono l’invio
in laboratori specializzati e l’impiego di tecnologie più o
meno complesse.
Del primo gruppo fanno parte i test BTA (bladder tumor
antigen) stat (frazione H del complemento), UBC (urinary
bladder cancer) rapid (citocheratine 8-18) e FDP (fibrinogen
degradation products), mentre nel secondo si trovano BTA
trak, NMP22 (nuclear matrix protein 22), telomerasi e FISH
(fluorescenza in situ di cromosomi). Non tutti i test sono attualmente utilizzati nel nostro Paese.
I valori globali di sensibilità e specificità dei vari marcatori
urinari più frequentemente utilizzati nella diagnostica delle
neoplasie vescicali ed i rispettivi intervalli di confidenza al
95% sono riportati in Tabella 1 (Lotan, Silvestrini:Linee
Guida per marcatori 2008).
Tab. I. Valori globali di sensibilità e specificità dei vari marcatori urinari più frequentemente utilizzati nella diagnostica delle neoplasie
vescicali ed i rispettivi intervalli di confidenza al 95%.
Marcatore
Citologia
BTA stat
FDP
UBC
BTA trak
NMP 22
Telomerasi
FISH
N.
studi
18
10
4
2
4
15
2
2
N. casi Sensibilità
%
1255
34 (20-53)
938
71 (57-82)
221
77 (41-93)
107
66 (50-79)
394
69 (55-80)
834
73 (47-87)
104
77 (53-91)
262
75 (71-80)
N.
casi
1512
1596
605
313
743
1579
168
262
Specificità
%
99 (83-99)
73 (61-82)
87 (77-94)
91 (84-96)
90 (38-88)
80 (58-91)
79 (46-99)
89 (85-94)
Occorre inoltre tenere presente che condizioni associate,
come ad esempio la presenza di infezione urinaria, pregresso
trattamento con BCG, ematuria, ipertrofia prostatica benigna,
possono rendere inaffidabili le informazioni fornite dai test.
Risulta dalla letteratura assai evidente che i nuovi test diagnostici superano in sensibilità, ma non in specificità, la
citologia urinaria, particolarmente per tumori di basso grado
e stadio. Per contro, la sensibilità dei vari marcatori urinari
non è sufficientemente alta da sostituire la cistoscopia come
standard diagnostico di riferimento. Effettuando simultaneamente vari test sugli stessi pazienti, quello che presenta la
migliore combinazione di sensibilità e specificità è la valutazione dell’attività della telomerasi nelle cellule del sedimento
urinario mediante RT-PCR (Ramakumar et al., 1999). In studi
recenti, pilota e confirmatori, è stata ribadita l’accuratezza diagnostica dell’attività telomerasica in termini sia di sensibilità
(90%) che di specificità (94%) nella popolazione maschile
con età inferiore a 75 anni (Sanchini et al., 2004). Tale accuratezza è stata osservata anche nei pazienti con citologia
negativa e con tumori a basso grado.
Ruolo della citologia. Pertanto, la citologia urinaria resta tuttora fondamentale per la diagnosi precoce di tumori ad alto
grado, mentre il saggio dell’attività telomerasica migliora la
sensibilità diagnostica nelle neoplasie a basso grado.
Le cellule esfoliate possono essere utilizzate anche per indagini in citometria a flusso, per valutare il grado di ploidia
(più frequentemente alterato nelle forme ad alto grado) o la
presenza di modificazioni cromosomiche, in citometria per
immagini (ICM) o a scansione laser. Attualmente i pazienti
117
sessioni scientifiche
affetti da neoplasie vescicali superficiali vengono sottoposti
a controlli cistoscopici periodici preceduti da esame citopatologico delle cellule del sedimento urinario (citologia urinaria),
su tre campioni. Esistono varie opzioni di frequenza delle
cistoscopie in assenza di recidive, generalmente effettuate ad
intervalli trimestrali per il primo anno, semestrali per i seguenti due anni ed annuali per altri due. Controlli trimestrali
più prolungati si rendono necessari in pazienti con neoplasie
di alto grado e nel carcinoma in situ.
La citologia urinaria presenta specificità e sensibilità elevate
nell’individuazione delle lesioni di alto grado, rappresentando
quindi lo strumento non invasivo d’elezione per la diagnosi
del cancro alla vescica.Possono anche essere utilizzati i marker urinari, che sembrano offrire una sensibilità più elevata,
soprattutto rispetto ai tumori di basso grado non infiltranti,
pur mancando della specificità della citologia. Gli svantaggi
principali della citologia e dei marker urinari sono che i risultati non sono immediatamente disponibili, sono altamente
dipendenti dall’operatore e non forniscono informazioni circa
la localizzazione e la gravità della patologia.
La processazione delle urine nei laboratori nazionali, a seguito
di una indagine condotta nella Regione Emilia Romagna nei
Servizi di Anatomia Patologica segue metodologie eterogenee
(Atti Convegno SICi Mirandola 2008) e lo stesso dicasi per
quello che riguarda la refertazione che, pur riflettendo omogeneamente le difficoltà diagnostiche riscontrate, usa terminologie diversificate e non sempre di univoca interpretazione.
Per quanto riguarda la RER (Tab. II) è stata eseguita una prima
indagine sulle modalità tecniche di esecuzione dei preparati:
Tab. II.
Città
Allestimento
Altri test
Cytoslip
–
X3
Cytoslip
–
X3
Pool (2/1 vetrino)
–
5.000
X3
Strato sottile
FISH (450)
Forlì
2.350
X3
Filtro Millipore
–
Imola
3.200
X3
Millipore/Pool
–
Mirandola
3.670
X3
Thin Prep
–
Cytospin
–
Bologna
Prestazioni
15.000
X3
Sant’Orsola
3.450
Cesena
3.000
Ferrara
(tutta)
Piacenza
Ra-Fa-Lu
10.000
X3
Filtro Millipore
–
Reggio E.
1.500
X3
Thin Prep
–
Rimini
3.450
X3
Filtro Millipore
–
Modelli di refertazione e ceck –list sono stati fornitiSant’Orsola
Maggiore: Dott.ssa R. Rapezzi Dott. P.Chieco
Cesena: Dott.sse E. Elegibili I. Lucchi
Ferrara: Dott.ssa M.D. Beccati
Forlì: Dott.ssa L. Medri
Imola: Dott.ssa D. Ghidoni
Mirandola Dottori C. Ghirardini, O. Raisi
Piacenza: Dott. Raoul P. Foroni
Ravenna
Reggio Emilia Dott.sse M. Ferrari I. Tamagnini
Rimini: Dott.ssa P. Fulgenzi
È pertanto nata la necessità di approfondire il confronto tra professionisti delle Società SICi, SIAPEC, SIURO
nell’intento di formulare all’interno di un gruppo di lavoro
intersocietario delle Linee Guida per la Citologia Urinaria.
Questa difformità di tecnica e refertazione supportata dalla
verifica di analoghe situazioni in altre regioni italiane ha portato quindi ad un primo incontro (2008) tra le suddette Società
Scientifiche, che occorre assolutamente riprendere, alla luce
peraltro di cambiamenti di metodica tecnica già avvenuti (uso
di urine prefissate), e verificati gli interessanti studi su fattori
prognostici e predittivi di recidiva tumorale.
Obbiettivo del gruppo di lavoro è di creare Linee guida che
portino alla identificazionedi un gruppo di pazienti ad alto
rischio, specialmente nelle forme iniziali, per intervenire più
efficacemente in questi e risparmiare da interventi terapeutici quelli a basso rischio, garantendo una migliore qualità
di vita. Inoltre consigliare la caratterizzazione biologica, con
parametri come P53, micro-satelliti, telomerasi EGFR, indici
di proliferazione, per fornire elementi nuovi da associare a
quelli clinico-istopatologici per definire in modo più personalizzato, cioè legato al singolo tumore, la aggressività e/o la
responsività al trattamento.
Presento come primo prodotto del Lavoro del gruppo i risultati
di un Questionario inviato a livello Nazionale. Quest’ultimo ci
fornisce un panorama e lo stato dell’arte su questo importante
argomento della diagnostica citologico. Pertanto occorrerà integrare il lavoro gia svolto, che seppure in questa fase iniziale
potrà fare chiarezza e fornire un percorso diagnostico il più
possibile omogeneo alla citologia urinaria.
Venerdì, 29 giugno 2012
ore 8.30-10.30
Statistica applicata agli screening
Chairmen: Lucio Torelli (Trieste), Arrigo Bondi (Bologna)
Statistical methods for the evaluation of
screening programmes: state-of-art
P. Pasqualetti
SeSMIT-Service of Medical Statistics and Information Technology,
AFaR-Fatebenefratelli Association for Biomedical Research, Rome,
Italy
Background/introduction. The appropriate statistical methods for the evaluation of screening programmes are long since
available. However some issues are still controversial and
recently proposed biostatistical methods could help to take
more correct decisions.
Materials and methods. Issues under debate were identified
through experts’ opinions, biomedical literature (PubMed and
118
Web-of-science) and specific web-sites (www.osservatorionazionalescreening.it, www.cancerscreening.nhs.uk, www.
cdc.gov). Methodological papers addressing such issues were
selected among those published in the biomedical literature
and particularly in biostatistical/epidemiological journals.
Results. A first issue is the effect size of screening on the
reduction of mortality. Although this topic has been largely
investigated, there is still a debate about the proportion of the
total reduction in the rate of death attributed to screening. The
comparison between two conflicting studies 1 2 is presented
and discussed with respect to the methodological approach
described by Seppänen J et al. Predicting impacts of massscreening policy changes on breast cancer mortality 3.
A second issue is how to optimize the identification of target
populations for screening. A method and its application to
colorectal cancer is presented 4.
A third issue concerns the comparison between multiple
diagnostic tests and sequential designs. The methodological
papers used for this purpose are Wruck et al. 5 and Qin and
Zhang 6.
Conclusion. Some recent and rigorous methods were presented and discussed to face three controversial issues about
evaluation and usefulness of screening programmes. This
review could help to properly address the problems and to
support the decisional processes.
References
1
Berry DA, Cronin KA, Plevritis SK, et al. Effect of screening and
adjuvant therapy on mortality from breast cancer. N Engl J Med
2005;353:1784-92.
2
Gøtzsche PC, Jørgensen KJ, Zahl PH, et al. Why mammography
screening has not lived up to expectations from the randomised trials.
Cancer Causes Control 2012;23:15-21.
3
Seppänen J, Heinävaara S, Hakulinen T. Predicting impacts of
mass-screening policy changes on breast cancer mortality. Stat Med
2008;27:5235-51.
4
Janes H, Pepe M, Kooperberg C, et al. Identifying target populations for screening or not screening using logic regression. Stat Med
2005;24:1321-38.
5
Wruck LM, Yiannoutsos CT, Hughes MD.et al. A sequential design
to estimate sensitivity and specificity of a diagnostic or screening test.
Stat Med 2006;25:3458-73.
6
Qin J, Zhang B. Best combination of multiple diagnostic tests for
screening purposes. Stat Med 2010;29:2905-19.
Mammography screening in Trieste: data quality,
assessment of indicators and the impact on the
population
L. Torelli, G. Barbati*, M. Borelli, F. Zanconati*, F. Giudici*
Department of Mathematics and Geoscience, University of Trieste; *
Department of Medical, Surgery and Health Sciences, University of
Trieste, Italy
Introduction. In this Section, “Statistics for screening”, we
want to emphasize that it is more and more important to use
statistics to understand, to analyze and to evaluate the data of
a screening, in order to describe the population and to take
decisions and, even before, to learn to set and design a screening itself.
It is important, though, to use this tool, statistics, properly.
It is not uncommon to find errors, even coarse ones, leading
to incorrect results: for instance when parametric statistic
methods are used instead of nonparametric ones, or when we
read μ ± σ in non-symmetric situations. At the same time, for
instance, it is important to know that the same diagnostic test
can lead to different predictive values Vp+ and Vp- when used
for a screening population or for a population with symptoms.
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
As highlighted by the title, using results from the project of
mammography screening in the Province of Trieste, we want
to focus in particular on the importance of
• the quality of the data: it is not possible to make correct statistics without it, and in this talk we will show an example
of the correction of the data concerning the blood group;
• the evaluation of the indicators: it is important to decide
which are the ‘informative indicators’, and how it is possible to read the output in a critical way;
• the impact of a screening on a population.
In this context it is evident the importance of a multidisciplinary work, and this is the experience started some years
ago in our local reality, with the involvement of pathologists,
surgeons, radiologists, radiotherapists, oncologists, physicists,
mathematicians, statisticians.
Materials and mathods. The analyzed data refer to women
(age 50-69 years) who have responded to the mammographic
screening program active in Trieste since 2006. As far as
radiological and cyto-histological data they are extracted
from computer databases of screening program and pathologic anatomy and they have been computerized. Also other
information, obtained from the questionnaire proposed to
the women during the screening exam, were converted in
Excel format. This interview gives information about the age,
weight, height, blood type, hormonal history, and lifestyle of
women aiming to assess the possible risk factors for breast
cancer.
The first problem in any statistical analysis concerns data
quality: correspondence between the data indicated by the patient and the ones found in computer databases was evaluated.
The evaluation of the screening program using appropriate
indicators was made only after the correction and integration
of the missing data. In particular, we determined sensitivity,
specificity and predictive values of diagnostic tests used in
screening in the different phases of the program: mammography (first level) and cytology (second level).
We wanted to compare the pre-screening period (biennium
2004-2005) with the second round of screening (period 20082009) with the aim to evaluate a possible positive impact in
terms of early cancer’s detection.
The information obtained from the questionnaire related to the
presence (cases) or absence (controls) of the tumor, was used
to assess the possible exposure to risk factors for breast cancer
in the female population of Trieste.
Results. The data quality is a necessary condition in order to
perform accurate and reliable statistical analysis: according to
our experience, without cleaning and integration of data, it is
possible to get to wrong conclusions. A significant example is
the following: before the revision of the data, women with the
factor Rh- seemed to be at risk for developing breast cancer,
indeed this result is wrong, because the correction of the data
has led to an opposite true result, that is, Rh + is a risk factor
(OR = 1.42, CI [1:11 to 1:48]).
Second, a statistical analysis without a good critical sense and
interpretation of the data is meaningless: a number, a percentage or a p-value, do not say anything if they are not related
to their specific context. The indicators (sensitivity, specificity, …) should be interpreted carefully: they give different
answers depending on the type of screening test to which the
women are subjected. Moreover, the predictive values are influenced by the prevalence of the disease and therefore should
be interpreted according to this factor.
The quantitative and qualitative impact of screening mammography is demonstrated by comparing breast cancer (and
their respective stages) in the period before the beginning
119
sessioni scientifiche
of the program (2004-2005), with those found in the same
population during the second round (2008-2009): the screening program has not only identified a greater number of breast
cancers within the same range of age (85 more malignant
lesions were found), but it has also allowed the very early diagnosis of breast cancer (in 2004-2005 tumors less than 1 cm
in diameter are 28% while in the second round of screening
they are increasing to 39%).
Conclusions. Statistics applied to screening aims to the evaluation of programs, reporting the results that express both their
potential and limits.
Only a multidisciplinary approach, in which each figure offers
their knowledge, can contribute to the success of a program,
and statisticians, together with the pathologists, surgeons, radiologists, radiotherapists and oncologists can provide a more
reliable interpretation of the analysis.
Venerdì, 29 giugno 2012
Ore 11.00-13.00
Versamenti
Chairmen: Ambrogio Fassina (Padova), Domenico Ientile (Palermo)
Diagnosis of serous effusions in cancer
B. Davidson
Senior Consultant, Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
Associate Professor, the Medical Faculty, University of Oslo, Oslo,
Norway
Background. The serosal cavities are a common site of cancer
metastasis, and essentially any tumor type has been detected
at this anatomic site. Ovarian, breast, lung and gastrointestinal
carcinomas are the most frequent organs of origin. In addition,
the serosal cavities are the site of origin of malignant mesothelioma and primary peritoneal carcinoma. The formation
of malignant effusions is generally a clinical manifestation
of advanced-stage cancer. Effusions are not infrequently the
only material available for diagnosis in recurrent cancer. The
detection of cancer cells in effusions based on morphology
alone has been shown to be associated with poor sensitivity.
Materials and methods. In recent years, others and we have
applied a battery of ancillary techniques as adjuncts to
morphology in effusion diagnosis, including immunohistochemistry, flow cytometry, colorimetric or fluorescent in situ
hybridization, PCR-based assays, Western blotting, traditional
cytogenetics, comparative genomic hybridization (CGH) and
gene expression arrays.
Results. All the above-techniques have been shown to be useful in this setting, and studies have identified a large number
of novel markers at the DNA, mRNA or protein levels which
may aid in confirming the presence of malignant cells in effusions, as well as provide information regarding the organ of
origin. These data will be discussed during this presentation.
Conclusions. Applying current technology, the large majority
of effusions may be correctly classified with respect to the
presence of malignancy and tumor type, provided satisfactory
material in terms of both viability and cell number is submitted for evaluation. Data regarding rare tumors are, however,
incomplete, and require multi-institutional studies in order to
generate meaningful data.
References
1
Davidson B. Malignant effusions: from diagnosis to biology. Diagn
Cytopathol 2004;31:246-54.
2
Davidson B. The diagnostic and molecular characteristics of malignant mesothelioma and ovarian/peritoneal serous carcinoma. Cytopathology 2011;22:5-21.
3
Davidson B, Firat P, Michael CW. Serous Effusions- Etiology, Diagnosis, Prognosis and Therapy. London, UK: Springer 2012.
Cytological features of primary effusion
lymphoma (PEL), pseudo-pel and HHV8unrelated effusion lymphomas: 18-year
experience from a single academic center
V. Ascoli
Dipartimento di Scienze Radiologiche, Oncologiche e Anatomo-Patologiche, Università Sapienza, Roma
Background. Primary effusion lymphoma (PEL) is a nonHodgkin’s lymphoma (NHL) characterized by recurrent effusions and liquid-phase growth of lymphoma cells within
serous body cavities, often in the absence of a solid tumor.
Since 2001, the WHO classification recognizes PEL as a
unique entity of NHL 1. In PEL, the tumor clone is infected by
human herpesvirus-8 (HHV8)–the etiologic agent of Kaposi’s
sarcoma (KS) and may be co-infected by Epstein-Barr virus
(EBV). Most cases of PEL arise in setting of immunodeficiency, usually AIDS. In the general population, PEL selectively
affects aged individuals, from geographical areas at high
prevalence of HHV8 infection such as the Mediterranean area,
including Italy 2. Intriguingly, a case of PEL ante-litteram in
a male patient with KS has been described in the Giornale
Italiano di Dermatologia e Sifilologia in 1930, 60 years before
the discovery of HHV8 within KS lesions in 1994.
Effusions containing HHV8-infected cells represent a heterogeneous group of lesions including PEL (monoclonal)3 and
pseudo-PEL (polyclonal) 4. PEL must be differentiated from
other liquid-phase lymphomas not related to HHV-8 3 5.
PEL and HHV8-unrelated effusion lymphomas are quite rare
and must be distinguished from secondary lymphomatous effusions occurring by direct extension or by lymphatic spread
from a tissue-based NHL.
The aims of this article are: 1) to describe the spectrum of primary effusion lymphomas and its mimics; 2) to highlight the
central role of cytology in the diagnostic identification of such
rare tumors; 3) to report the case series observed in a single
academic institution during about two decades.
Pel. The diagnosis of PEL is typically based on the cytological examination of body fluid. Detecting evidence of HHV8
viral infection within neoplastic cells is essential for a final
diagnosis. PEL tumor cells show the following characteristics: 1) large-cell morphology with immunoblastic/anaplastic
or plasmablastic/plasma-cell features); 2) negative stain for
B-/T-cell-associated antigens (null/indeterminate phenotype),
positive for CD45 (> 90% of cases), and several plasma
120
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
cell markers5, and positive stain for latent nuclear antigen-1
(LANA) with typical punctate intranuclear dots; 3) tumor
cell DNA exhibits clonal rearrangements of the heavy immunoglobulin (Ig) genes consistent with a monoclonal B-cell
population; 4) mutations of the Ig variable genes and of the
proto oncogene BCL-6, and lack of rearrangements of BCL-1,
BCL-2, BCL-6, and c-MYC proto-oncogenes; 4) latent HHV8
infection (20–50 viral copies per cell); 5) co-infection by EBV
(in about 70–80% of AIDS-PELs; rare in non-AIDS PEL).
The postulated normal cell(s) counterpart of PEL is presently
unknown but a B-cell that has reached a late differentiation
B-cell stage is supposed. Aberrant expression of T cell or B
cell markers can be observed in PEL.
Pseudo-Pel. Recurrent HHV8-positive body cavity effusions other than PEL may occur. Fluids are characterized
by: 1) presence of HHV8, with high viral load, and very
rare co-infection by EBV; 2) low fraction of LANA-positive
lymphomononuclear cells in association with polymorphous
inflammatory-type background of LANA-negative leukocytes
(abundant T-cells, few B-cells, monocytes/macrophages, and
plasma cells); 3) B-cell/T-cell policlonality by molecular
analysis 4.
HHV8-unrelated effusion lymphomas. After the discovery
of PEL, several reports have been published on the occurrence of lymphomatous effusions not related to HHV-8 and,
like PEL, lacking a tumor mass 3 5. They have been named
with different terms such as HHV8-negative body cavity
lymphomas, HHV8-unrelated PEL-like lymphoma, HHV8
independent PEL, HHV8-unrelated effusion lymphomas.
This entity is sometimes also referred to as HHV8-unrelated
large-cell B-cell lymphomas because of the differences
observed in its pathogenesis, morphology and immunophenotype (it expresses a B-cell phenotype unlike PEL). Alternatively, HHV8-unrelated effusion lymphomas may exhibit
Burkitt or Burkitt-like morphology, c-myc proto-gene rearrangements and CD-10 positivity 3 5. HHV8-unrelated effusion lymphoma cases may be associated with hepatitis C
virus (HCV) 3.
Materials and methods. Primary lymphomatous body cavity
effusions without a solid-tissue component diagnosed in a single academic institution (Anatomia Patologica, Azienda Ospedaliera Policlinico Umberto I, Università Sapienza, Roma)
in the last 18 years (period 1994 - April 2012) are included
in this study. An additional group of effusions (pseudo-PEL)
not fulfilling the diagnostic criteria of PEL are also included.
Results. Table I shows the clinical characteristics of the
twenty-three cases of this series: 14 cases of PEL, 6 cases of
pseudo-PEL and 3 cases of HHV8-unrelated effusion lymphomas.
PEL – These cases are characterized by: (i) presence of a
frank lympoma by cytomorphology with a spectrum of cytomorphologic features: cellsshow immunoblastic/anaplastic
or plasmablastic features (CD45 positive but B-cell- and Tcell-associated antigen negative); (ii) presence of a dominant
clone by B-cell clonality assessment (clonal rearrangement of
the IgH gene); (iii) presence of HHV8-infected cells (positive
LANA staining in the nuclei of neoplastic cells).
Pseudo-PEL – These cases are characterized by: (i) absence
of a frank lymphoma by cytomorphology; smears contain an
heterogeneous population of macrophages, neutrophils, eosinophils, small lymphocytes, mesothelial cells and atypical
lymphoid cells with plasmacytic/plasmablastic features; (ii)
absence of a dominant clone by B-cell clonality assessment;
PCR analysis discloses a polyclonal pattern of the IgVH (6
out of 6 tested), IgVL and TCR (3 out of 3 tested) genes; (iii)
presence of HHV8-infected cells; atypical lymphoid cells
co-express CD138 and the LANA-1 latent HHV8 nuclear
antigen.
HHV8-unrelated effusion lymphomas – These cases are characterized by: (i) presence of a frank lympoma by cytomorphology with a monomorphous population of medium-size
lymphoid cells with irregular, indented nuclei and light-blue
cytoplasm in 2 cases (CD3-negative, CD20-positive, and Ki67-pos (>80%)); the third case is characterized by small-size
noncleaved lympoid cells (CD20 positive, CD24 positive,
CD10 positive); this case shows t(8; 22) (q24; ql 1) translocation; (ii) presence of a dominant clone by B-cell clonality assessment (clonal rearrangement of the IgH gene); (iii) absence
of HHV8-infected cells (negative LANA staining)
Conclusions. The first suspicion of primary lymphomatous
effusions is by fluid cytology. The presence of a primary solid
NHL must be excluded. On a cytomorphologic basis alone, it
is impossible to differentiate primary lymphomatous effusions
from more frequent secondary lymphomas. When a primary
effusion lymphoma is suspected, HHV-8 detection is required
to distinguish between PEL (HHV8-related) from HHV8unrelated effusion lymphomas. The final diagnosis requires
the molecular proof of clonality.
PEL develops in the setting of immunodeficiency or in the
elderly. Since Italy is a country wherein HHV8 infection is
endemic in the general population (especially in some areas
such as Sardinia, Sicily and the Po valley), it is important to
know this rare entity and to recognize it by fluid cytology.
References
1
Banks PM, Warnke RA. Primary effusion lymphoma. In: World Health
Organization Classification of Tumours, Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues (ed. by Jaffe ES, Harris
NL, Stein H, Vardiman JW). Lyon: IARC Press 2001, pp. 179-80.
2
Ascoli V, Lo-Coco F, Torelli G, et al. Human herpesvirus 8-associated
primary effusion lymphoma in HIV-negative patients: a clinico-epidemiologic variant resembling classic Kaposi’s sarcoma. Haematologica
2002;87:339-43.
3
Ascoli V, Lo-Coco F. Body cavity lymphoma. Curr Opin Pulm Med
2002;8:317-22.
4
Ascoli V, Calabrò ML, Giannakakis K, et al. Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8-associated polyclonal body
cavity effusions that mimic primary effusion lymphomas. Int J Cancer
2006;119:1746-8; author reply 1749-50.
5
Carbone A, Gloghini A. PEL and HHV8-unrelated effusion lymphomas: classification and diagnosis. Cancer 2008;114:225-7.
Tab. I. Primary effusion lymphoma (PEL), pseudo-PEL and HHV8-unrelated effusion lymphomas.
Diagnosis
PEL (n=14)
Pseudo-PEL (n=6)
HHV8-unrelated effusion lymphomas5 (n=3)
Gender
Viral status of patients
M
F
HHV8-pos
HIV-pos
HIV-neg
HCV-pos
HBV-pos
14
4
2
0
2
1
14
6
0
10
43
0
4
24
3
2
0
1
4
0
1
Median age at diagnosis: 141 years; 276 years; 339 years; 447 years; 572 year
1
2
121
sessioni scientifiche
Detection of soluble mesothelin in pleural
effusion as a diagnostic marker of malignant
pleural mesothelioma: its contribution to
cytology
F. Fedeli1, P. Ferro1, P.A. Canessa2, E. Battolla3, P. Dessanti1,
B. Baccigalupo1, M.C. Franceschini1, V. Fontana4, M.P. Pistillo5, S. Roncella1,6
Division of Histopathology and Cytopathology; 2 Division of Pneumology, 3 Division of Clinical Pathology ASL5, La Spezia, Italy; 4 Epidemiology, Biostatistics and Clinical Trials Unit and 5 Tumor Genetics and Epigenetics Unit, IRCCS A.O.U. San Martino – IST– Genova,
Italy; 6 AIL F. Lanzone, La Spezia, Italy
1
Introduction. Malignant pleural mesothelioma (MPM) is
an aggressive tumor with poor prognosis showing a median
survival of less than one year post diagnosis.
Frequently, the differential diagnosis of MPM is often difficult because the clinical signs of MPM are non-specific.
The primary manifestation of MPM is often represented by a
pleural effusion (MPM-PE). However, PE is a common event
in pulmonary disorders which can be associated with a large
variety of diseases.
PE is obtained by performing pleural aspiration by thoracentesis and cytology (Cyt) using Papanicolaou staining which is
the most informative laboratory test for the diagnosis of PE.
Unfortunately, Cyt does not often lead to a precise diagnosis
of MPM-PE and its sensitivity is about 30% 1.
MPM is one of the major health priorities in our country. The
Province of La Spezia (Italy) is an area at high risk of MPM. It
is estimated that the incidence rate is about 15/100.000 against
about 5/100.000 inhabitants observed in other parts of Italy. In
addition, it is expected that the incidence of MPM in our area
will continue to increase for several years 2.
Therefore, it is extremely important for us to assess the clinical relevance of new biomarkers, in our local patients.
We previously studied Human mammaglobin (hMAM) as
a marker for diagnosis of malignant PE and MPM-PE. In
this regard, we developed a sensitive and specific nested
RT-PCR assay for amplifying the hMAM mRNA transcript
and demonstrated that addition of hMAM RT-PCR to the
Cyt increased the diagnostic cancer rate by 32%. We also
demonstrated that hMAM transcript was expressed in MPM.
Moreover, the patients with negative thoracoscopic biopsy,
but showing positivity of PE for hMAM mRNA, had an increased relative risk of cancer, including MPM, as compared
to the patients showing hMAM-negative PE 3.
Soluble mesothelin (SM) 4 originates from mesothelin, a 40-KD
cell surface glycoprotein, expressed by normal mesothelial cells
and over-expressed in various cancers such as MPM, pancreatic
adenocarcinoma, ovarian adenocarcinoma and others. SM can
be detected in peripheral blood and the evaluation of SM serum
levels has been approved by the U.S. Food and Drug Administration for disease diagnosis and monitoring in MPM.
In the present study, we assessed the SM levels in MPM-PE
by using an ELISA detection system and assessed whether it
can have an additional value to Cyt.
Material and methods. This study included 275 patients who
had developed a PE of unknown origin. All PE were analyzed
by Cyt and, when necessary for diagnosis, histology of pleural
biopsies, taken during medical thoracoscopy, was performed.
Fifty two samples were PE from MPM (MPM-PE), 129 were
from different benign diseases (B-PE) and 94 from different
non-MPM metastatic cancers (Mts-PE).
Histology, immunohistochemistry and Cyt were assessed by
standard protocols used in the Division of Histopathology and
Cytopathology (La Spezia, Italy). Cyt was evaluated on fixed
smears stained by Papanicolaou’s method.
SM levels were measured by the “MesoMark” ELISA assay
kit according to manufacturers’ instructions 5.
Diagnostic performance parameters were estimated through
the ROC analysis. Youden’s index was applied to obtain the
cut off level. The degree of correlation of SM in MPM-PE vs
other groups was estimated by diagnostic odds ratio (DOR)
and by P-value (P).
Results. The median SM levels were significantly higher in
MPM-PE (28.2 nM) than in patients with B-PE (3.2 nM), or
with Mts-PE (3.8 nM), as well as in the combination of B-PE
and Mts-PE (3.3 nM).
MPM-PE vs B-PE comparison yielded an area under ROC
curve (AUC) of 0.84 (P < 0.001), MPM-PE vs Mts-PE comparison an AUC of 0.80 (P < 0.001) whereas MPM-PE vs BPE+Mts-PE comparison yielded an AUC of 0.82 (P < 0.001).
The biomarker’s cut-off level of MPM-PE vs each patient
group was 9.30 nM. At this cut-off value, we established Se
= 75.0% with Sp = 93.0%, SP = 80.9%, and SP = 87.9%, for
MPM-PE vs B-PE, vs Mts-PE and vs all other PE respectively.
Using 9.30 nM as the cut-off value, we found SM-positive
cases in 38/52 (73.1%) MPM-PE, in 9/129 (6.9%) B-PE and
in 18/94 (19.1%) Mts-PE.
The higher correlation was seen for MPM-PE vs B-PE (DOR
= 40.0; P < 0.001), followed by MPM-PE vs all other PE
(DOR = 21.8; P < 0.001) and finally by MPM-PE vs Mts-PE
(DOR = 12.7; P < 0.001).
Cyt resulted positive in 15/52 (28.8%) cases, negative in
29/52 (55.8%) cases and it remained suspicious in 8/52
(15.4%) cases (Tab. I).
In the 38/52 (73.1%) MPM-PE showing SM levels ≥ cut-off,
Cyt was positive in 11 (29.0%) cases, negative in 20 (52.6%)
cases and it was considered suspicious in 7 (18.4%) cases
(Tab. 1).
Finally, in the 14/52 (26.9%) MPM-PE showing SM levels <
cut-off, Cyt was found positive in 4 (28.6%) cases, negative
in 9 (64.3%) cases and suspicious in 1 case (7.1%) (Tab. 1).
Tab. I. Comparison between mesothelin detection in mesothelioma
pleural effusion vs cytology.
Mesothelin
Cytology
(cut-off = 9.30)
Positive (%)
Negative (%) Suspicious (%)
Positive (n = 38)
11 (29.0)
20 (52.6)
7 (18.4)
Negative (n = 14)
4 (28.6)
9 (64.3)
1 (7.1)
Total (n = 52)
15 (28.8)
29 (55.8)
8 (15.4)
Discussion. SM detection in serum has been proposed for the
diagnosis of MPM. In our study, we found that SM is significantly elevated also in MPM-PE and most patients with MPM
have increased SM concentrations in PE.
The results of our study confirm that the diagnosis of MPMPE by Cyt has a low sensitivity (Se = 29%) and show that
SM detection in MPM-PE may provide a means for reducing
the delay in diagnosing MPM. In fact, SM detection helps
the diagnosis in 20/29 (70%) Cyt-negative cases whereas it
is confirmatory of the diagnosis in 7/8 (88%) cases of Cytsuspicious MPM-PE.
However, we also found that the levels of SM resulted
lower than the cut-off value in about 4/15 (27%) Cyt-positive
MPM-PE. This finding indicates that SM detection cannot
be alternative to Cyt, but it may offer an additional value for
MPM-PE diagnosis.
122
In addition, we found that positive cases for SM were also
observed in both B-PE (7%) and Mts-PE (19%). This finding
was consistent with previous reports who established that SM
may be found expressed at mRNA and protein levels in various
tumors and on the surface of normal mesothelial cells. Since the
mechanism of SM release is actually unknown, it might be possible that, in some cases of tumors or inflammatory conditions,
SM is released from the cells in the biological fluid. However,
referring to the SM positive cases observed among the B-PE
patient population, the follow-up of at least one year did not
show any clinical evidence of tumors in these patients.
Therefore, an increased level of SM in PE, would not be
regarded by itself as a diagnostic tool, but as a finding which
raises a strong suspicion of MPM-PE (or Mts-PE). Our finding suggests the need for further invasive investigations, such
as thoracoscopy and histology in MPM-PE diagnosis, when it
may be performed.
In conclusion, the PE on which the ELISA test is performed,
is routinely obtained through thoracentesis and represents the
same specimen on which Cyt, biochemical and microbiological
analyses are performed. As a consequence, undertaking SM
analysis, at the same time as other tests, is a simple and easy
event which does not cause further inconvenience to the patient.
Thus, considering the benefits that the quantification of SM
in PE could bring to the diagnosis of MPM, we conclude that
this test can be applied in the workup of patients with a PE of
unknown origin.
Supported by grants from Ricerca Sanitaria Regione Liguria
2009.
References
1
Rakha EA, Patil S, Abdulla K, et al. The sensitivity of cytologic evaluation of pleural fluid in the diagnosis of malignant mesothelioma. Diagn
Cytopathol 2010;38:874-9.
2
Gennaro V, Ugolini D, Viarengo P, et al. Incidence of pleural
mesothelioma in Liguria Region, Italy (1996-2002). Eur J Cancer
2005;41:2709-14.
3
Roncella S, Ferro P, Franceschini MC, et al. Diagnosis and origin
determination of malignant pleural effusions through the use of
the breast cancer marker human mammaglobin. Diagn Mol Pathol
2010;19:92-8.
4
Robinson BW, Creaney J, Lake R, et al. Mesothelin-family proteins
and diagnosis of mesothelioma. Lancet 2003;36:1612-6.
5
Beyer HL, Geschwindt RD, Glover CL, et al. MESOMARK: a potential
test for malignant pleural mesothelioma. Clin Chem 2007;53:666-72.
Biology and clinical relevance of serous effusions
in cancer
B. Davidson
Senior Consultant, Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Associate Professor, the
Medical Faculty, University of Oslo, Oslo, Norway
Background. The serosal cavities are commonly involved by
metastatic disease in ovarian, breast, lung and gastrointestinal
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
carcinoma, and are the site of origin of malignant mesothelioma and primary peritoneal carcinoma. The formation of
malignant effusions is generally a clinical manifestation of
advanced-stage cancer and the detection of tumor cells in effusions precludes a curative approach. Cancer patients with
malignant effusions consequently receive palliative care, and,
with the exception of ovarian/peritoneal carcinoma, benefit
little from conventional therapeutic modalities, such as radiation and chemotherapy. In view of this grim outlook, patients
with malignant effusions critically depend of the development
of targeted therapy. The large number of viable cells obtained
by effusion tapping is most suitable for studying biochemical
markers of tumor progression and for evaluating drug sensitivity and treatment response.
Materials and methods. In recent years, our group has
studied the biology of ovarian/peritoneal and breast carcinoma and malignant mesothelioma cells in effusions, with
focus on comparison of tumor cells in effusions with their
counterparts in primary carcinomas and solid metastases,
as well as pre-chemotherapy (primary diagnosis) vs. postchemotherapy effusions. The techniques applied included
immunohistochemistry, flow cytometry, mRNA in situ
hybridization, PCR, Western blotting, proteomics and gene
expression arrays.
Results. We repeatedly observed differences in the expression
levels of cancer-associated molecules between tumor cells
in effusions with their counterparts in primary carcinomas
and solid metastases, as well as pre-chemotherapy (primary
diagnosis) vs. post-chemotherapy effusions. Furthermore, the
prognostic role of many of these molecules differed at the
different anatomic sites, as well as between pre- and postchemotherapy specimens. In contrast, ovarian carcinoma cells
in peritoneal and pleural effusions appear to be practically
identical. These data will be discussed.
Conclusions. Since the presence of tumor cells in effusions
corresponds to tumor progression and is significantly associated with reduced chances for achieving cure, development
of molecular modalities for treating advanced disease must
include thorough characterization of these cells. By comparing anatomic site-related expression patterns of cancerassociated molecules in tumors affecting the serosal cavities, we can identify general and tumor-specific molecular
changes related to tumor progression and study their clinical
relevance.
References
1
Davidson B. The biological characteristics of cancers involving the
serosal cavities. Crit Rev Oncog 2007;13:189-227.
2
Shih IeM, Davidson B. Tumor-Associated Genes in Ovarian Cancer.
Future Oncol 2009;5:1641-57.
3
Davidson B, Reich R, Trope’ CG, et al. New determinates of disease
progression and outcome in metastatic ovarian carcinoma. Histol
Histopathol 2010;25:1591-609.
4
Davidson B, Firat P, Michael CW. Serous Effusions- Etiology, Diagnosis, Prognosis and Therapy. London, UK: Springer 2012.
123
sessioni scientifiche
Venerdì, 29 giugno 2012
ore 11.00-13.00
Linfonodi e mediastino
Chairmen: Luigi Ruco (Roma), Maurizio Lestani (Arzignano)
Diagnostic cytology of mediastinal masses
M. Lucioni, M. Nicola, G. Fiandrino, M. Paulli
Anatomic Pathology, Department of Molecular Medicine, University
of Pavia/Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
The mediastinum is a complex compartment in the thoracic
cavity, bounded laterally by the pleurae, anteriorly by the
sternum, posteriorly by the vertebral column, superiorly by
the thoracic inlet and inferiorly by the diaphragm. It contains
a large number of organs and structures and therefore can be
the site of a broad range of tumors, of which about 40% are
malignant, both primary and secondary. Most mediastinal
lesions, both benign and malignant, present as asymptomatic
masses found on chest radiography. When symptomatic, these
lesions may have a dramatic presentation, including pain,
dyspnea, cough and, at least in some cases, superior vena cava
syndrome.
Age plays an important role in the correct diagnosis of tumors arising in this site, because very different lesions are
expected in childhood as compared to adulthood. Most tumors
in children are primary mediastinal lesions, including neurogenic tumors, specifically neuroblastoma and ganglioneuroblastoma, germ cell tumors and lymphomas. In adulthood,
the most common mass lesions are metastases and cysts of
thymic, pericardial or enteric origin, followed by thymomas,
neurogenic tumors, lymphomas and germ cell tumors. Among
adulthood secondary mediastinal malignancies, small cell
carcinoma of lung is the most frequent metastasis, followed
by non-small cell tumours of the lung and breast carcinoma.
Knowledge of the precise tumor location within the mediastinal
compartments is useful to narrow the list of possible differential
diagnoses. Lesions of the anterior/superior mediastinum are
more likely to be thymomas, thymic cysts, lymphomas, thyroid
lesions and germ cell tumors; the middle mediastinum is the
site of lymphoma and benign pericardial or bronchogenic cysts,
whereas nerve sheath or neuronal tumors and bronchogenic
cysts are most commonly located in the posterior mediastinum.
Clinical and laboratory findings are often helpful. High levels
of circulating human chorionic gonadotrophin and alphafetoprotein are found in germ cell tumors. Numerous clinical
syndromes are associated with thymoma, including a range of
autoimmune diseases and haematopoietic disorders.
Although the clinical and radiological findings are useful to
define the origin of mediastinal masses, a tissue diagnosis is
needed in proper guidance of their management. Fine needle
aspiration (FNA) cytology has been used to investigate spaceoccupying lesions of the mediastinum and represents, in
many centers, the first line diagnostic method after imaging.
FNA aspiration is less invasive than bioptic and/or surgical
approach and it has a low incidence of complications, including pneumothorax, hemothorax, local hemorrhage, and
pain. Nevertheless the diagnostic role and value of FNA in
the work-up of mediastinal tumors is still a matter of debate
because of the complexity and difficulty in making an exact
histologic tumor subtyping even on surgical biopsy material.
As many diagnostic challenges may be encountered in this
specific field of cytopathology, the accuracy of the cytologic
identification of mediastinal masses largely relies on the cytopathologist’s experience. In order to make it easier, Geisinger
divided mediastinal tumors into categoriesaccording to the
cytomorphologic pattern observed in FNA aspirates. As expected small cell patterns were usually seen in lymphomas,
carcinoid, and small cell carcinoma. Polygonal or epithelioid
patterns were observed in thymomas, germinomas, embryonal carcinoma, and metastatic carcinoma. Based on a large
multi-institutional series of 189 cases, Powers et al. reported a
sensitivity of 87% and a positive predictive value of 97% for
a diagnosis of neoplasm. Singh et al reviewed materials from
the same institutions, focusing on cases (6%) with discordant
cytology and follow-up histology. These included small cell
carcinoma resembling lymphoma, Hodgkin disease and large
cell lymphoma with prominent spindle cell components, large
cell lymphoma diagnosed as Hodgkin disease, thymoma mimicking large cell lymphoma, ectopic epithelial tissues raising
the possibility of metastatic tumors, and clear cell adenocarcinoma resembling germ cell tumors.
Based on these data the subclassification of small cell malignancies, including the possible misdiagnosis of small cell
carcinoma for lymphoma, and the separation of germ cell
tumors from adenocarcinoma appear to be major areas of diagnostic challenge on FNA aspirate. These diagnostic dilemmas, whose therapeutic relevance is of paramount importance,
may be solved only by means of tissue architecture evaluation
and immunohistochemical characterization, based on many
different lineage markers.
Some lymphoid malignancies, including classic Hodgkin lymphoma, mediastinal large B-cell lymphoma, and T-cell lymphoblastic lymphoma, characteristically present with mediastianal
masses, and require a precise subclassification for therapeutic
purposes. However, according to the criteria proposed by 2008
WHO lymphoma classification, the differential diagnosis of
most lymphoproliferative diseases should be based on the integration of morphologic, phenotypic and molecular features.
Therefore, in most cases, sophisticated analyses need to be performed in order to asses diagnostic and prognostic criteria for a
correct lymphoma diagnosis. The lesional material obtained by
means of FNA may not be sufficient to perform all the investigations needed to make a correct diagnosis. The diagnosis of
spindle cell lesions may be very difficult on cytologic aspirates,
perhaps due to their rarity and lack of pathologists’ experience.
In addition sarcoma diagnosis is based also on architectural
features and phenotypic characterization.
On such bases many authors believe that core biopsies may
give higher rates of specific tumour typing for mediastinal
malignancies, characterized by small cell and epithelioid cytomorphology, as in most cases bioptic approach should yield
adequate tissue for ancillary diagnostic techniques, including
immunohistochemistry, ultrastructural analysis, cytogenetics
and molecular analysis. In some cases both FNA and bioptic
approaches may cause false negative diagnoses of malignancies, because of sampling errors, which are not uncommon
when tumors are fibrotic, or extensive necrotic. This frequent-
124
ly occurs with some mediastinal lymphoma such as Hodgkin
lymphoma, nodular sclerosis, and mediastinal large-B cell
lymphoma, with sclerosis. In these cases mediastinoscopy
and open thoracothomy appear to be still necessary to make a
definitive diagnosis.
In conclusion, we believe that FNA may still provide some
useful information in the characterization of mediastinal
tumours but in expert hands and in very selected cases. Otherwise a wide surgical bioptic approach should be preferred, if
allowed by the patients performance status, because only the
availability of a surgical specimen may ensure an adequate cytoarchitectural evaluation as well as material for biomolecular
investigations and biobanking.
References
Geisinger KR. Differential diagnostic considerations and potential
pitfalls in fine-needle aspiration biopsies of the mediastinum. Diagn
Cytopathol 1995;13:436-42.
Powers CN, Silverman JF, Gesinger KR, et al. Fine-needle aspiration
of the mediastinum. A multi-institutional analysis. Am J Clin Pathol
1996;105:168-73.
Singh HK, Silverman JF, Powers CN, et al. Diagnostic pitfalls in fineneedle aspiration biopsy of the mediastinum. Diagn Cytopathology
1997;17:121-6.
Wakely PE Jr. Cytopathology-histopathology of the mediastinum: epithelial, lymphoptoliferative, and germ cell neoplasms. Ann Diagn Pathol
2002;6:30-43.
Wakely PE Jr. Cytopathology-histopathology of the mediastinum II. Mesenchymal, neural, and neuroendocrine neoplasms. Ann Diagn Pathol
2005;9:24-32.
Lymphadenitis and lymph nodal metastases
A. Capitanio
Department of Cellular Pathology – University College London
Hospital, UK
The first application of Fine Needle Aspiration Cytology
was most like to investigate lymphadenopathy. It is a simple,
reliable and inexpensive technique that is often used as first
diagnostic approach to lymphadenopathies. Besides, many
ancillary techniques can be used to improve the effectiveness
of lymph nodal cytology. Immunocytochemistry, flow cytometry, in situ hybridization, PCR and other molecular investigations are now commonly and successfully used to increase the
reliability of lymph node cytological assessment.
Moreover, the material obtained by FNA can be centrifuged
and the pellet embedded to obtain cell blocks. In the last years
the development of liquid based cytology contributed to the
development and standardization of the ancillary techniques.
Here, after a quick look to the normal component of the
lymphoid tissue, we are going to examine a short review of
inflammatory diseases and metastatic conditions that can be
diagnosed by lymph node FNA cytology.
The cells of the normal lymph node. Mature lymphocytes are
small with dark nuclei and coarse chromatin pattern. A thin
pale-blue rim of cytoplasm surrounds the nucleus. B and T immunophenotype cannot be distinguished morphologically.
Plasma cells have oval shape with the nucleus eccentrically displaced. The nuclear chromatin is often described as
cartwheel-like and the cytoplasm shows a usually well visible
pale paranuclear area.
Centrocytes are slightly bigger than mature lymphocytes with
a more basophilic cytoplasm and fine chromatin. The nuclear
shape can be irregular and/or cleaved.
Centroblasts are bigger than centrocytes showing a larger
cytoplasm, which may contain some small vacuoles. Nuclei
are usually round with peripheral nucleoli.
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Immunoblasts are the larges lymphoid cells. The cytoplasm,
when visible, is basophilic. Nuclei are round with one or more
nucleoli.
Lymphadenitis. The reactive (non-specific) hyperplasia.
Two type of reactive hyperplasia can be identified: with follicular centre expansion and with interfollicular expansion.
The reactive hyperplasia with follicular centre expansion is
characterized by numerous centrocytes and centroblasts often
aggregates in fragments of germinal centres. Small mature
lymphocytes and a variable number of plasma cells and immunoblasts complete the cellular spectrum. Cases with high
cellularity and particularly rich in a single cell type (centrocytes or centroblasts) can mimic a Non Hodgkin Lymphoma.
The differential diagnosis is easily done identifying the immunoprofile of the lymphocytes. The traditional immunocytochemistry on the smear requires several slides and optimal
cytological preparations. Modern flow cytometry, instead, can
detect in a single cell suspension the presence of the centrofollicular cells and the polyclonal kappa/lambda light chain
distribution.
The reactive hyperplasia with interfollicular expansion. In
this kind of hyperplasia the smear shows a prevalence of
mature lymphocytes with a minority of large nucleolated immunoblasts and plasma cells. If the lymphocytic population is
monomorphous the picture is indistinguishable from a NHL
and the immunophenotype, which will show a dominance of
T lymphocytes, must be determined. The presence of macrophages with cytoplasmic tingible bodies, usually described as
a marker of benign hyperplasia, is of very little utility.
Viral and post-vaccinal lymphadenitis. Smears from FNA
show small lymphocytes and numerous centrocytes and centroblasts admixed with small mature lymphocytes, plasma
cells, plasmocytoid lymphocytes and immunoblasts. Capillaries, macrophages and eosinophils may also be seen.
HIV infection. The cytological picture may vary from a suppurative lymphadenitis to a granulomatous or non-specific
pattern. In presence of florid follicular hyperplasia the smear
shows centrocytes and centroblasts admixed with small
mature lymphocytes and plasma cells. The main differential
diagnosis is NHL.
Mononucleosis. The smears show a normal cell type phenotype including centroblasts and centrocytes. Numerous immunoblasts with large nucleoli and a rim of blue cytoplasm are
present. Macrophages and capillary structures may be present.
Differential diagnosis: Hodgkin lymphoma.
Suppurative lymphadenitis. FNA from lymph nodes draining organs with bacterial infections can show a cytological
picture characterized by a variable number of neutrophils and
lymphocytes on necrotic background. The diagnosis is usually
easy, but the possibility of metastatic carcinomas or, rarely,
Hodgkin Lymphomas must be considered.
Sinus Histiocytosis with Massive Lymphoadenopathy. This
lymphadenopathy (Rosay-Dorfman disease) is cytologically
characterized by large histiocytes with vescicular nuclei. The
presence of lymphocytophagocytosis (i.e. presence of well
preserved lymphocytes in the cytoplasm of some histiocytes)
is a characteristic of this condition. Numerous mature lymphocytes complete the cytological picture.
Sarcoidosis. Findings in FNA cytology are: cohesive clusters of epithelioid histiocytes, small lymphocytes, occasional
multinucleated giant cells and absence of necrosis in the
background.
A similar picture is seen in lymph nodes draining from organs
affected by primary carcinoma. This “sarcoid reaction” can
occur also in absence of metastasis in the lymph node.
sessioni scientifiche
Tuberculosis. Three possible major cytological features characterize the tuberculous lymphadenitis.
Epithelioid granulomata without necrosis. They are clusters of
histiocytes admixed with small lymphocytes. Langhans cells
are rare. Single histiocytes can be seen.
Epithelioid granulomata with necrosis. The cytology is similar
to the previous pattern, but amorphous necrotic material is
seen in the background.
Necrosis without granulomata. The amorphous necrotic material seen in the background contains polymorphs and occasional histiocytes.
Stain for acid fast bacilli or more sensitive techniques like
PCR are necessary for a final diagnosis.
Metastatic Epithelial Tumours. Squamous cell carcinoma.
Well differentiated squamous cell carcinomas (SCC) are usually not difficult to diagnose. Hyperchromatic nuclei, keratinized cytoplasms and a wide range of cellular atypia are helpful
features for the diagnosis. Poorly differentiated metastatic
SCC show cohesive groups of small polymorphic cells with
hyperchromatic nuclei.
Adenocarcinoma. Morphology of metastatic adenocarcinomas can be very heterogeneous. Cell cytoplasm is usually
large, pale and vacuolated. Round or oval nuclei with evident
nucleoli are common. In presence of these morphological
characteristics and if the smear shows groups of epithelial
cells with acinar or frankly glandular arrangement the diagnosis is easy, otherwise immunocytochemical investigations
are essential.
Small cell carcinoma. Cells from metastatic small cell carcinoma may resemble lymphoma cells especially if the lymph
node is widely substituted by the neoplastic cells. Cytological
features such as moulding, coarse chromatin, mitoses and necrotic background facilitate the diagnosis. Immunocytochemical positivity for CD56, TTF-1 and cytokeratin Cam 5.2 are
usually demonstrable.
Other metastatic malignancies. The list of the possible nonepithelial metastatic malignancies is too long to be enumerated. For some of them the morphological characteristics can be
helpful (for example: dark cytoplasmic pigment and nuclear
pseudo inclusion in melanomas). For many others without a
satisfactory immunocytochemical support the diagnosis will
be “metastatic malignancy”. Nevertheless, even if the cytological findings do not allow a final diagnosis concerning the
site of origin of the neoplasia they represent an important tool,
together with clinical and radiological data, in the diagnostic
process.
Flow cytometry in fine needle cytology
of lymphoproliferative processes
P. Zeppa, L.V. Sosa Fernandez*, M. Russo*, E. Vigliar*
Departments of Medicine and Surgery, University of Salerno, Italy;
* Biomorphological and Functional Science, University, of Naples,
Federico II, Italy
The double approach to cytological diagnosis of lymph
nodes and lymphoid organs with flow cytometry (FC) and
fine needle cytology (FNC), is an essential combination in
the evaluation of lymphoproliferative disorders. In fact the
current World Health Organization (WHO) classification
for lymphoproliferative disorders is based on morphological
and immunophenotypic criteria, determined by FC and or
immunohisto-cytochemistry (IHC/ICC). FC does not allow
the visualization of target cell as IHC/ICC does, but permits
the application of more markers than ICC.
125
FC can be generally performed in 1 or 2 days, allows the
detection of aberrant cells with a very high sensitivity at a
frequency of 1/1000 up to 1/10,000 cells using very sophisticate equipments. FC can also detect and quantify multiple
immunophenotypical markers on a single cell surface, using
a panel of different antibodies on suspended viable cells generating specific diagnostic algorithms. An accurate evaluation
depends first on the cellularity of the sample, which should
account for a minimum of 50,000 cells for each tube to obtain
10,000 events to analyze, second on fixation, lysis and permeabilization conditions. These conditions and perfect technical performances and procedures are essential to correlate
morphological and FC data. FC is usually performed using
a basic panel of antibodies to identify T or B-cell population
and their malignant counterpart. Diagnosis of B-cell, nonHodgkin lymphomas (NHL) is first based on the detection
of clonality and FC is a powerful technique to demonstrate
this feature by kappa and lambda light chain assessment. In
T-cell NHL, clonality assessment is less effective than in Bcell counterpart and may be assessed by the loss of specific
T-cell markers, such as CD2, CD3, CD5 and CD7 or by the
erroneous CD4/CD8 ratio. When a definitive diagnosis of
NHL is put forth by evaluating the combination of cytological features, light chain restriction or abnormal expression of
specific T antigens the cytological features are then matched
to the different expression and co-expression of different
antibodies such as CD5, CD10, CD23, FMC7, CD38, CD56
in different combined phenotypes, to classify, when possible,
the specific subtype. Therefore FC is extremely powerful
and effective in the diagnosis of NHL but also carries some
limitations and potential pitfalls. First any FC analysis should
be performed after a microscopic evaluation of corresponding smears to establish the real need for immunophenotypization, the technique (ICC and/or FC) and an appropriate
panel. FC does not allow the microscopic identification of
the analyzed cells; therefore the main antibody is first utilized
to identify the target cell population (i.e. CD20 or CD19 for
B-lymphocytes and CD3 for T-lymphocytes). The effective
expression of another antibody will be then evaluated on
the cell population identified by the main one. For instance
CD10 identifies follicular centre cells only if expressed on
CD19 or CD20 positive cells or CD56 identifies NK lymphocytes only when tested on CD3 positive cells. Therefore
the co-expression of two or more antigens is mandatory
for the evaluation of specific cell populations. Nonetheless
any co-expression has to be evaluated considering possible
artefacts and quantitative limits. The possibility that the coexpression of two antigens might be due to two cells tightly
attached each-other and identified by the laser beam as one
cell (doublets phenomenon), should always be considered. In
these cases the usage of a SSc-height vs. SSc–width dot plot
permits to identify doublets from single co-expressing cells
by the different widths of doublets compared to the single
width of each co-expressing cell. Quantitative features of
co-expressions are also important; for instance CD5/CD19
co-expression is indicative of small lymphocytic lymphoma/
chronic lymphatic leukaemia (SLL/CLL) or mantle cell lymphoma (MCL) but only if expressed in a quantitative significant proportion (10%-20%). Small clusters of cells showing
this co-expression in lower rates should not be considered
straightforward as malignant. Another example is furnished
by CD19/CD10; this co-expression identifies just follicular
centre cells but values of co-expression higher than 50% may
be considered the pathological expression of follicular lymphoma (FL) or lymphoblastic B-cell lymphoma (BL). Light
126
chain evaluation represents another paradigmatic example of
how FC values have to be carefully and critically evaluated. It
is generally reported that the kappa/lambda light chain ratio,
in non-neoplastic specimens, is usually in the range of 1:1 to
2:1. B-cell lymphomas generally express a single clonal light
chain therefore this ratio is generally increased or decreased
depending on the corresponding “restricted” chain. Overtime
more and more reports have described non-lymphomatous
conditions with values higher than those reported above,
involving small clusters of cells, mainly in specific clinical
context such as immunodeficiency or autoimmune diseases.
Therefore, using light chain restriction as sole parameter to
assess the clonality of a lymphoproliferative process, the rate
of light chains unbalancement has to be higher than 20% of
the gated cells. Finally FC report should not consist of a list
of CD markers with the percentages of positive cells instead
should be embedded in a comprehensive report together to
the microscopic features and any other technical data. This
comprehensive report should describe any aberrant populations detected by FC and summarize the salient features of
their phenotype. In conclusion FC applied to FNA samples
can support cytomorphological features provided of a critical
evaluation of the results and a logical merging with clinical
context and cytological data.
Diagnosis of lymphoma by fine-needle aspiration
cytology of lymph nodes
M. Ungari
Department of Pathology 1, Spedali Civili, Brescia, Italy
Fine-needle aspiration (FNA) is a rapid, cost-effective, and
accurate means for evaluating a wide variety of conditions
in most organ systems. FNA diagnosis of lymphoma remains
controversial. A review of the literature over the past 10 years
shows lymph node FNA to have modest to high sensitivity
(66%-100%, mostly > 80%), high specificity (58%-100%,
mostly > 90%), and high diagnostic accuracy (50%-100%,
mostly > 85%) for non-Hodgkin lymphomas (NHLs). FNA
evaluation of Hodgkin lymphoma (HL) has lower sensitivity
(48%-86%) but high specificity rate (98%-100%). Sensitivity
is generally better for recurrent disease than for a primary
diagnosis. The complementary use of cytomorphology, immunocitochemistry, and flow cytometry has proven more
accurate that any single modality alone. Often, a lymph node
FNA specimen provides sufficient information to decide
whether a patient requires simple observation, antimicrobial
therapy for an infectious process, radiation or chemotherapy
for malignancy, a staging operation for malignancy, or additional tissue in the form of a core biopsy or an excisional
lymph node biopsy. The application of immunocitochemistry
is essential for a lymphoma diagnosis. A simple combination
of CD20 and CD3 stains can group most processes into broad
categories of B-cell predominance, T-cell predominance, and
a mixed infiltrate. Additional more specific antibody panels
can then be applied to B-cell and T-cell processes for more
specific classification. Fluorescence in situ hybridization
(FISH) and polymerase chain reaction (PCR) are important
adjuncts to lymph node FNA. FISH analysis can be performed
on air-dried cytologic smears, cytospin prepared from cell
suspensions, or destained archival cytologic smears.
B-cell NHL of small cell type includes small lymphocytic
lymphoma (SLL), mantle cell lymphoma (MCL), follicular center cell lymphoma (FL), marginal zone lymphoma
(MZL), and lymphoplasmacytic lymphoma (LPL). All are
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
characterized by a smear dominated by small, lymphocytes,
with rare larger cells (particularly SLL and MZL), plasma
cells especially with Dutcher bodies (LPL), histiocytes (in
MCL), centroblasts, and dendritic cells (especially in FL).
The entities are distinguished by a combination of cytology,
immunophenotype, and cytogenetics (Tab. I). FNA from
SLL is characterized by a monomorphous population of
small, mature-appearing lymphocytes with smooth nuclear
borders, clumped chromatin, and scant cytoplasm, with scattered larger cells, with a prominent nucleolus (paraimmunoblasts). FNA features suggestive of transformation include
increased paraimmunoblasts, and increased mitotic figures.
An increased Ki-67 index by immunocytochemistry is also
useful to assess large-cell transformation. Rare cases will
transform to either a Hodgkin-like or true HL. FNA from
MCL is characterized by monomorphous small cells with
nuclear irregular outlines, fine and evenly dispersed chromatin, scant cytoplasm. Large transformed cells are usually
absent. Mitoses can be found. FNA from FL is characterized
by aggregates of mostly small lymphocytes with convoluted
nuclei, indistinct nucleoli, and scant cytoplasm (centrocytes),
with larger non-cleaved lymphocytes, with round or oval
nuclei, open chromatin and several small, membrane-bound
nucleoli (centroblasts). Tingible body macrophages are rare
or absent. Immunocitochemical stains demonstrate dendritic
cells (CD21+) within aggregates. Immunostain for bcl-2 is
less useful in FNA because localization of reactivity within
the follicle formations or centrocytes is necessary. Low-grade
lymphomas (grades 1 and 2 FL) usually show less than 20%
large cells. FNA smears from MZL are relatively polymorphous and contain small- to intermediate-sized lymphocytes
with moderate pale cytoplasm (monocytoid appearance). The
smear often contains plasma cells, follicular dendritic cells,
tingible body macrophages, immunoblasts, and lymphohistiocytic aggregates.
Tab. I. WHO Classification of Small B-Cell Lymphomas.
CD5
CD10/BCL6
CD23
Ciclina D1
SOX11
Cytogenetics
SLL
+
+
trisomy 12
MCL
+
+
+
t (11;14)
FL
+
- or +
t (14;18)
MZL
- or +
-
B-cell NHL of medium and large cell type includes DLBCL, grade 3B FL, Burkitt lymphoma (BL), lymphoblastic
lymphoma and blastoid variant of MCL, but also myeloid
leukaemia may enter in the differential diagnosis. FNA from
DLBCL and grade 3B FL show a predominance (usually >
20-40%) of large, uniform cells with large vesicular nuclei
(2 to 3 times the size of a reactive lymphocyte), with several
distinct nucleoli and moderate cytoplasm. There are variable
numbers of background tingible body macrophages. An important pitfall in the diagnosis of DLBCL is the T-cell- or
histiocyte-rich variant, which can mimic a reactive lymphoid
process by both FNA morphology and flow cytometry. FNA
from BL shows a uniform population of medium-sized cells
with round nuclei and course chromatin, prominent and multiple nucleoli, basophilic cytoplasmic with vacuoles. The background shows many tingible body macrophages (imparting
the “starry sky” appearance), mitoses, and apoptotic cells, indicating a high proliferation rate. Genetically, all BL harbors
a rearrangement of the c-myc proto-oncogene on chromosome
127
sessioni scientifiche
8. The most common translocation is t(8;14), which occurs in
80% of lesions and involves the immunoglobulin heavy-chain
gene on chromosome. The blastoid variant of MCL and
lymphoblastic lymphoma are characterized by intermediate
to large cells in size, with slightly irregular contours of the
nucleus, with fine evenly distributed chromatin and small
nucleoli. Immunocytochemistry (TDT, CD34 and Cyclin D1)
can differentiate blastic MCL from lymphoblastic lymphoma.
T-cell and NK-cell NHL compose approximately 10% of
NHL. The more common entities in cytology practice are
precursor T-cell lymphoblastic lymphoma (T-LL), peripheral
T-cell lymphoma, unspecified (PTCL), and anaplastic large
cell lymphoma (ALCL). Unlike B-cell processes, T-cell and
NK-cell lymphomas are generally not defined by specific immunophenotypes. FNA from PTCL are polymorphous, with
intermediate and large lymphocytes in a background of histiocytes, eosinophils, and plasma cells. Large clear cells and
RS-like cells may be present, leading to confusion with HL.
Other cases appear as pure large-cell lymphomas, whereas
still others show morphology similar to SLL. FNA of ALCL
is characterized by large, pleomorphic cells, often with a
horseshoe-shaped nucleus (hallmark cells). Variants include
the small cell type, resembling PTCL, the lymphohistiocytic
variant, in which a large number of histiocytes may mask the
large tumor cells, the rare signet cell variant, the neutrophilrich variant, and the sarcomatoid variant. ALCL often losts
CD3, CD5, and CD7; CD2 and CD4 are usually expressed.
ALCL also expresses CD30 (membranous or Golgi pattern),
in addition to EMA and TIA-1. Genetically, 60%–85% of
ALCLs are associated with the translocation t(2;5)(p23;q35),
which results in expression of the ALK protein (immunocytochemistry).
FNA smears from classic HL show rare RS cells (binucleate, with prominent nucleoli large, often at least 4 times the
size of a mature lymphocyte) in a polymorphous background
composed of nonneoplastic lymphocytes, eosinophils, neutrophils, histiocytes, plasma cells, and fibroblasts. The typical immunophenotype for the RS cells is CD15+ (usually),
CD30+, CD20- (usually), PAX5+ slight, CD45-, and EMA-.
Subtyping of classic HL by FNA has proven unsuccessful.
The RS cell-variant characteristic of nodular-lymphocytepredominant HL is the L&H cell (lymphocytic and/or
histiocytic RS cell variant). The L&H cell is mononuclear
and characterized by a large, multilobated nucleus with
irregular outlines (“popcorn cell.”), with smaller nucleoli
than those of classic HL. The typical immunophenotype for
L&H cells is CD15-, CD30-, CD20+, PAX5+, CD45+, and
EMA+. The cytologic differential diagnosis of HL includes
reactive hyperplasia, T-cell-rich LBCL, ALCL, acute lymphadenitis (rare examples), metastatic carcinoma, and metastatic melanoma. Pitfalls are many in the cytologic diagnosis
of HL. False-negative FNA may be due to the inability to
aspirate adequate numbers of diagnostic RS cells. On the
other hand, certain reactive and neoplastic conditions, such
as Epstein-Barr infection, NHLs, and immunosuppression
states, may contain large atypical cells, such as immunoblasts, that resemble RS cells.
References
Volmar KE, Singh HK, Gong JZ. The Advantages and limitations of the
role of core needle and fine needle aspiration biopsy of lymph nodes in
the modern era. Hodgkin and Non-Hodgkin lymphomas and metastatic
disease. Pathology Case Reviews 2007;12:10-26.
Serrano Egea A, Martinez Gonzalez MA, Perez Barrios A, et al. Usefulness of light microscopy in lymph node fine needle aspiration biopsy.
Acta Cytol 2002;46:364-8.
Chhieng DC, Cohen JM, Cangiarella JF. Cytology and immunophenotyping of low- and intermediate-grade B-cell non-Hodgkin’s lymphomas
with a predominant small-cell component: a study of 56 cases. Diagn
Cytopathol 2001;24:90-7.
Gong Y, Caraway N, Gu J, et al. Evaluation of interphase fluorescence in
situ hybridization for the t(14;18)(q32;q21) translocation in the diagnosis of follicular lymphoma on fine-needle aspirates: a comparison
with flow cytometry immunophenotyping. Cancer 2003;99:385-93.
Caraway NP, Gu J, Lin P, et al. The utility of interphase fluorescence in
situ hybridization for the detection of the translocation t(11;14)(q13;
q32) in the diagnosis of mantle cell lymphoma on fine-needle aspiration specimens. Cancer 2005;105:110-8.
Young N, Al-Saleem T. Diagnosis of lymphoma by fine-needle aspiration cytology using the Revised European-American classification of
lymphoid neoplasms. Cancer Cytopathol 1999;87:325-45.
Cytopathology and lymphoma diagnosis
A. Carbone
Dipartimento di Patologia, IRCCS – Centro di Riferimento Oncologico – Aviano, Istituto Nazionale Tumori
Background and methods. Fine-needle aspiration biopsy
(FNAB) today represents an effective procedure for investigating lymphoproliferative lesions in lymph nodes or inner
involved sites. In several instances FNAB procedure is associated with a tru-cut biopsy. The cell sampling by both FNAB
and tru-cut biopsy is guided by imaging analyses (ecography,
computed tomography, NMR, angiography). The cell material obtained by FNAB is usually suitable for preparing cell
blocks.
Results. These combined procedures, i.e. FNAB and tru-cut
biopsies, may provide cell material useful for: 1- diagnosis
and classification of the neoplasia; 2- prognostic factors; 3prediction of therapeutic response; 4- detection of molecules
that are possible target for therapy.
Special techniques applied on cell materials obtained by
FNAB (cell blocks, smears and cell suspensions) include:
immunocytochemistry, in situ hybridization, PCR and RTPCR, and flow cytometry. Combining these techniques, a
multidimensional diagnosis and classification of lymphomas
is feasible in several cases.
Cytological examination together with analysis of sections
from cell blocks or tru-cut biopsies by immunocyto-histochemistry may usually be sufficient for diagnosing. Molecular
data, as for example chromosomal translocations, may further
support the diagnosis, aid the differential diagnosis and assess
minimal residual disease.
Conclusion. Cytological examination alone is not a reliable procedure for a lymphoma diagnosis. However, when
a lymphoma or a lymphoid mass is investigated by FNAB,
and a cell block is feasible, most molecular analyses can be
performed. Special techniques successfully applicable on
cell block include immunocytochemistry, in situ hibridization, PCR and RT-PCR and flow cytometry. Adding tru-cut
biopsy to the FNAB provides further support to diagnosis
and makes possible the assessment of prognostic and predictive markers.
128
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Venerdì, 29 giugno 2012
ore 11.00-13.00
Citotecnici
Chairmen: Tiziano Zanin (Genova), Sandra Dudine (Trieste)
ASC-US: triage with HPV test
E. Isidoro, M. Di Napoli, A. Romano, F. Giudici, E. Nicastro*,
A. Brollo*, C. Bottin, L. Di Bonito, F. Zanconati, D. Bonifacio
UCO Anatomia e Istologia patologica, Università di Trieste; * UO
Anatomia patologica, ASS2 Isontina, Monfalcone (GO)
Background. A protocol for cervical cancer screening among
sexually active women 25 to 65 years of age was introduced in
Friuli-Venezia-Giulia in 1999. A pap test is performed with a
3-year-interval. In addition Human Papillomavirus test (HPV)
has been offered as a triage test for women with a diagnosis
of atypical squamous cells of undetermined significance
(ASC-US) since 2009. HPV testing was performed with HRHPV (Hybrid Capture II) like suggested by FDA. According
to protocol, HPV negative women were referred to regular
screening including a cytological exam every 3 years, while
HPV positive women were referred to colposcopy and closer
follow-up. We evaluated the implementation of the protocol
and the prediction of HPV testing as a triage tool for cervical
intraepithelial lesions in women of the screening program of
Trieste and Gorizia area with a cytological diagnosis of ASCUS after the first round.
Objective. The category ASC-US continues to be controversial, despite strict criteria laid down by the Bethesda 2001
Committee, as it does not have a histological counterpart and
it refers to cytological findings that may or may not be associated with a significant epithelial lesion of the cervix. The proportion of CIN2+ is very low among women with an ASC-US
diagnosis, so identification of those at higher risk would be
clinically useful (2). In this study we analyzed ASC-US cases
of Trieste and Gorizia diagnosed in the period 2009-2011 to
evaluate the presence/absence of lesions in the histological
follow up in women HR-HPV positives.
Material and methods. From 2009 to 2011, 49024 women
were screened with conventional cytology, from Trieste and
Gorizia provinces, 764 were reported as ASC-US (1.56%).
HPV detection was performed using HR-HPV tests (HCII, Digene) which detects 13 high risk oncogenic HPV types (16,1
8,31,33,35,39,45,51,52,56,58,59 and 68). HR-HPV DNA test
was detected on 710 women (710/764, 93%) with a median
age of 39 years (I-III 30,46). An HPV sample was considered
positive if it attained or exceeded of 1.0 pg HPV DNA ml-1,
which corresponds to 1.0 relative light unit (RLU/CO). We
had 206 positive tests (206/710, 29%) and 504 (504/710,
71%) negative ones.
Results. Among the 206 women with HPV positive test,
only 76 had no cytological or histological follow-up (76/206,
37%). At the end of the 3 years period, 83 had been given a
biopsy (83/206; 40.3%) with the following results: 25 high
grade lesions CIN2+ (25/83, 30%); 33 low grade lesions
(33/83; 40%) and 25 negatives (25/83, 30%). Considering the
histology results we evaluated PPV CIN2+ that was 30% with
a specificity of 14.7%. Among the women with HPV negative test none of them was found to have a lesion at follow
up. Only cytological follow up was carried out for 47 cases
(47/206; 23%); among them one high grade lesion was reported. ASC-US cases with following HPV positive test were
divided into four age groups: 25-34 years (76 women), 35-44
years (69), 45-54 years (44), and 55-65 years (17). HR–HPV
positivity decreased with increasing age from 47.8% among
women younger than 35 years to 21.4% in women older than
35 years (p < 0.0001). CIN2+ lesions were quite constant (3132%) in the first three groups to get close to zero in the last
one (Fig. 1).
Fig. 1. The graph is expressed in absolute terms (number cases).
Conclusion. Triage of ASC-US with HR-HPV testing showed
a high sensitivity for the detection of CIN2+ and a high negative predictive value on follow up. The results of this study are
in line with the current guidelines for triage of women with
ASC-US in the target age range of 25-65 1, and with other
studies on the same subject 2 3. Low positivity of ASC- US
cases for HR HPV test makes us notice several cytological
specimens reported as ASC- US should be considered negative; when HR HPV test is positive following histology shows
a lesion, mostly a low grade one, in 40% of cases. Dividing
patients for age groups makes us observe HR HPV test positivity decreases with the age increase; but finding of CIN2+
lesion is about constant till the age of 54; this can be explained
with the fact that in younger women, who have higher test
positivity, HPV infection is often transient, as described by
the literature 2. Efficacy of triage with HPV test is unquestionable, as it allows to select among women with ASC- US
diagnosis, a small number of women, who being positive to
the test may develop a squamous lesion, while for women
negative to the test chances to have a squamous lesions are
absent, so the colposcopy is not required.
References
1
Arbyn M, Sasieni P, Meijer CJ, et al. Clinical applications of HPV testing: A summary of meta-analyses. Vaccine 2006;24(Suppl 3):78-89.
2
Del Mistro A, Frayle-Salamanca H, Trevisan R, et al. Triage of women
with atypical squamous cells of undetermined significance (ASC-US):
Results of an Italian multicentric study. Gynecol Oncol 2010;117:77-81
3
Ibanez R, Moreno-Crespi J, Sarda M, et al. Prediction of cervical intraepithelial neoplasia grade 2+ (CIN2+) using HPV DNA testing after a diagnosis of atipical squamous cell of undetermined significante
(ASC.US) in Catalonia, Spain. BMC Infect Dis 2012;12:25.
129
sessioni scientifiche
Quality control of pap-test in “wide area
of Vicenza”
R. Corrado1, B. Graziani2, L. Corponi3, G. Ruzza4
Department of Anatomic Pathology of ULSS 3, 2 4, 3 5, 4 6 Wide Area
of Vicenza, Veneto, Italy
1
Background/introduction. The “wide Vicenza area” is the
department that analyse pap-tests in the area of its competence
and it counts four Pathological Anatomy divisions, 54 operators as pathologists, biologists and cytologists and an amount
of 60.000 pap tests screened every year. On each sample a
computer assisted examination (Imager, Hologic) is executed
with thin layer technique (Thin-Prep, Hologic).
As consequence, an necessity of constitute a quality control
has been born, and it has the aim of applying a strictly control
on this specific activity. Basically, we can consider it, after
its fifth year of life, as an unique reality in the italian public
health context.
In this report will explain the results achieved and the criticality found.
Materials and methods. Every year 40 pap-tests are chosen
(10 for each division) and they comprehend all the diagnostic
classes according to Bethesda classification. The clinical data of
each case were provided. The colouration of the samples was
absolutely standardised and it was realised on a single colouration machine, with standardised protocol for computer assisted
examination. The samples have been sent to the four divisions
with only the clinical data taken in the first phase. Every single
analysis has been collected and statistically evaluated. In particularly, the most frequent diagnosis for each case has been
taken as “gold standard”. For this type of control the computer
assisted examination was not used for organisational reasons.
The statistical analysis, comprehensive of the histological
comparison, has been done by the Veneto Tumor Register
(Dr. S. Guzzinati)
Results. The results regard both the analysis of the single
diagnoses and the cases coming from the single division.
They have been statistically elaborated with the exposure of
the concordance level (K of Cohen). A good/substantial level
of concordance has been found for the most part of diagnostic
classes, particularly for high risk categories (Fig. 1).
Furthermore, a constant performance has been verified across
the time (Fig. 2) with some improvements in concordance for
diagnosis of AGC, ADK/CA SG and CTM. The results of the
cyto-histological comparison are shown in Table I.
Tab. I.
Kappa
Value 2011
Value 2012
Simple
0,3931
N.D.
Weighed
0,5013
0,5026
Fig. 1.
Fig. 2.
Furthermore an audit of every single case with an high level
of discordance has been done with the aim of reach an unique
diagnosis.
Criticality was found with the time-consuming for the general
organisation and sending of the slides.
Conclusions. Our quality control has highlighted: a good
concordance even in remarkable heterogeneity of the cases.
The efficiency of the thin layer with standardised stain (even
if it was different from the usual one with more dark nuclei
which could be problem with glandular aggregates) was confirmed. Finally we conclude that our audit is very useful for
discordant cases.
Fish evaluated cellular aneuploidy as a second
level test in urinary and cervical cytology
C. Magnani
Clinica San Gaudenzio, Novara, the Cytology Service
Aims. A retrospective evaluation of some vaginal and urinary
cytology cases carried out over the last three years in our service has evidenced that alterations in the cell morphology does
not always allow for a conclusive diagnosis.
Indeed, not always have even repeated cytology controls of
some cases and their comparison with clinical and instrumental examinations – such as colposcopy and histological examination for cervical-vaginal cytology, ultrasound examination,
the CT scan and for some cases cytoscopy and scalpel biopsy
for urinary cytology – been able to provide the necessary information to formulate an indisputable diagnostic conclusion.
To increase the number of conclusive diagnosis in urinary and
vaginal cytology, we used a FISH technique for the detection
of aneuploidy in exfoliated cells
Methods. Our personal experience with the use of the FISH
technique for the identification of tumoural cells is fruit of
close collaboration with the Centro Diagnostico Italiano di
Milano. The cases selected for evaluation included cases with:
PAP-TEST with a diagnosis of ASC-US/HR-HPV+; followup for CIN-III post-surgical ablation (cervical conisation
with non-negative conisation margins); a cyto-histological
diagnosis of CIN-I/CIN-II; a dubious urinary cytology result
for haematuria.
Results. A FISH positive result for the presence of frankly aneuploid cell lines and, therefore, most likely tumoural, is an indication for surgery, even from a forensic medicine point of view.
FISH may be particulary useful for the detection of urothelial
tumours, as conventional cytology and cystoscopy have high
false negative rates, 50% and 30% respectively, making for an
insufficient diagnostic specificity and sensibility, leading to inconvenience to the patient and high national health service costs.
The FISH technique allowed for the diagnosis of a patient
with dubious clinical results as was the case for another patient under follow-up with a confirmed relapse by cystoscopy
and histological examination.
130
In conclusion, even if to date there is a limited number of
comparative cases, we may affirm that a “cautious” use of
other techniques as FISH, may be fundamental for some diagnosis, whilst, at the same time reiterating the conviction that
a “attentive” interpretation of the cell morphology remains at
the basis of identifying diagnostic strategies.
Risk management in cytology: analysis
and error handling
E.M. Nicastro
Anatomy and Pathology Dept., UO Hospital of Monfalcone (GO), Italy
Background/introduction. The Health system is a complex
organization. All system components must integrate and coordinate, to meet the care needs of the patient and assuring
the best results. Starting from the consideration that error is
an inevitable part of human condition, it becomes critical to
recognize that systems too can go wrong, favoring the occurrence of accidents and errors. It is therefore necessary to design
and identify risk containment system to prevent the occurrence of errors or, if they happen, to contain the consequences.
The error, seen as the results of the failure of a system, becomes a
source of learning to manage circumstances that lead to mistakes.
The Control techniques are mostly important for risk management. In reactive approach analysis follows harmful events. In
Proactive approach a prior organizational analysis allows the
identification of potentially harmful events. The FMEA (Failure Mode and Effect Analysis) is a qualitative analysis aimed
at defining what might happen (mode failure/error) due to any
defect, error or omission. The FMECA (Failure Mode Effect and
Critically Analysis) adds a quantitative path. In common usage,
FMEA embraces FMECA too. FMEA was developed in the
late ‘40s in the U.S.. The first document mentioning FMEA is a
military procedure, the MIL-P 1629, 1949. FMECA is an engineering technique used to identify, analyze, delete, monitor and
review the “failure”, and it refesr to the problems, faults, failures
and mistakes. There is no standard definition of Error in Anatomy Pathology so, to understand which are the errors in this area
is important to define which are needed features of a good and
right diagnosis, namely: exact identification of patient; correct,
complete, timely report, couched in a language both understandable and useful to the clinician who is treating the patient. The
work flow is divided into: Pre-analytical phase, Analytical phase
and Post-analytical phase. Some authors identify Pre-analytical
that one in which occurs the greatest number of errors.
The Unit of Pathological Anatomy and Cytopathology of
ASS. 2 “Isontina”, is splitted into two different locations, implying the need of people and samples sharing. All biological
samples from by wards, operating theaters, specialist clinics
into Monfalcone Hospital are sent for cytology and histology
to Monfalcone pathology unit.. Samples are matched with
paper forms. There is not support for electronic request or
automated matching of samples with patients.
Apsys, the software application in use, supports the administrative of the sample registration, reports items of the patho-
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
logic history of patient, and manages storage, recovery of
pathological data; it supports too database queries. The Unit
is not certified or accredited.
Since 2010, the cyto-Technician spontaneously began to report file errors identified when taking charge of the samples.
The most significant non-compliance has been classified as
“Sample not optimal due to fixation or preservation defects.”
Materials and methods. Previous experience has been
formally defined using the application FMEA / FMECA by
Biomedical Laboratory health technicians (TSLB) focused on
pre-analytical phase.
Results. The FMECA module, in addition to the activities and
modes of failure has been reported the professional responsibilities related to each activity, the causes, barriers/controls
(factors containing the error). To achieve these results it has
been used the technique of brainstorming. Following it was
evaluated these parameters and calculated the risk priority
index (IPR). In the next step we proceeded to the identification and analysis of the possible causes of adverse events.
Corrective actions will be undertaken on the basis of IPR in
accordance with the matrix of priorities.
Conclusions. The team work was crucial to allow a contribution of the various skills and experiences of individual practitioners and stimulated each operator to review the activities
carried out routinely to identify possible errors. The exact
mode of cyto-histologic specimen collection, the storage conditions and time elapsed between collection and delivery in
the laboratory is essential for a correct diagnosis and priority.
A not appropriate diagnosis or an error, that it may be identified during the later stages of the healing process, often involving hardship and risk to the patient’s health, especially as
regards the definition of an appropriate therapeutically plan.
Corrective actions identified for all operators involved were as
follows: dissemination of procedures and / or instructions on
samples collection, the mode of storage and transport wards of
the samples and the training staff on proper procedures, In addition, it is necessary to check the of shipping containers suitability, as well as training staff by considering the turn-over
of personnel transport. The re-assessment of the environments
regarding to noise reduction and the containment of the presence of disturbing factors can help to maintain a higher concentration especially when the data entry and acceptance of
information technology occur. To technical staff are directed
the re-assessment of the work environment and workloads.
It is imperative to identify risk factors that could lead to error.
Some risk factors are related to operators, including: difficulty
to execute instructions, missing or excessive supervision, and
lack in coordination, excessive workload and poor ability to
work in team. Other risk factors related to the system are: lack
of improvement or path work not well plant, high turnover of
personnel, procedures or protocols lacking and or poor or/and
not enforced documents. The complexity of health process, the
individual performance culture, the discomfort in discussing
errors and the lack of suitable indicators to measure safety are
the barriers to be overcome for developing a safety culture that
involve staff and patients.
131
sessioni scientifiche
Venerdì, 29 giugno 2012
ore 14.00-16.00
Ginecologia (I sessione)
Chairmen: Gaetano De Rosa (Napoli), Sonia Prandi (Reggio Emilia)
Cervical Cytology: quo vadis
C. Bergeron
Laboratoire Cerba, Cergy Pontoise Cedex 9, France
Virology and Cytology. Human PapillomaVirus (HPV) is
a highly infectious virus and presents in at least 10% of the
female population; most of infections are latent and regress
spontaneously without intervention. Neoplasia is a very rare
event and a complication of HPV infection.
The latent infection, even in case of the viral penetration in the
epithelium, by definition, is not associated with morphological modifications. The productive infection is associated with
the expression of the late viral genes L1 and L2 in the intermediate and superficial cells. It corresponds to a low-grade squamous intraepithelial lesion (LSIL) according to the Bethesda
terminology (TBS). A LSIL diagnosis is reproducible if it
is made on the presence of koilocytosis, the cytopathogenic
effect of a productive HPV infection. LSIL represent on average 1% of the smears but is more frequent in women younger
than 35. The transforming infection is associated with the
expression of the early viral genes E6 and E7 in the basal
cells. It corresponds to a high-grade squamous intraepithelial
lesion (HSIL) according to the TBS. A HSIL diagnosis is the
most reproducible and is based on the presence of abnormal
basal cells, hallmark of a precancerous lesion.The mean percentage of HSIL may vary between 0.3 to 1% of the smears
depending of the population.
In the TBS, the term “atypical squamous cells of undetermined significance” (ASC-US) is used for abnormal cytological changes that are suggestive of a LSIL, but lack criteria
for a definitive interpretation. Even with this definition,
the diagnosis of ASC-US is poorly reproducible and the
percentage highly variable among readers of the same set
of slides. The TBS suggests that not more than 3 % of the
smears should have the designation of ASC-US.
The HPV testing. The cytology primary screening approach
has been shown very specific but midly sensitive. To improve
sensitivity for detecting high-grade cervical intraepithelial
neoplasia (HGCIN), testing for high-risk types of HPV (HRHPV) has been investigated as an alternative or adjunct tool
for cervical cancer screening. Several studies have shown that
testing for HR-HPV DNA may provide a high level of sensitivity for CIN2+. However, overall diagnostic accuracy of
HPV testing is far from optimal due to the relatively low specificity, as most HPV infections are transient and cleared by
the women´s immune system. Only a relatively low proportion of infections persist and may progress into transforming
infections. Therefore, women with an HPV positive test need
a triaging with cytology before beeing sent to colposcopy.
There is also a substantial number of ASC-US or LSIL morphology for which no HGCIN can be confirmed on histology
specimens collected during subsequent colposcopy-guided biopsy sampling. Thus, it is important to implement an efficient
triage approach for ASC-US and LSIL to identify women
being at highest risk for underlying precancerous disease and
who require immediate further diagnostic follow-up. Testing
for the presence of HR-HPV has been incorporated into the
management of ASC-US in various countries. However, despite a high sensitivity for the detection of underlying HGCIN
within the ASC-US group, testing for HPV has its limitations
especially in terms of specificity. For the management of
LSIL, colposcopy remains standard for most patients.
Precancerous cervical lesions and p16. Over-expression
of the cell-cycle regulatory protein p16INK4a has been demonstrated in virtually all HGCIN and cancerous lesions of
the cervix uteri. At the molecular level, it has been shown
to be closely linked to the inactivation of the pRB-mediated
cell-cycle control machinery by the E7 onco-proteins from
high-risk HPV types in the basal epithelial cell layers. Thus,
p16 over-expression can be regarded as a surrogate marker of
the transforming activity of HR-HPV onco-proteins which is
required for the initiation and maintenance of the neoplastic
process. Various studies have been performed to evaluate the
potential utility of applying p16 immuno-cytochemical staining protocols especially for the triage of equivocal or mildly
abnormal Pap cytology. In most studies, a similar sensitivity
as HPV testing, but at a substantially higher specificity rate
has been reported for p16 cytology when used for the triage
of ASC-US, LSIL, or containing atypical glandular cells. This
effect was even higher in women aged less than 30 years
which is owed to the high prevalence rates of HPV infections
in the younger age groups. However, p16 single-staining
immuno-cytochemistry protocols required the morphologic
interpretation of immuno-reactive cells to distinguish abnormal cells and those occasionally over-expressing p16 such as
squamous metaplastic cells, or endocervical cells.
Simultaneous detection of p16 and Ki67 expression. The
clinical performance of a novel approach, i.e. the simultaneous detection of p16 and Ki-67 expression within the
same cervical epithelial cell as a morphology-independent
marker of cell-cycle deregulation protocol has been evaluated in the triage of ASC-US and LSIL. The results indicate
that p16/Ki-67 dual-stained cytology may identify CIN2+
with high sensitivity and high specificity. Concerning the
primary screening, p16/Ki-67 dual-stained cytology testing
can significantly increase the sensitivity for CIN2+ over Pap
cytology while maintaining the high specificity, irrespective
of age. The sensitivity of p16/Ki-67 dual-stained cytology approaches the sensitivity of HPV testing, whereas the number
of false-positive screening test results over all ages groups is
reduced by more than 50%. The other alternative of triaging
women with Pap negative/HPV positive screening test results
with p16/Ki-67 Dual-stained cytology has also shown that it
may identify women with a high probability of underlying
CIN2+ and may efficiently complement HPV-based screening
programs to prevent cervical cancer.
Vaccination and screening. The current vaccines provide
protection against 70% of cervical cancers. Also, they do
not protect women who are already infected by HPV 16 or
18. If women are vaccinated between 10 and 15 years old,
the time necessary to begin to see the impact on this vac-
132
cinated population on the incidence of cervical cancer will
be at least 20 years. This impact will also greatly depend
on vaccination coverage. The impact on cervical screening
programme by cytology will be much earlier and depends
of the catch up population concerned by the vaccination
programme (16-25 years). The vaccination will decrease
the percentage of abnormal smears with lowering the
probability of high-grade lesions but not much low grade
lesions or minor atypia. For the young vaccinated women,
primary HPV testing with triage by cytology and prolonged
screening interval would be probably the best scenario. Pap
screening or primary HPV screening for older non vaccinated women remains a debate. HPV screening on self sampled material in women who have not routine Pap screening
could become more important. Cervical cancer prevention
should be obtained in the future with the synergy of prophylactic vaccination for the young and adapted cervical cancer
screening for the older women.
The effect of the implementation of lbc in the
ppv for high grade dyskaryosis and its impact
on colposcopy multidisciplinary discussion
R. Dina
Dept. of Cellular Pathology, Hammersmith Hospital, Imperial
Following evaluation of the three UK pilot sites (Norfolk &
Norwich hospital; Southmead Hospital North, Bristol NHS
Trust; and Royal Victoria Infirmary, Newcastle) the NHSCSP
recommended that LBC is to be the preferred method for
cervical cytology in England and Wales with full conversion
within five years of the announcement made in October 2003.
When LBC was first introduced, it was believed to provide a
significant advantage over CC in terms of sensitivity for CIN
2+. However, the results from the numerous international
studies that have evaluated the effectiveness of LBC agree
that LBC improves specimen adequacy and reduces screening time compared to CC although there is still considerable
controversy regarding sensitivity and specificity of the two
methods (Bolanca KI and Vranes 2010).
UK pilot studies showed a statistically significant decrease in
the number of inadequate samples from 9.1% with conventional test to 1.6% with LBC (NICE 2003).
Our cytology laboratory has a very good Quality Control
(QC) in place. Multi Disciplinary Team meetings are part of
that QC. Over the past two years our laboratory has been collecting data of the colposcopic correlation and these are the
figures relating the patients discussed at MDT meeting during
2009: 78 patients were discussed because of discordance between cytologic, colposcopic or histological findings. Of these
61 patients had their cytology reviewed of which 13 (21.2%)
were found to be discordant and 78.8% (48/61) concordant. It
is to be noted that of the 13 discordant cytology cases as many
as 10 (77%) were interpreted as overcalls (Appendix 8).
These findings confirm our initial hypothesis that LBC is increasing the number of false-positive. However, this increase
is modest, therefore statistically not significant.
The aim of this study is to compare the accuracy and PPV
of moderate dyskaryosis or worse cytology in Liquid Based
Cytology and Conventional Cytology for the diagnosis of high
grade CIN, using colposcopy followed by histology as “gold
standard” for confirmation of positive cytology.
The data were extracted from the CoPath cytology database in
use at the Imperial Healthcare NHS Trust based at the Hammersmith Hospital, a computerised search identified all high grade
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
dyskaryosis (moderate and severe), including suspected invasive
carcinoma in the last year of conventional smear reporting from
1st April 2005 to 31st March 2006. 237 corresponding cases of
conventional cytology slides were available for this study.
The same selection method was used for LBC samples during
the 12-month period from 1st April 2007 to 31st March 2008.
267 cases of LBC with corresponding histology reports were
available for this study.
It was decided that a review of the second year of LBC screening would be used rather than the first year of conversion as
the learning curve effect tends to lead to overcalling lesions
during the initial phase of implementation.
Figure 2 (CC) shows the distribution of moderate and severe
dyskaryosis including the DTG category that accounted for
10.5% (25/237), while 43.5% (103/237) was reported as moderate and 46% (109/237) was severe dyskaryosis.
In table 3 (CC) is shown the cytology grading with corresponding histology. In 83% (197/237) of cases there was concordance
between cytology and histology. 9% (20/237) of cases cytology
was lower grade compared to histology and 8% (18/237) of cases
were discordant to histology. The first question that arises with a
situation like that is: are we looking at cytology false-positive results or false-negative result of histology? It is a well known fact
that biopsy can be taken at a different site from the cervical lesion. However, on a cytology review of those cases only 5 cases
(2.1%) were one grade lower but these were all positive/abnormal (Tab, III, Appendix 4). One of the reasons for the negative
histology could be simply that the punch biopsy missed the lesion
and for some reason we do not have a follow-up on those cases
possibly due to the transfer of these patients to the private sector.
As shown in figure 3 (LBC) the first thing worth mentioning is a big decrease in the DTG category from 10.5% in
conventional to 3.37% (9/267) in LBC due to the cleaner
background and monolayer of the cell presentation which allows most cases to be graded. Another, smaller decrease is in
the distribution of severe dyskaryosis to 32% (85/267) from
46% (109/237).
The distribution of results for the two categories (moderate and
severe dyskaryosis) from both methods, CC and LBC in this
project showed slight differences in numbers (Figs. 4, 5). The
distribution of the two categories failed to show any statistically
significant difference in both moderate and severe dyskaryosis.
Atrophy and dysplasia: a troublesome diagnosis
on both conventional and liquid-based pap-tests
B. Ghiringhello, G. Accinelli*, G. Alfonso*, S. Arnaud*, M.T.
Benenti, P. Burlo, V. Buratti, A. Coccia, C. Fiorito, D. Loche,
P. Luparia*, D. Maso*, S. Privitera, A. Sapino*
S.C. Anatomia Patologica, A. O. OIRM-S.Anna, Torino; * Centro
Unificato per lo screening cervico-vaginale città di Torino, A.O.U.
S.Giovanni Battista di Torino, Sede S.G.A.S.
Introduction. Pap-test examination in post-menopusal women is characterized by distinctive features when compared
with those performed in younger women, namely poor cellularity, dryness artefacts, blood material.
Cytological features of atrophy are often quite complex and
atypia in small cells may be responsible for misleading interpretations, even by pathologists of acknowledged experience.
The main diagnostic dilemma is represented by the differential
diagnosis between dystrophy and high grade squamous lesions
and sometimes even with glandular lesions. In addition, intraepithelial squamous lesions, typical of young women, are not rare
in post-menopausal state: false negative results are encountered
133
sessioni scientifiche
in up to 8% of cases and are partly due to scant exfoliation. The
percentage of false positive results is even higher and therefore
the “see and treat” approach constitutes mostly an overtreatment.
Material and methods. We have reviewed and compared a
series of matched conventional and liquid-based pap-tests.
Correspondent biopsies of the cervix were reviewed whenever available. A subgroup of cases had also been subjected
to molecular HPV test with a positive result. Liquid-based
specimens were crucial in solving cases with equivocal interpretation: indeed, a better fixation and a lower incidence of
not adequate specimens due to scarce material or to excess of
blood led overall to a higher quality of specimens.
Some authors have reported a higher rate of ASCUS lesions
in post-menopausal patients as assessed by liquid-based cytology. This is mainly due to HPV-alike artefacts because of
hormonal replacement therapy that leads to a higher content
of glycogen in the cytoplasm of squamous cells.
The positive predictive value of ASCUS lesions in post-menopusal patients is lower compared to that in younger women:
in fact, in women older than 50 years ASCUS lesions are less
frequently associated with dysplasia (2, 3 fold decrease) than
in young women. The positivity of the HPV molecular test
may be of help to sort out equivocal cases: it can be useful
to interpret some cytological features, such as higher nuclear
volume, hyperchromasia, ground-glass nuclei etc, as related to
L-SIL lesions or to “atypia” of post-menopausal age.
Even from a histological standpoint atrophy and dystrophy
may be responsible for interpretation issues, in particular
when the sample is constituted by an endocervical curettage,
which does not allow to evaluate the architecture of structures,
regardless of the positivity to the HPV molecular test. In such
a scenario immunohistochemical investigation for proliferation (ki-67) and for p16 are recommended.
Results. Atypia in squamous cells in pap-test of post-menopusal women often does not correspond to a dysplastic lesion
on the histological counterpart and more frequently represents
a reactive feature due to atrophy. Indeed, with the reduction
of oestrogen levels during menopause the cervico-vaginal
epithelium shows a lower grade of maturation leading to a
significant reduction of superficial cells and to a clearer exposition of cells from deeper layers mimicking a high-grade
dysplasia (parabasal cells with higher nucleus/cytoplasm
ratio, hyper- or hypo-chromasia, presence of small cells with
intense eosinophilic cytoplasm with pycnotic and irregular nuclei, endometrial cells undergoing degeneration, macrophages
mimicking squamous cells with high grade atypia).
The HPV molecular test and the comparison with the histological counterpart when available (the gold standard in the
diagnostic algorithm) allowed to re-evaluate several cytological diagnoses of H-SIL or AGC previously performed.
Conclusions. Some years ago it was proposed to identify and
report a subgroup of ASCUS lesions showing atrophy that
would have benefited from repetition following local or systemic therapy (as also recommended by the Bethesda system).
The introduction of the HR HPV test is responsible for a new
variable in the evaluation of triage pap-tests: cytologists are
aware on one side to be facing selected higher risk patients,
and on the other side to be dealing with patients undergoing a
stricter follow-up in case of negative results (1 year instead of
the conventional screening interval).
References
1
Confortini M, Carozzi F, et al. Prevenzione del carcinoma della cervice uterina. Dal Test HPV al vaccino. Elsevier srl 2012.
2
Montanari G, Parisio F, Accinelli G, et al. Syllabus a cura dei componenti del gruppo per il controllo di qualità in citologia cervico
vaginale “Prevenzione Serena “ di Torino. Quaderni CPO-Piemonte,
n. 13, Torino gennaio 2007.
3
Gorodeski GI. Vaginal-cervical permeability decreases after menopause. Fertility Sterility 2001;76:753-61.
4
Sanguedolce F, et al. Efficacia della citologia in strato sottile nel
paptest della menopausa: confronto con lo striscio convenzionale. 3°
Congresso Nazionale SIAPEC-IAP. Firenze, 26-30 settembre 2004.
Patologica 2004;96:299-300.
5
Vooijs GP. Benign proliferative reactions, intraepithelial neoplasia, and
invasive cancer of the uterine cervix. In: Bibbo M (ed). Comprehensive
Cytopathology. 2nd ed. Philadelphia: Saunders 1997, pp. 167-168.
6
Abati A, Jaffurs W, Wilder AM, et al. Squamous atipya in tha atrophic
cervical vaginale smears. Cancer 1998;84:218-25.
Venerdì, 29 giugno 2012
ore 16.30-18.30
Ginecologia (II sessione)
Chairmen: Cesare Gentili (Massa Carrara), Domenico Ientile (Palermo)
Quality control procedures in a cytopathology
laboratory: which ones are necessary and why
M.R. Giovagnoli
Dipartimento di Medicina Clinica e Sperimentale, Roma
The minimum accepted quality control (QC) procedures in
the United States and in some European countries will be
presented, besides International Guidelines.
Moreover, there will be analysed the results of an inquiry
on QC activities routinely performed in some cytopatology
laboratories of various Italian regions. These results will be
compared with the numerous QC procedures suggested by the
National and the European Guidelines; possibilities and difficulties of their application will be underlined.
Controllo di qualità interlaboratori: regolare peer
review nel programma di screening della regione
Piemonte
L. Viberti
Struttura Complessa di Anatomia Patologica Ospedali Martini e Valdese, Torino
In regione Piemonte la Prof. Gioia Montanari aveva coordinato sin dalla fine degli anni 90 i controlli di qualità per la
citologia cervicovaginale e fu la grazie a questa esperienza
che venne facile traslare l’organizzazione anche nel Centro
Unificato di Screening della Città di Torino. In tale sede
vengono regolarmente organizzate sedute di CdQ, che, in
totale, dal 2009, hanno comportato 10 giornate all’anno per
il confronto interlaboratori, destinate ai lettori del programma
134
di screening, con accreditamento delle riunioni. Il sistema
utilizzato è quello della revisione collegiale con microscopio
multi teste, quindi con numero limitato di partecipanti e con
la partecipazione di tutor esperti.
I centri regolarmente partecipanti sono 20 per il Piemonte
1 per la Valle D’Aosta e occasionalmente 2 dalla Liguria.
Queste riunioni sono state aperte a ospiti stranieri dello Zambia, della Bosnia, della Romania, nell’ambito di progetti di
sviluppo.
I casi possono essere presentati dai partecipanti, come casi
discordanti, o di maggior interesse, o pro diagnosi o proposti
dal Tutor, con richiesta di selezione di vetrini di una predefinita categoria diagnostica.
Anche le diagnosi di benignità quali le ACR sono state portare
in confronto e discussione, quando hanno costituito un incremento di richiami e ripetizioni.
In ogni riunione vengono registrati i casi studiati, formalizzate
le diagnosi proposte e la diagnosi formulata in concordanza.
Il punto critico nella scelta di questo controllo di qualità
interlaboratori consiste nella necessità di spostamento degli
operatori provenienti da altre città verso il Centro Unificato
di screening della Città di Torino, ma la tipologia della sede
degli incontri vicina alla stazione ferroviaria, riduce il disagio
della mobilità.
Peraltro l’accreditamento del corso con acquisizione di crediti
ECM (utili e finalizzati al miglioramento delle competenze
specifiche), la possibilità della visione diretta del preparato
con il microscopio (strumento consueto di lavoro), la possibilità per tutti di discussione aperta e diretta, la osservazione comparata con descrizione e quindi l’oggettivazione
dell’immagine diagnostica, riteniamo che siano elementi di
grande utilità nella formazione e nel mantenimento della
preparazione, anche in confronto alla odierna possibilità di
trasmissioni di immagini.
Molecular biology and immunocytochemistry
applied to cervical cytology: integrated report,
ancillary assay or diagnostic procedure?
M.D. Beccati, O. Bulzoni, C. Buriani, C. Cavicchi, A. Carantoni, A.L. Delazer, S. Immovilli, M.G. Pascale, R. Parolini
Diagnostica Citopatologica, Azienda Ospedaliero-Universitaria,
Ferrara
Introduction. The primary benefit of using HPV testing is
the high sensitivity and high negative predictive value, since
the absence of carcinogenic HPV indicates an extremely low
risk of CIN3/ICC for 5-10 years. The role of HPV DNA testing as a solitary primary screening test (to replace cytology)
or as an adjunct to cytological screening has been evaluated
in large randomized trials over the past decade. Results show
overwhelming evidence that HPV DNA testing has a higher
sensitivity in comparison with cytology for detection of CIN3.
Yet its utility is constrained by its lower specificity than cytology, since the majority of HPV infections are transient and
would not progress to cervical dysplasia. In many European
settings, a strategy with primary screening by HPV testing
followed by cytology triage of HPV DNA positives (‘sequential’ or ‘two-stage’ testing) was proposed and has been
evaluated in multiple RCTs. This strategy takes advantage
of the high negative predictive value of HPV DNA testing
and maximizes sensitivity, while reserving cytology for those
who have higher likelihood of dysplastic lesions. The reliance
on cytology, with subjective interpretation and substantial
inter-observer variability, along with potential for sampling/
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
collection errors, however, remains a challenge. Given limitations in use of both cytology and HPV DNA based approaches
as standalone tests for screening, the focus of cervical cancer
prevention research has been on development and validation of new disease-specific biomarkers of HPV-associated
transformation. For any biomarker to be useful, the test result
has to influence clinical management such as direct referral
for treatment, referral to colposcopy, increased surveillance
through more intensive screening or release to routine screening. Currently, a treatment threshold of CIN2 or worse lesions is widely used, despite the fact that a large percentage
of CIN2 lesions spontaneously regress. Furthermore, there is
increasing evidence that even CIN3 is a heterogeneous group;
only about 30–50% of large CIN3s are estimated to invade to
cancer over a long time period.
Biomarkers. An important area of cervical cancer biomarker
research focuses on the identification of markers for cervical
lesions that likely progress to cancer. The use of biomarkers
in both cervical cytology and histology has demonstrated
the ability to overcome issues with both false-positive and
false-negative results, leading to improved positive predictive
value of cervical screening results. Numerous biomarkers for
the detection of cervical disease have been identified. Many
of these are proteins involved in cell cycle regulation, signal
transduction, DNA replication, and cellular proliferation.
E6/E7 mRNA Detection. The progression from a transient
to a transforming HPV infection is characterized by a strong
increase of HPV E6/E7 mRNA and protein expression. Multiple studies have evaluated the role of detection of mRNA
transcripts in cervical scrapings to identify cervical precancer.
At least two commercial platforms are currently available:
PreTect® Proofer (Norchip [marketed as NucliSENS EasyQ®
by BioMerieux in some European markets]) and APTIMA®
(GenProbe). A ‘best evidence synthesis’ for E6/E7 mRNA
HPV testing accuracy was provided, that reflected a sensitivity ranging between 0.41 to 0.86 for the PreTect Proofer/
NucliSENS Easy Q assays while a higher range – from 0.90
to 0.95 – for the APTIMA assay. The specificity ranged from
0.63 to 0.97 and from 0.42 to 0.61 for the PreTect Proofer/
NucliSENS EasyQ and APTIMA assays, respectively. The
considerable difference in sensitivity (and specificity) between PreTect Proofer/EasyQ and APTIMA may in part be
explained by the difference in type coverage: the former tests
detect only five types (HPV16, 18, 31, 33, 45), while the latter
covers 14 types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56,
58, 59, 66, 68).
p16ink4a. The biomarker most widely evaluated is p16ink4a, a
cyclin-dependent kinase inhibitor that is markedly overexpressed in cancerous and precancerous cervical tissue. p16ink4a
is a cellular correlate of the increased expression of the viral
oncoprotein E7 that disrupts a key cell cycle regulator, pRb,
in transforming HPV infections. The disturbance of the Rb
pathway leads to a compensatory overexpression of p16ink4a
through a negative feedback loop. The resultant overexpression and cellular accumulation of p16ink4a is a specific marker
of cervical precancerous lesions and can be measured through
immunocytochemical staining of histology and cytology slide.
A commercially available CE-marked assay (CINtec®, mtm
Laboratories) has been widely validated. Liquid-based cytology systems such as ThinPrep®, SurePath™, CYTO-screen
system® and others have been used in these studies. p16ink4a
has been evaluated as a standalone test and as an adjunct to
cytology or HPV testing. There is a substantial heterogeneity
in methods used for defining p16ink4a positivity in the cytology application, including quantitative and morphologic ap-
135
sessioni scientifiche
proaches. The sensitivity has ranged between 0.59 and 0.96
and the specificity has ranged between 0.41 and 0.96 for the
detection of CIN2+ lesions in clinical studies, reflecting the
heterogeneity in test interpretation and analyzed populations.
Recently, a dual immunostain of p16ink4a with Ki-67 (CINtec®
PLUS) has been introduced that is supposed to substantially
simplify and standardize the evaluation of stained slides.
One study, nested in the NTCC randomized controlled trial
considered P16INK4A overexpression as determined by immunostaining. Samples were obtained in unselected HPV
DNApositive women and all of them had colposcopy. Among
HPV positive women age 35-60 the cross sectional sensitivity
of p16INK4A immunostaining was 92% (95%CI 79-98%) for
CIN2+ and 86% (95%CI65-97%) for CIN3+. Specificity was
57% (95%CI 51-63%) for <CIN2 and 56% (95%CI 50-61%)
for <CIN3. The relative detection vs. cytology (interpretable
as relative cross-sectional sensitivity) of HPV testing with
triage by p16INK4A immunostaining and no further repeat
was estimated to be 1.53 (95% CI 1.15-2.02) for CIN2+ and
1.32 (95% CI 0.88-1.95) for CIN3+ while the relative referral
to colposcopy was 1.08 (95% CI 0.96-1.21). Such results are
similar to those obtained with “cytological triage” as defined
above in the other RCTs. Therefore there is evidence (level II)
that HPV DNA testing with p16INK4A triage and no further
repeat is more sensitive than cytology-based screening with
no increase of referral to colposcopy. However some data
suggest low reproducibility of the interpretation of p16 positive cells. In addition, no longitudinal data on the subsequent
risk of high grade CIN in women who are p16INK4A negative are published to the moment. In conclusion, there is not
currently sufficient evidence to recommend the routine use of
p16INK4A over-expression for triaging HPVpositive women
(strength C)
Markers of Aberrant S-phase Induction. The cell cycle
activation mediated by HPV oncogenes in transforming
infections is characterized by aberrant S-phase induction.
An assay detecting two proteins indicating aberrant S-phase
induction, topoisomerase IIA (TOP2A) and minichromosome
maintenance protein 2 (MCM2) is commercially available
(ProEx™ C by Becton Dickinson). Few clinical studies with
limited sample size have shown that it has a sensitivity ranging between 0.67 and 0.99 and specificity ranging between
0.61 and 0.85.
Conclusion. Cervical cancer prevention is at a transition from
cytology-based screening programs to HPV-based prevention.
New biomarkers will be important to decide who among the
HPV-positive women needs to be referred for further evaluation or treatment. Large studies are currently underway for
various triage biomarker candidates. It will be important to reserve treatment for those women who are at risk of developing
cancer, rather than treating any high-grade lesion. Prospective
biomarkers may play an important role in these therapy decisions, made easier by an integrated report to support the clinician in a patient-risk-tailored decision making process.
Disease into the cervical canal: cytobrush,
curettage or conization? The adequacy
of the sampling
C. Gentili
Associazione Culturale Enea Carrara
Objective. The 2006 ASCCP consensus guidelines for managing women with abnormal cervical cancer screening tests
describe “endocervical sampling” as a “preferred” management when colposcopy is inadequate or colposcopic findings
do not explain an abnormal Pap test result 1.
The ASCCP guidelines do not distinguish between endocervical curettage (ECC) and endocervical brush cytology (EBC)
for the endocervical sampling. There is growing evidence that
EBC has advantages over ECC in terms of increased sensitivity for detection of lesions beyond colposcopic view, as well
as higher adequacy rates. The aim of this revue is to evaluate
and compare the efficacy in obtaining an adequate endocervical sampling using the endocervical brush and the endocervical curettage and their contribution to the management of
patients with abnormal cytology.
Study design. A retrospective revue of 29 articles on PubMed
searches from 1983 to 2010.
The articles were divided into three groups
1) The evaluation of cervical canal with Endocervical Curettage (ECC) and its contribution to the management of
patients with abnormal cytology
2) The evaluation of the cervical canal with the endocervical
brush (EBC) and its contribution to the management of
patients with abnormal cytology
3) Comparison of endocervical curettage and endocervical brusching
Results. Some studies identified a lower specificity and adequacy of EBC compared with ECC 2 3, but subsequent studies
showed, that using a more accurate sampling technique with
sleeved cytobrush and intensive endocervical brushing and
subsequent examination with LBC and cell block, both systems
were equivalent with regard to the specificity and sensibility 4 5.
EBC showed more adequacy, was easier to use, less costly and
painful 4 5, and furthermore could be used in pregnancy 6.
Conclusions. EBC is acceptable for endocervical evaluation
as substitute for endocervical curettage at colposcopic biopsy.
Both methods are not predictive of invasive carcinoma.
References
1
Wright TC Jr, Massad LS, Dunton CJ, et al. 2006 consensus guidelines
for the management of women with abnormal cervical screening tests
Am J Obstet Gynecol 2007;197:346-55.
2
Hoffman MS, Sterghos S Jr, Gordy LW, et al. Evaluation of the cervical
canal with the endocervical brush. Obstet Gynecol 1993;82(4 Pt 1):573-7.
3
Klam S, Arseneau J, Mansour N, et al. Comparison of endocervical
curettage and endocervical brushing. Obstet Gynecol 2000;96:90-4.
4
Boardman LA, Meinz H, Steinhoff MM, et al. A randomized trial of
the sleeved cytobrush and the endocervical curette. Obstet Gynecol
2003;101:426-30.
5
Maksem JA. Endocervical curetting vs. endocervical brushing as case
finding methods. Diagn Cytopathol 2006;34:313-6.
6
Stillson T, Knight AL, Elswick RK Jr. The effectiveness and safety
of two cervical cytologic techniques during pregnancy. J Fam Pract
1997;45:159-63.
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I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Venerdì, 29 giugno 2012
ore 14.00-16.00
Immunocitochimica
Chairmen: Claudio Clemente (Milano), Roberto Fiocca (Genova)
Immunocytochemistry for lung cancer
subtyping
M. Papotti, V. Monica, L. Righi
University of Turin at San Luigi Hospital, Orbassano (Torino), Italy
Background. Lung cancer classification was specifically
designed for surgical specimens and recognizes four major histological subtypes, namely squamous cell carcinoma
(SQC), adenocarcinoma (ADC), large cell (LCC) and small
cell carcinomas (SCLC). Other rare primary tumors include
carcinoids, salivary gland type carcinomas and non epithelial
neoplasms. Metastatic tumors are an additional source of diagnostic problems. Lung neoplasm classification is more difficult to apply on biopsy and cytology specimens, especially
in the case of poorly differentiated tumors, or heterogeneous
cellular patterns, or technical problems or artefacts. An accurate differential diagnosis and precise subtyping was less
relevant in past years and the generic separation of SCLC
from all other non-small cell lung cancers (NSCLC) was well
accepted by oncologists. 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 differentiated SQC or ADC. However,
a percentage of poorly (or “less-well”) differentiated cases are
still simply diagnosed as NSCLC, in the lack of morphological 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 now mandatory for personalized treatments.
The immunocytochemical detection of specific markers is
useful to identify squamous or glandular or neuroendocrine
differentiation and characterize poorly differentiated NSCLC
in almost all types of cytological material, including smears
(either unstained or de-stained after conventional cytomorphological analysis). Besides a diagnostic role, immunocytochemistry may contribute to investigate markers having
a prognostic significance (eg Ki67) or being predictive of
response to chemotherapy. To this purpose, molecular tests
may be applied also in cytological (cell block) and biopsy
materials, and may help to implement the diagnosis and define
the therapeutic strategy.
Lung cancer histological subtypes that are morphologically
recognizable on small biopsy fragments or cytological samples are basically three, i.e. ADC, SQC and SCLC. The latter
usually requires immunocytochemical confirmation using
a more or less extensive panel of neuroendocrine markers.
Large cell carcinoma and its variants (including large cell neuroendocrine carcinoma, LCNEC), and sarcomatoid carcinoma
can be definitely diagnosed on surgical specimens, only. The
group of LCC has an heterogeneous immunocytochemical
profile, probably reflecting divergent differentiation mainly
along squamous and glandular lineages (and more rarely
have a neuroendocrine phenotype). Most adenocarcinomas
are positive for TTF-1, napsin-A, cytokeratin 7 (CK7) as well
as for surfactant apo-protein A (SPA) and MUC5AC; they
are generally negative for CK5/6, CK20 and p63. Indeed,
this latter is occasionally found in up to 30% of adenocarcinomas, although with a focal and weak reactivity. p40 is a
truncated isoform of the p63 gene and is much more specific
of squamous lineage than p63 protein itself, being completely
unreactive in pulmonary adenocarcinomas, and is therefore
to be preferred in the differential diagnosis marker panel.
Finally, regarding the above immunoprofile, mucinous adenocarcinoma of the lung is an exception, since CK20 (and even
the intestinal marker CDX2) rather than TTF-1 or napsin A
may be expressed. Squamous cell carcinoma consistently and
strongly reacts with p63 (and also p40), CK5 and desmocollin-3, and never for TTF-1 and napsin A. Conversely, CK7
is less specific and may be detected in some SQC, as well.
Neuroendocrine large and small cell carcinomas are known
to express chromogranin A, synaptophysin and CD56, but not
high molecular weight cytokeratins.
In the daily practice, a panel of immunocytochemical markers
is generally employed, in variable numbers, based on availability of reagents, financial resources and the cytopathologist’s personal experience. Once a neuroendocrine phenotype
has been excluded, the most commonly used marker panel for
NSCLC sub-classification includes TTF-1, p40 (p63), CK7
and CK5. The former two are nuclear markers and seem more
reliable in cytological materials, which are sometimes poorly
cellular and/or may have poor cytoplasm preservation in isolated tumor cells.
In the case of poor cellularity, the use of cocktails of antibodies was explored [Righi et al. Cancer 2011; 117:3416-23],
to reduce the number of necessary glass slides. Despite the
results were largely depending on the cellularity and cell
arrangements of each single case, in general, it seemed that
combinations of two different-by-lineage antibodies (eg TTF1
with desmocollin-3 or p63 with napsin A) were easier to interpret than homogeneous-by-lineage antibody cocktails (TTF1
and napsin A or p63 and desmocollin-3) in the tested NSCLC
cell blocks obtained by fine needle aspiration biopsy.
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). A fraction of TTF-1 negative ADC cases can be
correctly classified by napsin A expression, although the two
markers together do not reach a 100% sensitivity. Conversely,
p63 (and p40) expression in SQC is robust and not affected by
tumor grade, although p63, but not p40, immunoreactivity has
been observed in a subset of ADC. Problems of interpretation
can occur in the presence of ambiguous phenotypes or discrepant marker reactivity. This event may be related to aberrant
marker expression on the one side and heterogeneous tumor
phenotypes on the other, as in the case of adenosquamous carcinoma, which is extremely difficult to recognise in cytology or
biopsy specimens. Additional antibodies may be used in such
cases, a final report of dual differentiation or “null” phenotype
can be expected in a small fraction of “NSCLC” cases.
sessioni scientifiche
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 defining a personalized
therapy, followed by other targets of specific drugs including
ALK, c-MET on the side of tyrosine kinase inhibitors and
specific DNA repair & synthesis enzymes, such as ERCC1,
thymidylate synthase or topoisomerase II for the purposes of
a chemotherapy strategy. Such a range of different molecular,
hybridisation or immunocytochemical tests and opportunities
highlights the absolute need of saving as much material as
possible during the diagnostic step of each individual tumor
(immunophenotypic markers), since this same material could
be the only tissue available for further analyses of predictive
factors in the case of inoperable lung tumors.
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, immunocytochemistry can be helpful, if
not mandatory, in confirming the neuroendocrine phenotype
(SCLC and LCNEC), and in defining the histotype (or the
most likely differentiation lineage) of poorly differentiated
tumors; 3) TTF-1 & napsin A on the one side, and p40 &
desmocollin-3 on the other, seem to date the most valuable
markers for ADC and SQC, respectively; 4) in cytological
specimens, prognostic and predictive markers can also be
investigated for the purposes of better defining the therapeutic strategy. These also include molecular tests for EGFR
and KRAS mutations and other oncogene alterations (ALK,
cMET, etc). Cell blocks or better core biopsies are more
suitable for the purpose of molecular tests, but smeared cells
can also be successfully scraped for individual mutational
analyses; 5) immunophenotyping will ultimately allow to
abandon the “NSCLC” category, thus reducing as much as
possible the number of unclassified cases even in cytological
specimens.
Immunocytochemistry of bronchoalveolar lavage
A. Capitanio
Department of Cellular Pathology, University College London Hospital, UK
The Bronchoalveolar Lavage (BAL) technique using a flexible fibreoptic bronchoscope was described for the first time
on the Journal of Laboratory and Clinical Medicine in 1974
by Dr. Reynolds and Dr. Newball. Beacause of its possibility
to sample large areas of the distal lung regions, BAL evolved
from research tool to diagnostic method to identify lung infections and to diagnose interstitial lung diseases.
Immunophenotyping of BAL fluid cells, mainly of lymphocytes, applies to a wide range of diseases and conditions.
Excluding the malignancies, the list includes sarcoidosis,
hypersensitivity pneumonitis, asthma and many infectious
diseases including tuberculosis, Pneumocystis Carinii, CMV,
HIV infection and Hepatitis C. Lung and Bone marrow transplantation, alcoholic cirrhosis, bronchiolitis and some pneumoconiosis can also been investigated with this technique.
In many of these conditions the number of lymphocytes in
BAL is increased. Examples are sarcoidosis, tuberculosis,
HIV, berylliosis and hypersensitivity pneumonitis.
137
A typical example of lymphocytic Immunophenotyping concerns the CD4 and CD8 T lymphocytes. Their distribution is
abnormal in some diseases like hypersensitivity pneumonitis
and sarcoidosis (30-70% of total cell count) while it is normal
in tuberculosis.
CD4/CD8 ratio can be also 20:1 in sarcoidosis, while it is
often reversed in hypersensitivity pneumonitis.
Immunocytochemistry, both in light and fluorescence microscopy and flow cytometry have been used to recognize and
enumerate the cells of BAL fluid.
The use of immunocytochemistry in all its possible technical variations (immunoperoxidase, alkaline phosphatase–anti
alkaline phosphatase and immunofluorescence) is common
and widely established, but the reliability of the results depends on technical and professional factors. Quality of the
immunoreaction, number of cells counted and experience of
the observer play in fact an important role for the quality of
the results.
Flow cytometry is able to analyse a huge number of cells in
the unit of time. This characteristic is certainly very important
in the statistical validation of the results. Nevertheless, some
important issues can bias flow cytometric performances. First
of all, while for flow cytometry of blood cells there are a number of well-known guidelines, which ensure a high degree of
standardization and reproducibility, for BAL fluid cells analysis there are not widely accepted guidelines. The lack of common rules makes difficult the comparison of results between
various reports. The lymphocytic gate chosen for the analysis
represents a good and important example of this problem.
Literature data show at least five different gating methods:
scatter characteristics of blood lymphocytes; light scatter
alone (side scatter by forward scatter); light scatter by CD3
positive cells; side scatter by CD45; light scatter by CD14CD45 combination. Considering that “gating” is probably the
most important operation identifying different cell population,
it is easy to understand the relevance of the problem.
A second issue rises from the heterogeneity of BAL cell population as well as from the presence of clusters and overlapping
cells in the fluid itself. This can lead to the exclusion of interesting cells and, on the contrary, to the exclusion of important
cells from the final count. Finally, cell autofluorescence can
modify the light scatter and alter the result. Finally, also the
presence of dead apoptotic cells, erythrocytes and naked nuclei can give false and/or unwanted light signals.
If these technical issues are not considered during the sample
preparation and analysis, the accuracy of the results can be
seriously biased.
The immunoprofile analysis of some Interstitial Lung Disease
and Tuberculosis follows.
BAL immunoprofile in some Interstitial Lung Disease.
Acute Interstitial Pneumonia (AIP). BAL is difficult to perform in patients with AIP and the cellular profile of the fluid
is non specific: increased number of neutrophils, occasionally
increased lymphocytes and atypical reactive pneumocytes.
The immunoprofile is non-specific.
Cryptogenic Organizing Pneumonia (COP). Increased
content of T lymphocytes and decreased CD4/CD8 ratio is
common. Increased number of neutrophils and eosinophils
can occur. Treatment with corticosteroids normalizes the cell
count and the CD4/CD8 ratio. This should be considered
monitoring a patient treated for COP.
Non Specific Interstitial Pneumonia (NSIP). Two different
types of NSIP can be considered: the cellular NSIP and the
fibrotic NSIP. Independently from the subtype, the CD4/CD8
ratio is decreased with an increment of neutrophils. Overall
138
cellularity is variable and usefulness of BAL for NSIP monitoring is questionable.
Desquamative Interstitial Pneumonia (DIP) and Respiratory Bronchiolitis Interstitial Lung Disease (RB-ILD).
Both the diseases are smoking related and the BAL fluid usually shows pigmented macrophages. Eosinophils number can
be increased in DIP. Only some cases show lymphocytosis
without peculiarity in the immunophenotype.
Lymphocytic Interstitial Pneumonia (LIP). LIP is a chronic
ILD usually associated with collagenopaties such as Sjögren
syndrome. BAL fluid usually shows lymphocytosis. Some
studies report an increased CD4/CD8 ratio, but the absence
of clonality is the main clue for the differential diagnosis with
lymphomas.
Idiopathic Pulmonary Fibrosis (IPF). IPF is a progressive
fibroproliferive disease histologically associated with Usual
Interstitial Pneumonia (UIP). BAL fluid shows a low number
of lymphocytes and a clear correlation between the CD4/CD8
in BAL fluid and in lung parenchyma.
Sarcoidosis. The diagnosis of sarcoidosis is based on three
main criteria: an overall radiological picture consistent with
the diagnosis, histological evidence of non caseating granulomata and the exclusion of any other possible disease with the
same or similar clinical or histological features. At diagnosis
time about 90% of patients show a lymphocytic alveolitis in
BAL fluid. In contrast, the overall cellularity of BAL is slightly elevated if compared with the high cellularity of BAL fluid
in Hypersensitivity Pneumonitis or Extrinsic Allergic Alveolitis. Other cellular characteristics of BAL in Sarcoidosis are
a usually normal proportion of eosinophils and neutrophils
and the lack of plasma cells and foamy macrophages. An
increased CD4/CD8 ratio is commonly considered a typical
feature of Sarcoidosis. Even if some patients (about 15%) can
show a decreased ratio, an elevated ratio in the proper clinical
and radiological settings can avoid the need of lung biopsy
for the final diagnosis of Sarcoidosis. Unfortunately, BAL
cellularity and immunophenotype are not useful to assess the
activity of the disease or its prognosis.
BAL cellular pattern and immunoprofile in Tuberculosis.
Recent findings demonstrate that neutrophils are more abundant than macrophages in patients affected by Tuberculosis.
Besides, Mycobacteria are found more frequently in neutrophils cytoplasm than within macrophages. This suggests that
neutrophils play an essential role in Mycobacteria replication
especially in the first phase of the disease.
Moreover, the number of lymphocytes and eosinophils is increased in the BAL of children with tubercular infection with
an increment of CD4 lymphocytes.
Standardizzazione delle metodiche
immunocitochimiche: problematiche
preanalitiche, tecniche e interpretative
F. Tallarigo
Anatomia Patologica ASP Crotone
L’immunocitochimica (ICC) è una tecnica di identificazione
degli antigeni nei campioni citologici mediante anticorpi
specifici per gli antigeni stessi. Il legame antigene-anticorpo,
e di conseguenza il riconoscimento della presenza dello
specifico antigene in questione, nella maggior parte dei casi
si ottiene utilizzando un anticorpo secondario (solitamente
marcato), un enzima o un sistema complesso enzimatico ed
un substrato cromogeno. Questa tecnica viene utilizzata per
la diagnosi dei diversi tipi di tumori e degli agenti infettivi,
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
nonché per l’identificazione dei marcatori di proliferazione e
della differenzazione cellulare.
Pertanto si ritiene che l’analisi immunocitochimica sia uno
strumento ormai irrinunciabile nel campo della diagnostica
isto-citopatologica. Essa viene applicata sia come metodo
di completamento diagnostico che consente di identificare
espressione di particolari geni correlabili con particolari
caratteristiche di valenza prognostica o predittiva (ad esempio
l’indice di proliferazione cellulare con Ki67, la valutazione
semiquantitativa dell’espressione di recettori ormonali e di
Her2 nel carcinoma della mammella), sia nella validazione
di ipotesi diagnostiche basata sull’identificazione di specifici
“profili immunofenotipici”. In base a quanto detto si evince
che l’impiego primario della ICC è quello di contribuire
alla diagnosi delle neoplasie, anche se attualmente non esiste
alcun marcatore immunocitochimico che consente di differenziare le cellule neoplastiche da quelle non neoplastiche.
Difatti l’ICC viene eseguita solo dopo aver formulato una
diagnosi di neoplasia maligna utilizzando le valutazioni morfologiche di routine. Questa metodica, d’altronde come tutte
le altre è definita in termini di sensibilità e specificità che nel
caso dell’immunocitochimica dipendono da diversi fattori.
La sensibilità di un particolare anticorpo può essere molto
elevata, in particolare quando si impiegano anticorpi monoclonali, la specificità è simile alla sensibilità, poiché dipende
tecnicamente da quella di un dato anticorpo per un particolare
marcatore. Solitamente più elevata per gli anticorpi monoclonali rispetto ai policlonali, ma è anche determinata dalla
specificità di un determinato marcatore per un particolare
istotipo neoplastico.
Dal punto di vista tecnico la metodica consta di quattro fasi:
(pre-analitica; analitica; post-analitica; interpretativa).
Prima di descrivere singolarmente le vari fasi, è importante
sottolineare che l’indagine può essere effettuata sia su preparati allestiti con metodica tradizionale, sia allestiti mediante
strato sottile, utilizzando vetrini già colorati (EmatosilinaEosina o Papanicolaou) o in bianco.
La fase pre-analitica è caratterizzata principalmente dalla scelta dei vetrini. I vetrini devono essere cellulati e ben strisciati.
Importante evitare quei vetri in cui è presente sovrapposizione
cellulare in cui le cellule non sono ben distribuite. Altro
criterio fondamentale è che la popolazione cellulare risulti
ben fissata. Questa fase è considerata di cruciale importanza
per l’utilità diagnostica dell’immunocitochimica. Nella fase
pre-analitica se si utilizzano vetri già colorati, questi dapprima vengono messi a smontare, successivamente si elimina
l’eccesso di montante facendo dei passaggi in “Xilolo”; “
Etanolo”; “Etanolo al 95%”; Acqua distillata. Giunti in Acqua
distillata i vetrini sono sottoposti allo smascheramento antigenico, eseguito immergendo gli stessi in un tampone liquido
ad alto pH, alla temperatura di 98°C, per 20 minuti. Terminato
lo smascheramento antigenico, i vetrini vengono fatti raffreddare a temperatura ambiente per 20 minuti.
La fase analitica prevede l’applicazione del principio attivo
della metodica: il legame antigene-anticorpo primario. Questo
legame è strettamente influenzato dalla sensibilità e specificità
dell’anticorpo, che a sua volta è dipendente dalla titolazione,
e dal tempo di incubazione dell’anticorpo. In questa fase i
vetrini sono inseriti nell’immunocoloratore, che provvede a
dispensare l’anticorpo primario, che si lega al sito antigenico
di interesse, conteggia il periodo di incubazione ed effettua
vari lavaggi, prima di distribuire l’anticorpo secondario, che è
legato al sistema di rivelazione.
Nella fase post-analitica i vetrini sono immersi in acqua distillata e controcolorati in un bagno di ematossilina per 2 minuti,
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sessioni scientifiche
passati rapidamente in acqua corrente ed infine disidratati prima
dell’applicazione del vetrino copri-oggetto. A questo punto i
vetrini sono pronti per essere esaminati al microscopio ottico.
Nella fase interpretativa entra molto la soggettività caratterizzata dall’esperienza di colui che effettua la valutazione dei
preparati citologici. L’operatore deve soprattutto conoscere
che esistono alcune strategie che possono aiutare a distinguere
una colorazione immunocitochimica specifica da una non
specifica o legata ad artefatti tecnici. Le cause di colorazioni
non specifiche dovute ad artefatti sono molteplici: presenza
di perossidasi o fasfatasi non adeguatamente inibite; crossreattività dell’anticorpo primario con un antigene diverso da
quello in studio; legame aspecifico dell’anticorpo alla cellula
o tessuto (attraverso il frammento Fc o per carica elettrica);
inadeguata fissazione delle cellule. Scarsa fissazione provoca
un eccesso di “fondo”, eccesso di fissazione provoca una
ridotta sensibilità.
In conclusione possiamo affermare che anche in citologia
la valutazione e la descrizione della immunoreattività di un
singolo marcatore immunofenotipico o di diversi marcatori
in una popolazione cellulare si traduce in una valutazione
qualitativa e quantitativa e quindi tale da poter consentire una
sintesi diagnostica dei risultati nel contesto di una diagnosi
differenziale.
Venerdì, 29 giugno 2012
ore 16.30-18.30
Tiroide
Chairmen: Lucio Palombini (Napoli), Francesco Nardi (Negrar)
The FNC classification SIAPEC/IAP in thyroid
diagnosis
L. Palombini
Anatomia Patologica, Università di Napoli Federico II partecipante
al Consenso Italiano
In the October of 2006, on proposal of Prof. Aldo Pinchera,
the “Società Scientifiche italiane di Endocrinologia (SIE),
“Associazione Italiana della Tiroide (AIT)” and of “Anatomia Patologica e Citopatologia (SIAPEC/IAP – International
Academy of Pathology – Italian Division)” represented by
the Authors: Paolo Vitti, Teresa Rago, Rossella Elisei, Enrico Papini, Fabio Orlandi, Antonino Belfiore, Furio Pacini;
and of the Participants: Aldo Pinchera, Enio Martino, Paolo
Miccoli, Giancarlo Di Coscio, Fabio Monzani, Alfredo Pontecorvi, Concetto Regalbuto, Fulvio Basolo, Arrigo Bondi,
Gianni Bussolati, Anna Crescenzi, Guido Fadda, Oscar
Nappi, Francesco Nardi, Lucio Palombini, Mauro Papotti,
Gianluigi Taddei, developed the “Gestione clinica del paziente con patologia nodulare tiroidea: Consenso Italiano” 1.
The Consent was, in the intention of the Authors, a “clinical”
document into which a classificative system was “included”.
This system was supposed to be “operative” of the fine-needle
cytopathology report, and it had to be simple, concise, easily comprehensible. It could, properly introduced into the
clinical-laboratory-instrumental presentation of the patient,
permit the clinical-therapeutical management of the same
patient. The extract of the cytological part of the “consensus”
of the thyroid nodule has subsequently been published autonomously organized by the group SIAPEC/AIP 2 and, however,
after that of the Papanicolaou Society of Cytopathology 3. The
Italian consent of the management of the thyroid nodule is
also, chronologically, next to similar consents of the American Association of Clinical Endocrinologists-Associazione
Medici Endocrinologi (AACE-AME, ATA -2006) 4 and of
the American Thyroid Association (ATA -2006) 5 but it is
contemporary to that of the British Thyroid Association (BTA
-2007) 6 and of the National Cancer Institute (NCI -2007) 7.
The group SIAPEC/IAP was not requested and did not want
to impose diagnostic microscopical criteria but, preferably,
only contributed to compile a document of connection among
cytologist-clinician-patient, so limiting, however, the report
discretion and giving guidelines of operativity, of quality control, and of suggestions.
The cathegories codified by the group SIAPEC/IAP are
five but the diagnostic cathegories are only four, all of them
indicated by the abbreviation TIR followed by a numerical
code from 1 to 5. The TIR 1 is not diagnostic; it indicates,
in fact, a cathegory that does not consent a diagnostic evaluation because of the not-representativity of the report and/or
because of the quality inadequacy of the smear; this cathegory
should not be superior to the 10% of the cases. The TIR 2 is
the cathegory into which neoplastic cells are not found but,
however, we can find cells from degenerative pathologies of
the thyroid (colloid goiter) or sub acute and chronic inflammatory pathologies. In the TIR 2 the expectation of False Positives should be beneath the 3% of the cases. The cathegory
TIR 3 does not indicate a conclusive diagnostic assessment
but an opinion of in conclusive/indeterminate (some prefer a
qualification to the other and this is, maybe, the only margin
of tolerance of the consensus SIAPEC/IAP). With the TIR 3
we declare the limit of the FNC test which does not conclude,
does not determine a definite diagnostic assessment to which
nonetheless should correspond a benign lesion in the 80% of
the cases. The TIR 4 is the cathegory of the clinical suspicion into which we consider all the cytologic presentations
not including every carachteristic necessary and sufficient to
formulate a conclusive assessment of malignity. A malignant
lesion should correspond to this assessment in about the 80%
of the cases. The TIR 5, at last, is the cathegory into which
neoplastic cells, primitive or secondary, are found. The expectation of the False Negatives in this cathegory should be
beneath the 3% of the cases.
We have associated to every cathegory an operative suggestion which can be both the repetition or the therapeutical
indication not, however, omitting the suggestion that, because
of the sensibility and the specificity of the FNC test, it remains
a screening test and thus the conclusive diagnosis remains the
histological one.
140
References
1
Gestione Clinica del paziente con patologia nodulare tiroidea: Consenso Italiano SIE 2007. L’Endocrinologo 2008;Suppl. 4:S1-S16.
2
Cytological Classification of Thyroid nodules. Proposal of the SIAPECIAP Italian Consensus Working Group. Pathologica 2010;102:405-6.
3
Guidelines of Papanicolaou Society of Cytopathology for the examination of Fine-Needle Aspiration Specimens from Thyroid Nodules.
Modern Pathology 1996; 9(6) 710-715.
4
AACE-AME medical guidelines for clinical practice for the diagnosis
and management of thyroid nodules. Endocr Pract 2006;12:63-102.
5
ATA management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2006;16:1-34.
6
Guidelines for the manegement of thyroid cancer British Thyroid Association. Royal College of Physicians 2007.
7. The National Cancer Institute Thyroid fine needle aspiration state of
the science conference: A summation. CytoJournal 2008;5:6.
Comparison of SIAPEC/IAP System and Bethesda
System in thyroid cythology
S. Crippa
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
References
1
Fadda G, Basolo F, Bondi A, et al. Cytological classification of thyroid
nodules. Proposal of the SIAPEC-IAP Italian Consensus Working
Group. Pathologica 2010;102:405-8.
2
Ali SZ, Cibas ES. The Bethesda System for Reporting Thyroid Cytopathology: Definitions, Criteria and Explanatory Notes. New York:
Springer 2010.
3
Krane JF, Nayar R, Renshaw AA. Atypical cells of undetermined
significance. In: Ali SZ, Cibas ES, eds. The Bethesda System for Reporting Thyroid Cytopathology: Definitions, Criteria and Explanatory
Notes. New York: Springer 2010, pp. 37-49.
4
Crippa S, Mazzucchelli L, Cibas ES, et al. The Bethesda System for
reporting thyroid fine-needle aspiration specimens. Am J Clin Pathol
2010;134:343-4; author reply 345.
5
Bongiovanni M, Crippa S, Baloch Z, et al. Comparison of 5-tiered and
6-tiered diagnostic systems for the reporting of thyroid cytopathology: A multi-institutional study. Cancer Cytopathol 2011 Oct 13. doi:
10.1002/cncy.20195.
Tab. I.
SIAPEC/IAP
Locarno, Svizzera
Tir1 Nondiagnostic
Over the past decade, several classification schemes for thyroid
gland FNA have been proposed by various professional organizations. Most of these schemes consist of 4 to 6 diagnostic
categories, which are not always comparable with each other.
This has led to confusion and differences in perceptions of diagnostic terminology in cytopathology reporting of thyroid FNA
between cytopathologists and clinicians. The main difficulty is
represented by “borderline” lesions characterized either by atypia
of undetermined significance and/or by a microfollicular pattern.
In this context, it is interesting to compare two systems for the
reporting of thyroid cytopathology: the 5-tiered SIAPEC/IAP
system 1 and the 6-tiered Bethesda system 2.
Both the classification systems provide a category for nondiagnostic FNA, a category for benign lesions and a category
for malignant lesions. However, there are also notable differences. The SIAPEC/IAP system provides a single category
for all borderline lesions, named indeterminated/follicular
proliferation. Conversely, the Bethesda system, as illustrated
in Table I, introduces two categories for borderline lesions,
namely “atypia/follicular lesion of undeterminated significance” (AUS/FLUS) and “follicular neoplasm or suspicious
for a follicular neoplasm”. The AUS/FLUS category is a
heterogeneous one and the Bethesda system outlines a variety
of scenario for which this category is appropriate3. It is worth
noting that, in many instances, a predisposing condition for
an AUS/FLUS diagnosis is a compromised specimen due to
scant cellularity, partial air-drying, or obscuring blood. We
discussed some doubts with the authors 4.
We compared the two systems in a multi-institutional study.
Sensitivity was equally high in the 5-tiered and in the 6-tiered
systems (98,3% and 99,2%, respectively), whereas specificity (54,3% and 22,8%) and diagnostic accuracy (76,3% and
56,5%, respectively) were much lower. Particularly, based on
the finding of the current study, the 5-tiered and the 6-tiered
reporting systems share similar negative predictive values for
benign cases, similar positive predictive value for malignant
cases and, above all, similar rates of false-negative and falsepositive case. Differences exist between the two systems with
regard to the percentage of cases categorized as benign and as
follicular proliferation/neoplasm.
Differences in the medicolegal environment of European
countries compared with the United States may influence the
way in which the two thyroid FNA reporting systems are used
to guide the clinical management 5.
Tir2
Negative for malignant
cells
Tir3
Indeterminate
(follicular proliferation)
Tir4
Suspicious of
malignancy
Tir5 Malignancy
Bethesda
I.
Nondiagnostic
II.
Benign
III.
Atypia/Follicular lesion of
undetermined significance
Follicular neoplasm/
IV. Suspicious for follicular
neoplasm
V. Suspicious for malignancy
VI. Malignant
Cytopathology and immunocytochemistry
of thyroid nodules processed by liquid-based
cytology
G. Fadda, E.D. Rossi
Division of Anatomic Pathology and Histology, Università Cattolica,
“Agostino Gemelli” School of Medicine, Rome, Italy
Introduction. The fine-needle aspiration biopsy (FNAB) was
widely appreciated as a diagnostic tool during the 1950s in
Sweden: since then it has spread worldwide because of its
simplicity, safety and the possibility of repetition.
FNAB is regarded as the most accurate method for the selection of patients with thyroid nodules for surgery and a very
cost-effective diagnostic test.
The thin-layer or liquid-based cytology (LBC) technique,
originally developed for application to gynecologic cervical smears, has progressively gained consensus after being
applied to both non-gynecologic and fine-needle aspiration
cytology.
Materials and methods. The LBC procedure includes twosteps which are the fixation of the totality of the material in
an alcohol-based solution (methanol or ethanol depending on
the technique); and the automated processing of the material
to obtain a thin layer of representative cells. A computerassisted device allows the transfer of the fixed and partially
disaggregated cells onto a single slide The two most common methods for processing the cytologic samples use an
alcohol-based fixative solution. In the first (ThinPrep2000
and 5000TM, Hologic Co., Marlborough, USA), the cells are
aspirated from a methanol-based solution (CytolitTM) then
filtered and transferred onto a positively charged slide with a
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sessioni scientifiche
gentle positive pressure. In the second, the cells are collected
in an ethanol-based solution (CytoRichTM), centrifuged twice
then slowly sedimentated onto a poly-L-lysinated slide and
eventually stained with a specific hematoxylin-eosin stain.
(SurePathTM, TriPath Imaging, Burlington, USA). The final
result for both methods is one slide for each lesion where all
cells are concentrated in a thin layer on the central area of the
slide measuring 20 square mms for ThinPrep and 13 square
mms for PrepStain LBC. The LBC method enables the storage
of a variable amount of cells in a preservative solution for up
to 6 months after the biopsy. The remaining material can be
used for the application of special techniques such as immunocytochemistry and molecular biology.
Results. The FNA cytology plays a key role in the preoperative diagnosis of thyroid neoplasms, because of the possibility: a) to select patients who should be addressed to surgery
from those who can be simply followed-up; b) to define the
surgical approach and/or c) to give to the patient correct information regarding the management of their lesion. The diagnostic accuracy of the cytology cannot equal that of histology
since the aspiration cytology may yield a diagnosis of “follicular neoplasm” which defines those lesions composed of
follicular aggregates of follicular cells which may correspond
to a follicular adenoma or to a differentiated carcinoma. These
differential diagnoses rely on histologic details (invasion of
the capsule and of the vessels, different patterns between inner
and outer portions of the nodule) rather than on the atypical
features of the follicular cells. Although a diagnosis of follicular neoplasm warrants the surgical removal of the lesion,
the possibility that a benign lesion can be removed affects the
figures of specificity and sensitivity of the procedure cited in
the literature. Nonetheless, FNA cytology is a fundamental
tool in the management of thyroid nodules.
Immunohistochemistry was introduced in the early 1970s
for the definition of the nature of the lesions, expecially
in the differential diagnosis between follicular and C-cell
derived neoplasms and in the identification of primary or
metastatic thyroid neoplasms (e.g. malignant lymphoma)
even if the main role is the identification of the markers of
malignancy which may distinguish malignant from benign
lesions regardless of the presence of capsular and vascular
invasion.
There have been only few reported experiences in literature
dealing with ICC applied on LBC. The use of ICC on the
cells stored in the preservative LBC solution yields excellent
results with most immunoreagents in terms of staining pattern,
intensity of the reaction and less amount of reagent due to the
clear background and smaller size of the LBC slide.
In our practice and based on the data in the literature, no
“magic single marker” may be useful but only the concordance of a panel should be considered especially in cases of
follicular lesions. The use of more than one immunomarker
is a further guarantee for a correct diagnostic approach, especially when a concordant panel is expressed and with excellent results also in the differential diagnosis between benign
and malignant follicular neoplasms.
HBME-1, Galectin-3 and RET proto-oncogene have shown
the best specificity and sensitivity in discriminating benign
from malignant differentiated tumors. These data emerged
from one of the paper of our group, in which the combination
of nuclear pleomorphism and positivity of the panel resulted
in 75% specificity and 89% diagnostic accuracy of FNAB. In
another study the complete immunocytochemical panel (made
up of HBME-1 and Galectin-3) was positive in 83.3% of malignancy and negative in 87.5% benign histological cases. In
the group of FN (TIR 3 according to the SIAPEC-IAP Italian
classification), the expression of HBME-1 and Galectin-3 on
LBC can effectively distinguish lesions which need immediate surgery (high risk FN) from those which can be followedup (low risk FN).
Conclusions. LBC-processed fine-needle biopsies represent
a valid alternative to conventional cytology. The possibility
of applying special techniques enhance the efficacy of the
cytological diagnosis of thyroid follicular-patterned lesions.
References
1
Fadda G, Basolo F, Bondi A, et al. Cytological classification of thyroid
nodules. Proposal of the SIAPEC-IAP Italian Consensus Working
Group [Classificazione citologica dei noduli tiroidei. Proposta del
Consensus Working Group italiano della SIAPEC-IAP]. Pathologica
2010;102:405-8.
2
Bishop-Pitman M, Abele J, Ali SZ, et al. Techniques for thyroid FNA:
a synopsis of the National Cancer Institute thyroid fine-needle aspiration state of the science conference. Diagn Cytopathol 2008;36:40724.
3
Rossi ED, Raffaelli M, Zannoni GF, et al. Diagnostic efficacy of
conventional as compared to liquid-based cytology in thyroid lesions.
Evaluation of 10,360 fine needle aspiration cytology cases. Acta Cytol
2009;53:659-66.
4
Rossi ED, Raffaelli M, Minimo C, et al. Immunocytochemical evaluation of thyroid neoplasms on thin-layer smears from fine-needle
aspiration biopsies. Cancer Cytopathology 2005;105:87-95.
5
Cochand-Priollet B, Dahan H, Laloi-Michelin M, et al. Immunocytochemistry with cytokeratin 19 and anti-human mesothelial cell
antibody (HBME-1) increases the diagnostic accuracy of thyroid fineneedle aspirations. Preliminary report of 150 liquid-based fine needle
aspirations with histological control. Thyroid 2011;21:1067-73.
6
Fadda G, Rossi ED, Raffaelli M, et al. Follicular thyroid neoplasms
can be classified as low-and high risk according to HBME-1 and
Galectin 3 expression on liquid based fine needle cytology. Eur J Endocrinol 2011;165:447-53.
Molecular diagnostics in thyoid cytology
F. Basolo
Pisa
Background. Thyroid cancer is the most common endocrine
neoplasm. Papillary thyroid carcinoma (PTC) is the most
frequent type of endocrine malignancy. Surgery can be curative in well-differentiated thyroid cancer mainly when detected before the establishment of local or distant metastases.
Preoperative diagnosis of thyroid nodules is based on fine
needle aspiration (FNA) cytology. Cytological examination
of FNA by an expert pathologist provides the most reliable
information. However, in some situations, the cytological
examination cannot be conclusive either because of insufficient material or overlapping/undefined (indeterminate) morphological criteria. In the last year, our understanding of the
molecular biology of PTC has made a step forward with the
discovery that roughly 45% of PTC harbor one specific activating point mutation in the BRAF gene. The high prevalence
combined with the PTC specificity render BRAF an attractive
molecular marker for PTC diagnosis. Since 2004 our group
suggested that BRAF mutation and RET/PTC rearrangements
are molecular markers of PTC that can be applied to FNA in
adjunct to traditional cytology. More recently, we evaluated
the diagnostic use of protein expression of CXC chemokine
receptor 4 (CXCR4) and galectin-3 (gal-3) that were found
to be upregulated in papillary thyroid carcinoma compared to
normal thyroid and of mesothelial cell surface protein recognized by monoclonal antibody Hector Battifora Mesothelial
cell (HBME)-1 in thyroid tumors. In this study we showed
that CXCR4 and HBME-1 were significantly associated with
142
FV-PTC and seemed useful in differentiating the benign from
the malignant follicular patterned lesions, although no statistical difference was observed. Moreover, the NPV of this combination of immunomarkers was very good even in addition
to gal-3, suggesting that it could potentially be used safely to
spare patients surgery.
Materials and methods. In this work, 286 consecutive thyroid FNACs were analyzed for the presence of BRAF mutations to support cytological diagnosis, in particular, for inadequate or indeterminate cases. In addition, in selected samples
we investigated on a differential gene expression profiles with
the aim to further improve the diagnostic approach. A syringe
with a 22-gauge needle was used and samples were prepared
on a slide glass for cytological examinations, and then leftover cells inside the needle were flushed into a 1.5-ml tube.
Total cellular DNA and RNA were extracted and purified by
a standard commercial methods and then conserved. DNAs
was analyzed for the presence of BRAF mutations by pyrosequencing using a commercial CE IVD test. Gene Expression
analysis was performed on retro-transcribed RNA using the
Human Extracellular Matrix & Adhesion Molecules RT² Profiler PCR Array profiles the expression of 84 genes important
for cell-cell and cell-matrix interactions.
Results. Out of the 286 analyzed samples 4 nodules resulted
mutated on BRAF gene. In detail, all the four mutations were
c.1799T>A p.V600E. According to British Thyroid Association guidelines for FNAC, cytological results were Thy1
(inadequate) in 104 cases, Thy2 (benign) in 152 cases, Thy3
(indeterminate) in 26 cases, Thy4 (suspicious of PTC) in 2
cases and Thy5 (PTC) in 5 cases. The 4 mutated samples were
Thy4 in one case and Thy5 in 3 cases. Noteworthy, none of
the indeterminate or inadequate samples resulted mutated on
BRAF gene. Up to now the expression profiling experiments
are still running.
Conclusions. On the basis of these data we can speculate that
BRAF analysis on indeterminate cytological thyroid samples
is not an adequate diagnostic tool, considering the overall low
percentage of mutated cases. Therefore we suggest the addition of other molecular markers analysis, such as RAS family and RET/PTCs rearrangements, or more comprehensive
evaluation of gene expression levels.
The Bethesda system: does it really help
my practice?
F.C. Schmitt
Professor of Pathology, Medical Faculty of Porto University Director
of the Unit of Molecular Pathology – IPATIMUP
The Bethesda Thyroid System (BTS) for reporting thyroid
cytopathology is a six-tiered classification that was proposed
considering some possible advantages: 1) it is based on a
literature analysis; 2) it is linked with a risk of cancer value
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
for each category; 3) clinical management is recommended
for each category; 4) it is a developing system that should
be modified following the results obtained with further studies; 5) it is accompanied by an atlas with images, diagnostic
criteria and explanations leading to homogeneous diagnoses.
Therefore, the BTS seems to be an opportunity for a “standardization” of diagnoses and reports of thyroid FNA. In
2009 during the ECC in Lisbon, the scientific committee of
EFCS organized a working party to discuss the implementation of this system in Europe. At the end of this meeting,
disagreements persisted amongst the participants concerning
the six-tiered systems and the Follicular Lesion of Unknown
significance (FLUS) as well as the Follicular Neoplasm (FN)
categories, but the great majority of the participants agreed
with the need for standardization. Recently, the scientific
committee of EFCS tested the reproducibility of the system
among four cytopathologists in 116 cases. As expected, most
of the diagnostic disagreements were for the FLUS and FN
categories. The criteria to distinguish these two categories
have been well described but each cytopathologist needs a
specific time of training and also to have the opportunity to
compare with the histological controls, when available, in
order to determine the level between these two categories.
Although recognizing the advantages of harmonization, in
our perspective, to reduce cytological diagnosis to broad
categories (benign, malignant, suspicious, etc.) can be a disadvantage for the method as a diagnostic method. I always
used descriptive reports for thyroid FNA. Thyroid diagnoses
have to be immediately understandable to the local clinicians
and thyroid FNA cytology is a diagnostic procedure. It is
interesting to observe that in the recommendations of BTS,
each category should be further described and classified
as appropriate, with an attempt to place the findings into a
specific pathologic entity. I think that when it is possible to
diagnosis a colloid goiter, thyroiditis or carcinoma on FNA,
this is not should replaced by categories of benign or malignant. Standardization is quite useful in a series of situations of
the pathology practice, but to restrict the thyroid cytological
diagnosis in some categories, like on gy cytology (a screening
and not a diagnostic method) can not bring clear advantages
to the clinical practice. Is important to emphasise that the
implementation of any terminology only will be helpful if all
personal involved on the procedure (cytopathologists, radiologists, endocrinologists, surgeons.) be aware of that.
References
1
NCI Thyroid Fine Needle Aspiration State of Science Conference.
Diagn Cytopathol 2008;6:388-448.
2
Kocjan G, Cochand-Priollet B, de Agustin PP, et al. Diagnostic terminology for reporting thyroid FNAC: EFCS thyroid working party
symposium, Lisbon 2009. Cytopathology 2010;21:86-92.
3
Cochand-Priolet B, et al. Bethesda Terminology for thyroid FNAs:
from the theory to the practice at a European level. Acta Cytol
2011;55:507-11.
143
sessioni scientifiche
Venerdì, 29 giugno 2012
ore 16.30-18.30
Citologia aspirativa degli organi profondi
Chairmen: Marco Santucci (Firenze), Ambrogio Fassina (Padova)
Radiological features of deep organ lesions:
indications for cytologic diagnosis
M.A. Cova, R. Pizzolato
Unità Clinica Operativa di Radiologia, Dipartimento di Scienze Mediche, Chirurgiche e della Salute, Università di Trieste
An increasing number of lesions of different organs, such as
lung, liver, pancreas, kidney are detected because of a more
frequent use of cross-sectional imaging techniques such as
computed tomography (CT) and magnetic resonance imaging (MR).
Both CT and MR are excellent modalities in detecting and
describing morphologic features of the lesions and in many
cases also allow to characterize the lesions. However, in some
cases it is not possible to provide a specific diagnosis. Fine
needle aspiration (FNA) may be of value in cases that are
indeterminate at cross-sectional imaging.
In chest CT findings suggestive for malignant lesion are: lesion size > 11 mm, predominantly ground-glass opacity, air
bronchogram, no concave margin, not poligonal shape. CT
findings suggestive for benignancy are: lesion size < 11 mm,
predominantly ground-glass opacity, no air bronchogram,
predominantly solid, concave margin, not poligonal shape.
Nodules with CT findings suggestive for benignancy do not require needle aspiration. In patients with suspected malignancy
on chest radiograph and CT with indeterminate bronchoscopy,
patients with a hilar mass following negative bronchoscopy,
patients with a new or enlarging solitary nodule or mass at CT
that it is unlikely to be accessible by bronchoscopy, patients
with indeterminate solitary pulmonary nodules on chest CT
(i.e. nodules without typical findings of malignancy or of
benignancy), patients with multiple nodules without known
malignancy or in whom a prolonged remission occurs, and in
patients with infiltrates, either single or multiple, for which no
diagnosis has been made by sputum or blood culture, serology
or bronchoscopy, FNA may be indicated.
Persistent focal ground-glass opacity (GGO) on high resolution chest CT might also be an indication to FNA. GGO is
defined as hazy increased attenuation of the lung with preservation of bronchial and vascular margins. It is a non-specific
finding, and several different diagnoses must be considered,
including inflammatory diseases, focal fibrosis, atipical adenomatous hyperplasia, bronchoalveolar carcinoma, and adenocarcinoma. Some reports have suggested that focal GGO
lesions with a solid component are statistically more likely to
be associated with malgnancy. FNA might be a useful diagnostic technique, even for small and deeply located lesions.
CT and MRI today play an important role in evaluating the
mediastinum. While plain film gives limited results, a specific diagnosis is sometimes possible with these techniques
and, if not, at least a circumscribed differential diagnosis can
be made. Tissue components, as shown by CT or MR scans,
together with the size, shape, and precise location within the
mediastinal compartments, are the most important parameters
to be considered for the diagnosis of a mediastinal mass. The
diagnosis can be at least partly suggested on the basis of the
major component of a mass: fatty, cystic, or solid tissue. In
most cases calcifications are also detectable, but this pattern is
not as useful as the previous ones in the differential diagnosis.
Additional information can also be obtained from the degree
and type of vascularity of the lesion by means of contrast
enhancement. However, sometime differential diagnosis by
imaging may be difficult due to the broad spectrum of both
malignant and non malignant lesions that can be found in the
mediastinum, some of them with similar imaging findings. In
these cases percutaneous FNAB of the mediastinum can be
performed. FNAB is a helpful, minimally invasive procedure
that can be performed as a part of the evaluation of a mediastinal mass lesion.
Considering the liver, despite technological improvements,
the accuracy of US (without and with contrast), CT and MRI
for the diagnosis of small hepatocellular carcinomas (HCCs)
remains unsatisfactory. Small and well-differentiated HCCs
may not fulfill the formal diagonstic criteria recommended
in the American Association for the Study of Liver Diseases
practice guidelines, that is increased contrast enhancement
relative to liver during the arterial phase followed by expedite
washout during the hepatic venous and/or delayed phases.
For these small undeterminate lesions FNA may be performed,
although false negatives may occur caused by the difficulty of
targeting smaller lesions and by the complexity of distinguishing well-differentiated HCCs from nonmalignant tumor precursors. Therefore imaging follow-up has been advocated as
the most cost-effective strategy in the management of small,
indeterminate liver lesions in patients with cyrrhosis. FNA
may be of value for the diagnosis of malignancy in candidates
to non surgical treatment, i.e., radiofrequency ablation.
CT and MRI are excellent diagnostic modalities for the initial
detection of cystic pancreatic lesions. The initial evaluation
of a pancreatic cyst should be directed toward the exclusion
of a pseudocyst. The postinflammatory pseudocyst is preponderantly the most common cystic pancreatic mass. However,
a cystic mass is not always a pseudocyst. In general, pseudocysts are unilocular cystic masses that have a thick enhanced
wall and no gas bubbles. Mucinous cystic neoplasms, as well
as anaplastic carcinoma and cystic islet tumors, may occasionally have a similar appearance.
Multilocular cysts include mucinous cystic neoplasms, serous
cystadenoma/cystadenocarcinoma, echinococcal cysts and,
occasionally, postinflammatory pseudocysts. An imaging
classification system for pancreatic cystic lesions based on the
morphologic features of the lesion has been proposed.
This system can be helpful in characterizing lesions, narrowing the differential diagnosis. MRI with MR colangiopancreatography is particularly useful because it accurately depicts
the morphologic features of the cyst and has the advantage
of demonstrating the relationship of the cyst to the pancreatic
duct.
However, in the majority of cases, it is not possible to provide
a specific diagnosis on the basis of imaging because of the
substantial overlap of imaging features. In the appropriate
144
clinical setting, a reasonable differential diagnosis can be
formulated by combining the findings of CT, US and MR imaging. FNA is useful in cases that are indeterminate at crosssectional imaging or that require observation.
FNA is safe and accurate also for the evaluation of patients
with pancreatic cancer initially deemed unresectable on the
basis of imaging findings, if chemotherapy or radiotherapy
is planned, because a histopathologic diagnosis is required
before cytotoxic treatments are begun, when imaging findings
are suggestive for a rare malignant tumor (e.g., lymphoma)
that would be better managed with an alternative protocol,
when imaging findings are suggestive for a focal pancreatic
lesion in a patient with a history of a previous malignant disease, because nonoperative therapy may be not appropriate for
metastatic disease, and when imaging findings not allowing
a differential diagnosis between neoplastic disease and focal
pancreatitis.
Considering renal lesions, FNA may be considered in patients
with advanced stage renal lesions detected by imaging who
cannot tolerate resection for medical reasons and in patients
referred for percutaneous ablations of suspected renal cell
carcinoma.
Actually, characterization of small renal masses by imaging
alone bears various limitations. Enhancement may be equivocal in small masses. It may be difficult to distinguish renal cell
carcinoma from an angiomyolipoma with minimal fat, Moreover, approximately 5% of angiomyolipomas do not contain
visible fat on CT or MRI and are indistinguishable from small
renal cell carcinomas. Some studies in the literature showed
that about 30% of small renal masses who underwent surgery
were found to be benign and one study reported that more
than one third of patients referred for cryotherapy of suspected
renal cell carcinoma had a benign renal mass. FNA could be
of help in avoiding that patients with benign masses, such as
angyiomyolipoma, oncocytoma and metanephric adenoma,
undergo percutaneous ablation or to surgery.
FNA of focal renal lesions might be also advocated in evaluating the indeterminate cystic renal mass (Bosniak category 3
and 2F) on CT or MR imaging, although this is a controversial
topic in the literature. Some Authors claim that truly indeterminate cystic masses require surgery regardless of whether
biopsy specimen is positive or negative.
FNAC of lung lesions: correlation with molecular
techniques
A. Assi, L. Roncoroni
U.O. Anatomia Patologica, Ospedale Civile di Legnano, Legnano (MI)
Introduction. The emergence of new treatments with different activity or limited indication in subtypes of Non-SmallCell Lung Carcinomas (NSCLC) has put emphasis on the
importance of an accurate diagnostic subtyping (i.e. Adenocarcinoma vs Squamous Cell Carcinoma). Moreover, some
advanced lesions are unresectable, therefore making it impossible to establish an exhaustive diagnosis on a tissue sample.
As a consequence, the majority of patients with NSCLC are
managed on the basis of a diagnosis made by FNAC or of a
small tumor biopsy.
FNAC of lung lesions, either transthoracic needle aspiration
(TTNA) or transbronchial needle aspiration (TBNA) is often
used for the diagnosis of peripherical lung nodules and hilarmediastinal lymph nodes.
Methods. A total of 3662 FNACs (TTNA) were performed
by Pathologists at the Legnano Hospital from 1985 to 2011.
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Another 200 TBNAs were performed by pneumologists from
2008 to 2011. Computed Tomography-Guide TTNA was
performed in patients with peripherical lung nodules using a
22-gauge needle, whereas Endobronchial Ultrasound-guide
TBNA was performed in patients with hilar-mediastinal
lymph nodes with a 22-gauge needle.
Two needle passes were always performed, and from two to
four fresh smears were prepared. Part of the cell samples was
put in 4% formaldehide for paraffin embedding, including the
residual material left within the needle bore.
The smears was stained with May Grunwald –Giemsa and Papanicolau stainings. All cases underwent cytomorphological
evaluation. Cases not meeting the cytomorphological criteria
were also submitted to IHC for TTF1 and p63 expression, to
confirm the diagnosis. From 2010 on, 53 cases were also studied for EGRF mutations. Mechanical microdissection using
a 30-gauge needle was performed to obtain samples suitable
for molecular analysis of EGFR mutations by real-time PCR
or sequencing.
Results. Out of the original 3862 FNACs, 32% were diagnosed as Adenocarcinoma, 13% as Squamous Cell carcinoma,
6% as NSCLC, 13% as other histotypes, 33% as negative for
malignant cells and 3% as inadequate, respectively. Seven
hundred patients subsequently underwent surgical resection of
the lesion, with 97% concordance between FNAC and histological findings. In such cases, FNAC yielded 96.6% sensitivity and 97.3% specificity. IHC reanalyis of the 476 NSLCLC
cases allowed a correct reclassification, with 219 cases only
confirmed as NSLCL. Molecular techniques showed EGFR
mutations in 14 cases out of 53 (25%).
Discussion. FNAC is a minimally invasive, relatively safe
and effective diagnostic procedure, so that it supports the
vast majority of lung neoplasm diagnoses. In expert hands
lung FNAC allows a sensitive and accurate first-line typing
of NSCLCs. IHC and molecular techniques may provide
diagnostic refinement and sometimes generate a more specific diagnosis to suit newly developed therapeutic regimens.
Prognostic markers can be studied using molecular techniques
on small tumor cell samples, both on fresh smears and on
paraffin-embedded material.
Reference
1
Travis W, Brambilla E, Noguchi M, et al. International Association
for the Study of Lung Cancer/American Thoracic Society/European
Respiratory Society International Multidisciplinary Classification of
Lung Adenocarcinoma. J Thorac Oncol 2011;6:244-85.
Fine needle cytology / core needle biopsy
in pancreatic solid neoplasms
G. Zamboni
Department o Pathology, University of Verona, Ospedale Don Calabria, Negrar, Verona, Italy
Introduction. The extensive utilisation of imaging has increased the discovery of pancreatic masses. Unfortunately,
most (80 to 90 percent) of the clinically detected masses in
the pancreas are adenocarcinomas. With the exception of
functioning endocrine tumors, characterised by a specific
clinical picture, the other pancreatic tumors manifest with
either non-specific symptoms, or symptoms similar to pancreatitis. Although a correct diagnosis is mandatory to plan
the therapeutic approach and establish a prognosis, accurate
preoperative diagnosis sometimes is very difficult to achieve.
Methods. The ideal diagnostic test, as in other disease, should
be the tissue diagnosis with a trucut biopsy, that allows a boro-
sessioni scientifiche
ader use of ancillary studies. The less invasive methods, like
the FNAB with US guidance or the EUS-guided biopsies have
a better level of safety and are considered reliable in detecting
the presence of malignant cells. Direct bile duct or pancreatic
duct brushing cytology can also be performed; however it
seems that this method results in a high false positive and
negative rate. Independently on the used sampling method the
accuracy of pathologic evaluation is higher when a pathologist
makes the procedures or cooperates to them.
Whether a lesion should be punctured or not it depends on
many different aspects, and most of them are basically clinical
and radiological ones. In many centers, if a mass is resectable
the surgeons perform a pancreatectomy, because of the false
negative results that we can have with both cytology and
biopsy (10 to 30 percent). One thing has to be clear is that a
morphological diagnosis has to be served to all patients, either
on the primary or secondary lesions, before to plan chemotherapy. In that respect, the diagnosis of ductal carcinoma versus endocrine or acinar carcinoma it makes a lot of difference.
Diagnostic features. Ductal carcinoma: the cytologic smears
are characterized by high celluarity, and the presence of
relatively pure neoplastic cellular component. The presence of
normal ductular-like aggregates serves by comparison. Major
criteria of malignancy are considered: the presence of nuclear
crowding and overlapping, the irregular chromatin distribution and the irregular nuclear contour, whereas minor criteria
are the nuclear enlargement, the presence of single malignant
cells, necrosis and mitosis.
In the biopsy interpretation of a primary pancreatic lesion
eight features are proposed as particularly helpful in the
differential diagnosis with chronic pancreatitis: the loss of
lobular architectural, resulting in a hapzard distribution of
glands, the variation in nuclear area greater than 4 to 1 from
cell to cell, prominent and multiple nucleoli, the presence of
incomplete glands, intraluminal necrosis, glands immediately
adjacent to muscular artery, perineural invasion by glands and
vascular invasion.
Endocrine Neoplasms. The smears are hypercellular with
a clean background, except for high grade neoplasia. The
cells can be individually dispersed or arranged in clusters.
The nuclei are frequently uniform, round to oval, with a salt
and pepper pattern (coarse, finely distributed chromatin) but
sometimes they can be pleomorphic and hyperchromatic.
Acinar Cell Carcinoma and Pancreatoblastoma. The loosely
cohesive groups of cells show the typical acinar differentiation with a cytoplasm filled with deeply eosinophylic granules. The cells show signs of atypia, with prominent nucleoli
and mitoses; necrotic debris are frequently found. Pancreatoblastoma also have distinctive squamoid corpuscles and may
present with hypercellular stromal component. Both acinar
cell carcinoma and pancreatoblastoma may have endocrine
cell component.
145
Pitfalls. The pitfalls that a pathologist has to avoid in the
interpretation of both cytology and biopsy are many. The
best way to avoid them is to know them. The point is what
a pathologist should know? He should know in first istance
all the clinical and radiological relevant features. The most
important technical informations he has to know differ in
solid and cystic lesions. In solid lesions the most important
differential diagnosis is between ductal carcinoma and chronic
pancreatitis, expecially the tumor-forming pancreatitis, like
the autoimmune pancreatitis.
Immuncytochemistry and genetic methods. In ductal adenocarcinoma, the tumor cells, like the normal intralobular small
ductules, consistently expressed MUC1, whereas MUC2,
which is not found in the normal pancreas, and MUC5AC,
normally expressed by the gastric mucous surface cells, are
lacking. Other duct cell markers that are typically found in
adenocarcinomas are the cytokeratins 7, 8, 18, 19 and occasionally 20, CA19.9, DUPAN-2 and CEA. K-ras mutations,
an early events, and p53 and DPC4 inactivation, relatively late
genetic alterations, can be detected in aspirates. An elevated
serum IgG4 level, a high number of IgG4-positive plasma
cells (more than 20 to 50 cells per HPF), with a high IgG4/
IgG ratio, have been suggested as highly specific for the diagnosis of autoimmune pancreatitis.
In endocrine neoplasms, the diagnosis may be confirmed by
the immunohistochemical demonstration of endocrine markers such as chromogranin A and synaptophysin and the detection of hormone production. The Ki67 proliferative index
has a great importance to differentiated low grade endocrine
neoplasms (G1 and G2 according to the WHO classification)
to high grade neuroendocrine carcinoma (G3).
In acinar cell carcinoma, the epithelial cells immunihistochemically stain positively with at least one of the acinar
markers trypsin, chymotrypsin, and lipase, whereas they
are negative for, CD10 and beta-catenin. The neuroendocrine markers may also be present in a minor component
of cells.
References
1
Bosman FT, Carneiro F, Hruban RH, et al. Who classification of tumours of the digestive system. Lyon: international agency for research
on cancer (iarc) 2010.
2
Deshpande V, mino-kenudson M, brugge WR, et al. Endoscopic ultrasound guided fine needle aspiration biopsy of autoimmune pancreatitis:
diagnostic criteria and pitfalls. Am J Surg Pathol 2005,29:1464-71.
3
Hruban RH, pitman MB, Klimstra DS. Tumors of the pancreas. Afip
atlas of tumor pathology. Series 4, vol. 6. Washington, dc: Armed
Forces Institute Of Pathology 2007.
4
Kojima M, Sipos B, Klapper W, et al. Autoimmune pancreatitis:
frequency, igg4 expression, and clonality of t and b cells. Am J Surg
Pathol 2007,31:521-8.
5
Stelow EB, Bardales RH, Stanley MW. Pitfalls in endoscopic ultrasound-guided fine-needle aspiration and how to avoid them. Adv Anat
Pathol 2005,12:62-73.
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I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Sabato, 30 giugno 2012
ore 9.00-10.30
Mammella (I sessione)
Chairmen: Leonardo Resta (Bari), Anna Sapino (Torino)
Recent developments in breast molecular
cytopathology
P. Vielh
Institut de cancérologie Gustave Roussy, Villejuif, France
Gene-expression arrays have generated molecular predictors
of relapse and drug sensitivity in breast cancer. We aimed to
identify exons differently expressed in malignant and benign
breast lesions and to generate a molecular classifier for breastcancer diagnosis.
A series of 165 breast samples were obtained by fine-needle
aspiration cytology. Complementary DNA was hybridised
on splice array. A nearest centroid prediction rule was developed to classify lesions as malignant or benign on a training
set, and its performance was assessed on an independent
validation set. A two-way ANOVA model identified probe
sets with differential expression in malignant and benign lesions while adjusting for scan dates. The 165 FNAC samples
included in the study consisted of 120 breast cancers and 45
benign lesions. A molecular classifier for breast-cancer diagnosis with 1228 probe sets was generated from the training
set (n = 94). This signature accurately classified all samples
(100% accuracy, 95% CI: 96-100%). In the validation set
(n = 71), the molecular predictor accurately classified 68
of 71 tumours (96% accuracy, CI: 88-99%). When the 165
samples were taken into account, 37 858 exon probe sets
(5.4%) and 3733 genes (7.0%) were differently expressed in
malignant and benign lesions (threshold: adjusted p < 0.05).
Genes involved in spliceosome assembly were significantly
overexpressed in malignant disease (permutation p = 0.002).
In the same population of 165 samples, 956 exon probe sets
presented both higher intensity and higher splice index in
breast cancer than in benign lesions, although located on
unchanged genes.
In conclusion, many exons are differently expressed by breast
cancer and benign lesions, and alternative transcripts contribute to the molecular characteristics of breast malignancy.
Development of molecular classifiers for breast-cancer diagnosis applicable to the minute material obtained from FNAC
samples are under development.
Nipple discharge in breast pathology
I. Castellano, F. Montarolo, L. Macrì, R. Coda, L.Righi,
A. Sapino
Department of Biomedical Sciences and Human Oncology, Breast
Unit San Giovanni Hospital, Turin, Italy
Background. Cytological examination of nipple discharge
(ND) is a non-invasive diagnostic and easy to perform test.
Although ND usually has a benign aetiology (such as ductal
ectasia and papilloma), previous studies have found that the
incidence of in situ carcinoma ranges from 9.3% and 21.3%
in the presence of ND 1-5. However, due to its low sensitivity
ND has little complementary diagnostic value.
ND is clinically considered as pathologic if unilateral, bloody,
persistent and spontaneous, and if it arises from a single duct.
The aim of this study is to investigate the sensitivity, specificity, positive predictive value (VPP) and negative predictive
value (VPN) in a subset of patients in which ND is clinically
classified as pathologic.
Material and methods. We have recorded a series of 329
smears collected from December 2004 to December 2007 in
the Breast Unit of the San Giovanni Battista Molinette Hospital, Turin. We selected a subset of 88 cases with clinical
features of pathologic ND. For these cases radiological data
(mammography [Mx] and ultrasound [US]), exact localisation
of the lesion in the breast and colour of the ND were collected.
Cytological results of ND were than compared to those of
Mx/US and with histology. In addition we compared the ND
colour with histological result and specificity, sensitivity, VPP
and VPN were then calculated.
Results
1) Clinically pathologic ND was associated to either invasive
or in situ carcinomas in 21% of cases, with a sensitivity of
68%, and a specificity of 97%, (VPP of 89% and VPN of
88%).
2) In 55% of cases bloody ND is related to an atypical or a
malignant lesion at histology, however, the sensitivity and
specificity of bloody ND is 70% and 41% respectively,
with VPP of 30% and VPN of 78%.
3) Mx and US were negative respectively in 40% and 58% of
patients with pathologic ND. Malignant ND cytology was
reported in 10% and 52% of cases with negative RX and
US diagnosis.
4) All cytological malignant ND in which the Mx/US lesion
was localized in Q5 revealed an in situ carcinoma at histology.
5) An over diagnosis of cytological malignant ND was made
in about 10% of cases, maybe caused by artefacts of cell
morphology due to air drying in Giemsa staining.
Conclusions. Cytological examination of clinically pathologic ND is a non invasive reliable test and its usefulness
is further demonstrated by the fact that identifies high risk
lesions in 10% and 52% of cases with negative Mx and US
imaging respectively. Bloody ND should be always examined,
because in half of cases it may be related to a high risk or
malignant lesions.
References
1
Dawes LG, Bowen C, Venta LA, et al. Ductography for nipple discharge: no replacement for ductal excision. Surgery
1998;124:685-91.
2
King TA, Carter KM, Bolton JS, et al. A simple approach to nipple
discharge. Am Surg 2000;66:960-5; discussion 965-6.
3
Murad TM, Contesso G, Mouriesse H. Nipple discharge from the
breast. Ann Surg 1982;195:259-64.
4
Florio MG, Manganaro T, Pollicino A, et al. Surgical approach to nipple discharge: a ten-year experience. J Surg Oncol 1999;71:235-8.
5
Kalu ON, Chow C, Wheeler A, et al. The diagnostic value of nipple
discharge cytology: Breast imaging complements predictive value of
nipple discharge cytology. J Surg Oncol 2012 Mar 6. doi: 10.1002/
jso.23091.
147
sessioni scientifiche
Role of needle aspiration cytology in the
preoperative diagnosis of the head and neck
tumors
S. Fiaccavento
Anatomia Patologica sezione di Citologia Diagnostica, ICSA – Istituto Clinico Città di Brescia, Brescia, Italy
The complexity and heterogeneity of tumors of the head and
neck region makes extremely difficult a precise preoperative diagnosis, essential step for proper treatment planning.
Furthermore the complexity of this anatomic compartment
indicates the use of minimally invasive diagnostic techniques
and Fine Needle Aspiration Cytology (FNA), among the various techniques, appears to be particularly suitable for its high
diagnostic accuracy. In this context is shown to pay attention
to the importance of ultrasound evaluation in all lesions that
require cytological evaluation with collaboration between the
radiologist and pathologist, keeping the pathologist always in
account at the time of sampling and possibly executor of the
smear.
However, sensitivity and specificity, in relation to the diversity of the targets, may change so that, in certain circumstances, to distinguish between benign and malignant may be
sufficient while in other cases you can also perform a more
accurate histological diagnosis.
Finally, in view of a correct differential diagnosis can be
useful to use immunocytochemical investigations especially
when the problem is to determine if the location site of the
tumor is primary or secondary, important fact especially in
tumors of this anatomical region.
Therefore, we propose a series of cases usefull in diagnosis
and in differential diagnosis of all primary and secondary
neoplasms of the head and neck, with the exclusion of those
located in the thyroid.
Sabato, 30 giugno 2012
ore 11.00-12.30
Mammella (II sessione)
Chairmen: Francesco Feoli (Bruxelles), Fabrizio Zanconati (Trieste)
Cyto-radiological disagreement: management
F. Zanconati , F. Giudici , A. Bianco , D. Bonifacio , C.
Bottin1, S. Dudine4, F. Martellani4, E. Ober4, A. Romano4,
A. Zacchi4, M. Pinamonti1, L. Zandonà1, T Al-Omoush1, M.
Tonutti5, M. Bortul1 6, A. Assante5, M.A. Cova1 5, C. Gasparini5, F. Frezza5, M.P. Bortolotto5, C. Cressa5, R. Perrone5,
E. Makuc5,G. Petz7, PL. De Morpurgo8, L. Torelli2, L. Di
Bonito1 4
1 4
1 2
3
1
Dep. of Medical, Surgery and Health Sciences, University of Trieste,
Department of Mathematics and Geoscience, University of Trieste,
3
Department of Life Sciences, 4 Pathology Unit, University Hospital
of Trieste; 5 Radiology Unit, University Hospital of Trieste; 6 General
Surgery Unit, University Hospital Trieste, 7 Radiology Unit of Salus
Trieste, 8 Radiology Units of Sanatorio Triestino, Italy
1
2
Background. In Trieste, pathologists have been actively
involved in breast diagnostics working alongside radiologists in the sampling time in order to set up together the most
adequate approach in case-solving basing on the clinical/
radiological and rapid stain cytological findings. Based on
our own experience we prefer, whenever possible, to use the
fine needle aspiration sampling method (FNA), almost always
under ultrasound guidance, even for palpable lesions, reserving tru-cut biopsy only for a limited number of cases and vacuum-assisted biopsy (VAB) only in not ultrasound detectable
lesions (id: isolated microcalcifications). The collaboration
between radiologist and pathologist is essential to correctly
evaluate identified lesions: the more coincident the results of
radiological and cytological exams are, the more accurate the
final diagnosis will be. Nevertheless there are some cases in
which radiology and cytology do not agree. We decided to
reassess the entire database, focusing on the management of
discrepant cases in the light of the final histological diagnosis
for surgical-treated cases or referring to the follow-up exams
in the cases that did not undergo surgery.
Materials and methods. The two-year period considered for
this study is 2008-2009. The database comprises 1643 women
with breast abnormalities of whom 1338 underwent FNA,
for a total amount of 1995 nodules of which 1756 examined
with FNA (88%). To each lesion examined through FNA the
radiologist assigns a score for mammographic and ultrasound
suspicion (R1-5/U1-5) according to BIRADS classification.
Then the pathologist performs a rapid evaluation of adequacy
of the sample with the possibility of repeating the exam in
the same session or integrating it with other more invasive
methods; this allowed a rate of inadequacy of 4.4% to be mantained. Cytological diagnoses are coded using the diagnostic
categories (C1-5) proposed by the European Guidelines.
To each lesion is assigned a triplet R-U-C (mammography/
ultrasound/ cytology) allowing a diagnosis to be defined.
Excluding incompletely coded radiological lesions (Rx, Ux)
and inadequate cytological lesions (C1),we selected for the
analysis 1553 cases that are subdivided, on the bases of the
radiologic characteristics, into the following groups:
1. Benign or probably benign lesions: R2-3 and U2-3.
2. Lesions defined as benign or likely benign by only ultrasound (U2-3) in a dense breast or in young women, in
which mammography is not indicated (R0-1).
3. Lesions defined as malignant or likely malignant by only
ultrasound (U4-5) in a dense breast or in young women, in
which mammography is not indicated (R0-1).
4. Lesions characterized as suspect/malignant by both mammography and ultrasound: R3 & U5, R4 & U4-5, R2-U4,
R5 & any U.
5. Radiological inconclusive lesions: R3 & U4, R4 & U2, R2
& U4 (mammographic reports discrepant with ultrasound
orientation).
The lesions belonging to these different groups are associated with the cytological diagnosis. In most of the cases the
radiological and cytological analyses were in agreement. In
148
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
discrepant cases we reviewed the diagnostic procedure for
each lesion.
On the basis of histological outcome or the follow-up, we
determined the sensitivity, specificity, number of false positives or negatives, by each method alone or by both methods
combined, trying to determine the best combination in order
to reach the most accurate diagnosis.
Results. The correlation between radiology and the cytological-histological evaluation gave the following results:
Radiologically benign lesions (209 cases): 186 lesions classified as benign by both radiology and cytology and confirmed
histologically or by follow-up (89.9%); 17 malignancies
(C4-C5) (radiological false negatives), of which 16 histologically confirmed, and one relapse not surgical-treated, (7.7%);
4 cases (1.9%), correctly identified by radiology as benign
lesions, that had been classified as suspect (C4) by cytology
(false cytological suspects) were further examined through the
use of 3 benign nodulectomies and of 1 benign tru-cut.
2 false negatives C3 (0.96%) (double false negative), the former had undergone nodulectomy and subsequent quadrantectomy and the latter malignant tru-cut and mastectomy.
Lesions with benign ultrasound diagnosis and R0/1 (606 cases): 569 benign lesions with concordance (94%) histologically
or follow-up confirmed; 28 malignancies (C4-5) (radiological
false negatives), histologically confirmed (4.6%), of which
only 2 in 28 were examined with tru-cut before surgery; 6
lesions correctly identified benign by radiology had been labeled as suspect by cytology (false cytological suspect) (1%)
had been treated with four nodulectomies and two quadrantectomies; 2 false negatives (C3) (double false negative)
treated with one mastectomy (both lesions in the same patient)
(0.3%). 1 case (C4) with no follow-up available.
Lesions with ultrasond diagnosis of suspect or malignancy
and R0/1 (135 cases): 92 cases of concordance between
malignant radiology and cytology (68.9%) histologically or
follow-up confirmed; 38 cytologically benign lesions (28.1%)
of which 27 identified with cytology alone, 3 with cytology
plus tru-cut and 8 with cytology plus nodulectomy (radiological false suspect); 1 cytological false negative (C3) (0.7%) in
which radiology correctly identified the malignancy, treated
with nodulectomy and subsequent quadrantectomy; 4 double
false positive (C4) (3%) treated with one quadrantectomy and
three nodulectomies.
Lesions identified as malignant by radiology (ultrasound and
mammography) (533 cases): 485 cases of malignancy with
agreement between radiology and cytology (91.0%) of which
467 histologically confirmed and 18 elderly women without
histology; 35 lesions identified as benign by cytology (6.5%)
(radiological false suspect) of which 20 were confirmed as
negative by cytology alone and follow-up, and 15 with be-
nign nodulectomy; 9 cytological false negatives (1.7%), of
which 8 cases (C3) were solved with three nodulectomies,
one quadrantectomy, three malignant tru-cut, of which one
underwent mastectomy, one quadrantectomy and one not operated, one tru-cut lymphoma, and one case (C2) solved with
quadrantectomy (patient with another C5 lesion in the same
quadrant); 4 double false positive (0.7%): one (C5) treated
with quadrantectomy (atypical adenosis), one (C4) followed
by tru-cut with inconclusive results turned out to be benign
with follow-up and the latter (C4) treated with quadrantectomy (atypical hyperplasia) and one (C4) without histology
with benign follow-up.
Radiologically inconclusive lesions (70 cases): 32 malignancies (C4-5) identified by cytology (45.7%) and histologically
confirmed (28 only cytology and surgery); 31 benign cytological cases (C2-3) (44.3%); 10 were confirmed with
follow-up, 3 with tru-cut, 12 with nodulectomy, 6 were lost;
4 false negative (C3) (5.7%) whose pathology was resolved
with one nodulectomy, one malignant tru-cut with subsequent
quadrantectomy, one nodulectomy and subsequent quadrantectomy, one nodulectomy and subsequent mastectomy and
3 false positive C4 (2.9%), one treated with tru-cut (B3) and
subsequent quadrantectomy, one with nodulectomy and one
not underwent surgery but benign follow-up.
Quality Indicators’ assessment from the radiological database
(excluding the group of 70 radiologically inconclusive lesions
and 29 C4-C5 lesions not histologically confirmed) on 1454
total lesions, demonstrated for radiology alone a sensitivity of
92.7%, and a specificity of 90.1%.
Independently by radiological outcome out of 842 citologically benign lesions (C2-3), 1.6% were demonstrated histologically malignant, while on 612 citologically malignant lesions
(C4-5) 2.8% were defined as benign with histology. Sensitivity and specificity determined on 1454 lesions (excluding
inadequate cases C1, inconclusive lesions and C4-C5 cases
not histologically confirmed) for cytology was respectively
97.7% and 98.0%
Sensitivity and specificity for both radiology and cytology
alone or combined are summarized in Table I.
Conclusions. From our experience, thanks to a consolidated
collaboration between radiologists and pathologists lasting
for about twenty years, we can support the central role of
needle aspiration cytology (FNA) in breast diagnostics. With
a comparison between the independent outcomes of the two
diagnostic methods, it was possible to decide, in cases of
disagreement, the right choice of non-invasive diagnostic
methods (radiological follow-up alone) or other increasingly
invasive investigations (repetition of the FNA, Tru-cut or surgical diagnostic biopsy).
Tab. I.
N° Cases
Sensitivity
Specificity
FP
FN
Radiology Alone
Diagnostic
1454
92.7%
90.1%
84
44
Cytology Alone
1454
97.7%
98.0%
17
14
Rad. B + Cit. B
Rad. M + Cit. M
1326
99.29%
99.2%
6
4
Rad. B + Cit. B
Rad. M + Cit. M e B
1374
99.30%
94.5%
44
4
Rad. B + Cit. B
Rad. M e B + Cit. M
1380
99.34%
97.9%
16
4
149
sessioni scientifiche
Sabato, 30 giugno 2012
ore 9.00-10.30
Nuove tecnologie
Chairmen: Stefano Pizzolitto (Udine), Gian Luigi Taddei (Firenze)
Molecular diagnosis of fine needle aspiration
(FNA) thyroid specimens
D. de Biase, M. Visani, V. Cesari, A. L. Pession, G. Tallini
Facoltà di Medicina, Università di Bologna, Anatomia Patologica,
Ospedale Bellaria, Italy
Several studies, many of which published in the past several
years have shown that molecular diagnosis of fine needle
aspiration (FNA) thyroid specimens is both feasible and useful 1-11. In fact, the type of genetic alterations that occur in
thyroid cancer, the clinical context and the very nature of FNA
samples, make the latter ideally suited for molecular analysis.
This is the case for several straightforward reasons. Thyroid
tumors enjoy a remarkable correlation between phenotype
(i.e. tumor type) and genotype (genetic alterations), a correlation that is more robust than that observed in many other
epithelial tumors. To the point, carcinomas with papillary
architecture (classic papillary carcinoma or its tall cell variant) are characterized by a high prevalence of two specific
molecular alterations: BRAF mutation (BRAFV600E), in
30-70% of cases and RET/PTC rearrangement, in 20-40%.
Encapsulated tumors with follicular architecture (follicular
adenomas, follicular carcinomas and follicular variant papillary carcinoma) are characterized by Ras mutations (usually
N-Ras codon 61) 12 13 in 20-50% of cases or by PAX8/PPARɣ
rearrangement in 5-50% of cases. In spite of the sometimes
remarkable variability in their reported prevalence, these
genetic alterations are mutually exclusive. One of them is
present in ~70% of carcinomas of follicular cell origin. The
detection of these molecular alteration is easily accomplished
with assays based on the analysis of DNA (BRAF and Ras)
or RNA (RET/PTC and PAX8/PPARɣ). Thyroid nodules are
extremely common in the general population. Although in the
vast majority of cases they are benign, a small subset of them
is malignant. Even if conventional cytologic examination with
FNA is highly cost-effective, given the high prevalence of
nodular thyroid disease, any additional tool that can add specific information is of significant value. An additional consideration is that FNA samples are an ideal source for molecular
analysis. In fact, aspirated cells can be immediately placed
in special solutions to preserve nuclei acids at the time of
the FNA procedure. An FNA passage dedicated to molecular
analysis is preferred, but even the lavage fluid obtained from
rinsing the needles after the preparation of the smears can
give adequate results 10. Moreover, alcohol-based fixation and
staining (e.g. PAP) used for diagnosis preserve nuclei acids
well, so that molecular analysis can be easily performed from
the routinely processed slide after removal of the coverslip 3.
This procedure has the obvious advantage of the selection of
the slide (or the slide area) where the cytologic alterations
are located, allowing a very precise correlation between morphology and genetic changes. The archival slide is lost, but
diagnostic areas can be photographed for documentation and
future review before removing the coverslip and dissecting the
cells for molecular analysis.
The initial studies on the molecular analysis of FNA material
were mostly retrospective. Furthermore, comparison of their
results and interpretation of the data presented were made
difficult – if not impossible – by the lack of a standardised
nomenclature for cytologic diagnoses. More recent work has
been careful in precisely defining the diagnostic categories
used. This is to be credited to the application of the six-tiered
Bethesda system for reporting thyroid cytopathology 14 and
to its incorporation into the Guidance on the Reporting of
Thyroid Cytology Specimens document by the Royal College of Pathologists (Guidance on the reporting of thyroid
cytology specimens, November 2009, http://www.rcpath.org/
Resources/RCPath/Migrated%20Resources/Documents/G/
g089guidanceonthereportingofthyroidcytologyfinal.pdf, accessioned may 2, 2012) 15. In addition, several of the most
recent studies have been prospective rather than retrospective 1 11. Some of the studies, particularly those of Nikiforov
and collaborators 8 11, have reported nearly 100% specificity
in diagnosing malignancy in thyroid nodules, with a higher
sensitivity compared with the cytologic FNA diagnosis. What
these studies are showing is that molecular testing is not to
replace conventional cytologic evaluation, but that molecular analysis plus cytologic diagnosis increases the ability to
identify malignant thyroid nodules. They have also shown
that this increase is particularly significant for those very
cases were cytologic evaluation is equivocal or indeterminate.
These are cases with the FNA diagnosis of Atypia of Undetermined Significance (Follicular Lesion of Undetermined
Significance– AUS/FLUS of the Bethesda classification, corresponding to the Neoplasm possible – atypia/non-diagnosticThy3a category of the Royal College of Pathology) 11.
Currently much of the work has focused on the four simple
genetic alterations commonly detected in thyroid carcinoma (BRAF, RET/PTC, N-Ras, PAX8/PPARɣ, see above).
Among these molecular markers, testing for the BRAFV600E
mutation is at the moment the most cost-effective. Virtually
all BRAF mutations in thyroid cancer are the result of a T
to A transversion (in exon 15, at nucleotide 1799, that results in substitution of a Valine with a Glutammate residue)
therefore the mutation can be very easily tested, at low cost,
even in laboratories with limited equipment. The test can be
performed using a variety of methods, of which allele-specific
PCR is probably the most rapid and least expensive 4. Our
group has recently developed ASLNAqPCR, a novel allelespecific quantitative test to accurately detect and quantify hot
spot mutations, including those of BRAF 16. It is important to
recognize that to date a handful of cases “false positive” for
the BRAFV600E mutation have been reported. Kim et al. 5
has shown five false positive cytology specimens in a large
prospective study that included 279 cases where the cytology
diagnosis was followed by the histologic examination of the
resected nodule. FNA material was analyzed using a highly
sensitive method. Direct sequencing of DNA extracted from
the five resected nodules, that were diagnosed as follicular
adenoma (one case) and hyperplastic nodules (four cases),
failed to confirm the presence of the mutation.
150
Given the high specificity and positive predictive value of the
BRAFV600E in cytology specimens (close to 100%) 17, a thyroidectomy can be proposed to a patient whose FNA has been
diagnosed as AUS/FLUS with positive BRAF testing, without
the need to repeat the FNA 1 17. Another diagnostic category
that would benefit is that of lesions diagnosed as Suspicious,
since a thyroidectomy, without the need for frozen sections
or a diagnostic lobectomy, can be directly recommended for a
patient with a Suspicious FNA that is BRAF positive 1 17. Many
(but not all) studies have shown that BRAFV600E is an indicator of poor prognosis: papillary carcinoma with BRAFV600E
mutations behave more aggressively, with higher rates of extrathyroidal extension and lymph node metastases, higher rates of
persistent disease and higher rates of tumor-related death 18 19.
Therefore the identification of BRAFV600E in cases diagnosed as Malignant (or Suspicious) may modify the surgical
approach to include central compartment lymph node dissection, although this should probably wait for more definite data
regarding the prognostic role of BRAF, particularly in the case
of small papillary carcinomas 20.
Several prospective studies have shown that the other molecular alterations common in thyroid carcinoma and mentioned
above (RET/PTC, N-Ras, PAX8/PPARɣ) can be tested as
a panel, together with BRAF. These studies, both from the
United States and Europe, have shown that FNA often provides enough material for all the tests to be performed and that
sensitivity can be significantly improved by adding markers
like RAS mutations and the PAX8/PPARɣ rearrangement
that are particularly relevant for follicular-patterned lesions
i.e. follicular adenoma, follicular carcinoma and the follicular
variant of papillary carcinoma 2 6 8 11.
There is, however, one problem with the molecular analysis of
BRAF, RET/PTC, N-Ras, PAX8/PPARɣ in FNA. Although
they may have a high positive predictive value (particularly
BRAFV600E), the negative predictive value is not high. Thus,
if the mutation is not there the nodule can still be malignant.
A number of studies are threfore focusing on different approaches. These include expression profiling to generate tests
with a high negative predictive value 21 or the analysis of
micro RNA (miRNA) 22. The results of these studies, although
encouraging are still preliminary.
In summary, data currently reported in the literature point to
a role for molecular diagnostics as a useful complement to
conventional FNA cytology, with the greatest impact in the
AUS/FLUS (Thy3a) and the Suspicious (Thy4) categories.
References
1
Adeniran AJ, Theoharis C, Hui P, et al. Reflex BRAF testing in thyroid
fine-needle aspiration biopsy with equivocal and positive interpretation: a prospective study. Thyroid 2011;21:717-23.
2
Cantara S, Capezzone M, Marchisotta S, et al. Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules
improves the diagnostic accuracy of cytology. J Clin Endocrinol Metab
2010;95:1365-9.
3
Cohen Y, Rosenbaum E, Clark DP, et al. Mutational analysis of BRAF
in fine needle aspiration biopsies of the thyroid: a potential applica-
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
tion for the preoperative assessment of thyroid nodules. Clin Cancer
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Jin L, Sebo TJ, Nakamura N, et al. BRAF mutation analysis in fine
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2006;15:136-43.
Kim SW, Lee JI, Kim JW, et al. BRAFV600E mutation analysis in
fine-needle aspiration cytology specimens for evaluation of thyroid
nodule: a large series in a BRAFV600E-prevalent population. J Clin
Endocrinol Metab 2010;95:3693-700.
Moses W, Weng J, Sansano I, et al. Molecular testing for somatic mutations improves the accuracy of thyroid fine-needle aspiration biopsy.
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Nam SY, Han BK, Ko EY, et al. BRAF V600E mutation analysis of
thyroid nodules needle aspirates in relation to their ultrasongraphic
classification: a potential guide for selection of samples for molecular
analysis. Thyroid 2010;20:273-9.
Nikiforov YE, Steward DL, Robinson-Smith TM, et al. Molecular
testing for mutations in improving the fine-needle aspiration diagnosis
of thyroid nodules. J Clin Endocrinol Metab 2009;94:2092-8.
Salvatore G, Giannini R, Faviana P, et al. Analysis of BRAF point mutation and RET/PTC rearrangement refines the fine-needle aspiration
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Troncone G, Cozzolino I, Fedele M, et al. Preparation of thyroid FNA
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Ohori NP, Nikiforova MN, Schoedel KE,et al. Contribution of molecular testing to thyroid fine-needle aspiration cytology of “follicular
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Basolo F, Pisaturo F, Pollina LE, et al. N-ras mutation in poorly differentiated thyroid carcinomas: correlation with bone metastases and
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Garcia-Rostan G, Zhao H, Camp RL, et al. ras mutations are associated with aggressive tumor phenotypes and poor prognosis in thyroid
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Cibas ES, Ali SZ, Conference NCITFSotS. The Bethesda System For
Reporting Thyroid Cytopathology. Am J Clin Pathol 2009;132:658-65.
Tallini G, Gallo C. Fine-needle aspiration and intraoperative consultation in thyroid pathology: when and how? Int J Surg Pathol
2011;19:141-4.
Morandi L, de Biase D, Visani M, et al. Allele Specific Locked Nucleic
Acid Quantitative PCR (ASLNAqPCR): an Accurate and Cost-effective
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Kim SK, Hwang TS, Yoo YB, et al. Surgical results of thyroid nodules
according to a management guideline based on the BRAF(V600E)
mutation status. J Clin Endocrinol Metab 2011;96:658-64.
Elisei R, Ugolini C, Viola D, et al. BRAF(V600E) mutation and outcome of patients with papillary thyroid carcinoma: a 15-year median
follow-up study. J Clin Endocrinol Metab 2008;93:3943-9.
Xing M, Westra WH, Tufano RP, et al. BRAF mutation predicts a
poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 2005;90:6373-9.
Soares P, Sobrinho-Simoes M. Cancer: Small papillary thyroid cancers--is BRAF of prognostic value? Nat Rev Endocrinol 2011;7:9-10.
Chudova D, Wilde JI, Wang ET, et al. Molecular classification of
thyroid nodules using high-dimensionality genomic data. J Clin Endocrinol Metab 2010;95:5296-304.
Nikiforova MN, Tseng GC, Steward D, et al. MicroRNA expression
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151
sessioni scientifiche - Comunicazioni libere
Sabato, 30 giugno 2012
ore 11.00-12.30
Qualità e risvolti medico-legali
Chairmen: Alfredo Fabiano (Roma), Claudio Clemente (Milano)
La gestione del rischio diagnostico in
citopatologia. Dai principi alla pratica
G. Santeusanio
Università di Roma “Tor Vergata”, UOC di Anatomia e Istologia Patologica, Ospedale S. Eugenio – CTO, Azienda USL Roma C, Roma, Italy
Il tema del rischio diagnostico in Anatomia Patologica si pone
come argomento di rilevante interesse nel processo clinicoassistenziale, data la fondamentale importanza che ha una
corretta, completa e tempestiva diagnosi ai fini dell?output
clinico del paziente.
Programmi di assicurazione e miglioramento della qualità
(Sistema di Gestione per la Qualità) e un approccio sistemico
alla prevenzione e gestione degli errori (Risk Management)
diventano parte integranti della “gestione del servizio clinico
di Anatomia Patologica” per garantire la qualità delle prestazioni e la sicurezza del paziente.
La gestione del rischio diagnostico in citopatologia è un “processo multistep” che prende in considerazione (a) il controllo
del processo (preparazione, conservazione e invio del materiale biologico; identificazione e accettazione del campione
biologico; verifica della compilazione della richiesta di esame
e delle notizie cliniche; rilevazione delle non conformità;
allestimento e colorazione; osservazione microscopica, refertazione e comunicazione della diagnosi; tempo di refertazione-TAT; utilizzo di check-list, linee guida, sistemi automatizzati e identificazione con supporti automatici dei vetrini;
semplificazione e riduzione del numero dei passaggi; carichi
di lavoro; conservazione e archiviazione) e (b) le strategie per
la riduzione dell’errore interpretativo (doppia lettura; utilizzo
di check-list per reporting; correlazioni esami precedenti e
citologico/istologico; consultazione intra-laboratorio, per organo o patologia e revisione % di casi random; consultazione
extra-laboratorio, con riferimento ad esperti; consultazione
in telepatologia; consultazione esterna istituzionale). Sia il
controllo del processo che le strategie per ridurre l’errore
interpretativo migliorano la qualità diagnostica e promuovono
la sicurezza paziente.
In questa relazione, sarà presentata una revisione della letteratura sulla riduzione dell’errore con accenni al valore della
seconda opinione e alle diagnosi critiche / urgenti nella diagnostica citopatologica
Sabato, 30 giugno 2012
Ore 9.00-10.30
Comunicazioni Libere
Chairmen: Attilio Leotta (Catanzaro), Ferdinando Quarto (Castellammare di Stabia)
Spontaneous screening and colposcopy:
is useful the association with HPV-DNA test
or other test?
D. Antonini1, C. Magnani2, I. Rostan3, R. Navone3
ASLTO 1 di Torino, Ospedale Martini, UO di Anatomia Patologica,
Clinica san Gaudenzio di Novara, Servizio di Citologia, 3 Dipartimento di Scienze Biomediche e Oncologia Umana dell’Università di
Torino (Sez. di Anatomia Patologica), Italy
1
2 Aims. Even if there is a lack of statistical data, spontaneous
(“opportunistic”) screening of cervical carcinoma by Pap test
is now commonplace in Italy, at the same time of organised
screening. Only in spontaneous screening colposcopy is also
performed along with the Pap test in some cases and not only
after a positive Pap test.
Methods. A total of 146,020 cytological cervical samples
of spontaneous screening of San Gaudenzio Clinic (Novara,
Italy) in a ten-years period (2000-2009) 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
%). Any correlation amongst cytology, histology and colpos-
copy in a group of 21.451 cases, where colposcopy was done
at the same time as the Pap test was evaluated. In the presence
of grade I or higher ATZ, a biopsy was also carried out.
Results. A total of 21,451 Pap tests were done along with
colposcopies, resulting in: 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 they were both positive
in 1.3% of cases. However, there were 8.8% of patients with
positive colposcopy where both 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
152
negative. The discordance between the colposcopy and histocytology results 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 always require
anatomo-pathological confirmation. The HPV-DNA test is
not useful in this context. Other tests (p16, Ki67, DNA ploidy,
LOH, MSI, etc.) have to be evaluated in particular cases. The
Pap smear is the most useful first level test also when it is
performed along with the colposcopy
Correlation between p16 immunopositivity and
presence of HPV DNA in selected pap tests with
ASCUS and LSIL diagnosis
M. Pastormerlo, G. Campagnone, A. Sanzone, S. Rosso, S. Erra
SOC Anatomia Patologica, ASL AL, Casale Monferrato, Italy
Introduction. In recent years several techniques have been
developed in order to detect the presence of HPV DNA in
cervical smears, both to explain the etiopathogenesis of the
cervical cancer, and to provide therapeutic and prognostic
indications. In the present study, we correlate p16 immunohistochemical evaluation with HPV DNA detection in selected
PAP tests with ASCUS and LSIL diagnosis.
Materials and methods. 50 PAP smears, referred as ASCUS
and LSIL in the Surgical Pathology Department of Casale
Monferrato Hospital, have been considered. The ThinPrep
specimens have been investigated with double p16-Ki67 immunostaining and HC2 (HybridCapture 2), a direct test of
screening of HPV genome.
Results. 48% of our selected cases resulted positive both
with p16 immunostaining and HC2 test, while the remaining
52% had alternate results, with positive correlation between
p16 immunostaining and PAP test in 36% of these cases. The
comparison of HC2test and PAP test morphology presented
correspondence in 78% of all the cases considered.
Conclusions. The aim of our study is to compare the three
mentioned tests in order to verify the reliability of ASCUS
and LSIL diagnosis on PAP test morphology. Positive correlation between morphology, p16 immunostaining and HC2
test in 48% of selected cases and a higher concordance (78%)
between morphology and HC2 test lead to deduct that the association between PAP test morphology and HC2 is the most
reliable for the diagnosis.
May the cervico-vaginal cyto-screener
competently work on extra-vaginal cytology?
F. Pietribiasi, M. De Manna
Anatomia Patologica Ospedale S. Croce ASL TO 5 Moncalieri (TO),
Italy
Our Unit ran cervico-vaginal cytology for 15 years (20000
cases/year) with 3 cyto-technicians (CT). One year ago, two
of them moved to another hospital because the screening program has been centralized. Being the extra-vaginal cytology
one third of the workload of our Unit, we tested the hypothesis
that a skilled CT may be appropriately hired for extra-vaginal
citology.
A training plan has been set, consisting of 3 phases:
1) A one to one tutorship by FP, with the examination of 1000
cytology cases from different sources (fna, serous effusions, urine, sputum, etc) at the double viewer microscope,
and with theoretical lessons.
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
2) A review of our Unit archives (1000 cases randomly
selected),where the CT made the description, marked the
microscopic fields to be reviewed and put a diagnosis
by herself. Each case has been critically reviewed and
discussed with FP, getting topics for further theoretical
deepening.
3) The CT started to see as first the routine cases, (controlling
also the correctness of the anagraphic and clinical data).
She writes the microscopic description (type and quantity
of cells, other materials present in the smears) and proposes
a diagnosis. Each single case is reviewed with FP.
4) Appropriateness of FNA (this training is still on the way):
the CT prepare the smears from the sample taken by the
radiologist, makes a rapid toluidine- blu staining and
screens by the microscope if the material is enough for the
diagnosis, together with the pathologist.
Every step has been successfully reached in the planned timing.
The CT is now able to give the first look of every extravaginal case: she prepares the work for the pathologist, writes
the microscopic description and marks the significant microscopic fields. At the double microscope every case is then
closed together with FP.
The CT is now very skilful also in the difficult cytology of
serous effusions, helping the pathologists of the Unit in this
part of the work.
Our successful experience shows that the skill acquired during
years of screening cervico-vaginal cytology, together with the
theoretical learning is useful also for other kind of cytology;
in addition an intensive practical training at the microscope
allows to reach a very good skills.
The pathologist and FNA procedures:
technical and training-related problems
A. Bellomi, S. Negri
A.O. Carlo Poma, Mantova
The Pathologic Anatomy Unit at Mantova Hospital has been
equipped with a FNA ambulatory since 1984. At first, with
palpable nodules only and then with instrumentally-detected
lesions in collaboration with radiologists.
The diagnostic quality of our work enabled us to treat 2.000
cases per year by 1990 and this figure has gone up 3.000 since
2.000, with an ever increasing number of FNA on ultrasound
guidance.
The spread of breast screening has helped to raise awareness
and appreciation of fine needle aspiration cytology for the
point to bepassed the examen intraoperative. The reliability
of this diagnostic procedure has led to a very high number of
requests for instrumentally-guided exams also in relation to
other organs, booth deep and superficial.
This increase, not followed by a parallel increase in the
number of pathologists, often diverts the activities to other
professionals needle aspiration (ultrasound technicians, clinicians, radiologist)
The need to limit the number of non diagnostic samples and
the impossibility for pathologists to be present every time
FNA is performed, has made it necessary to develop training
paths with a view to improving the quality of the exam in the
absence of a pathologists.
In 2011 with the help of the “ Servizio di Sviluppo Organizzativo e Formazione Aziendale” we organized two editions of
a training course called “Instrumentally-guided FNA. From
theory to practice”.
Comunicazioni libere
The aim of the project was to uniform and optimize the instructions and methodology of FNA on ultrasound guidance in
dealing with both palpable and non palpable lesions.
The expected changes were:
• improvement in the quality of FNA on ultrasound guidance;
• improvement in the quality of diagnosis;
• reduced discomfort for patients.
The aim of the training course was:
1) to provide extensive knowledge of:
• ultrasound diagnostics
• cytologic diagnostics
• FNA procedures for cytologic diagnostics
2) to provide technical abilities in the management of FNA
on ultrasound guidance.
The lectures were radiologists in regard to ultrasound diagnostics, and pathologists as far as cytologic diagnostics and FNA
procedures were concerned. The methods and results of FNA
performed by pathologists were compared with those carried
out by radiologists and with those reported in the literature.
The course will be repeated in 2013 with another two editions.
Statistical comments about the impact of the course are still
premature but we observed a reduction (from 13% to 8%)
in the number of non diagnostics samples in the first three
months of the year, compared with the same period in 2.011,
when FNA was performed by non pathologists.
Discussion. FNA on ultrasound guidance is the most widespread procedure in the field of oncological diagnostics. However, its nationwide use is hampered by the lack of professional figures to be entrusted with the preparation of the material.
What’s more, the need to integrate the professional skills of
the cytologist with those of the radiologist is hindered by the
fact that these two figures work in two different Departments.
The lack of collaboration between pathologists and radiologists leads to poor results, which makes it necessary to use
more expensive and invasive methodologies with an increase
in discomfort for Patients.
The Pathologist and radiologist must collaborate blending
their professional skills with the purpose of providing patients
with a quick, reliable diagnosis essential for effective treatment.
“Siapec” and its own Cytology committee should address
the issue of FNA with proposals, based on their competence,
to organise training courses which must find new ways of
enhancing effective interaction between radiologists and cytopathologists.
US guided fine needle aspiration cytology
with cyto-assistance: experience of a peripheral
hospital
S. Erra, S. Modena, E. Mazzoni, N. Manoiero, S. Barbero*,
L. Spagnolo*, G.V. Salmaso*
SOC Anatomia Patologica ASL Al Casale Monferrato; *SOC Diagnostica per immagini ASL Al Casale Monferrato, Italy
The term cyto-assistance back to 25 years ago by a group
of Italian pathologists. This practise was thinked to reduce
pitfalls in diagnosis. Cyto-assistance provides the presence of
cytopathologist during all the stages of preparation of cytological specimens from the preliminary clinical–anamnestic
discussion to the final agoaspirative withdrawal and sample
microscopic evaluation.
In the present study, we report the first three months experience of our US-guided FNA cytology with cyto-assistance in
collaboration with radiologists.
153
Matherials and methods. We report the results of one hundred fine needle aspiration champions obtained during cytoassistance in collaboration with ultrasound ambulatory.
Seventy percent of our cases are represented by thyroid and
breast nodules equally, ten percent are both salivary gland
lesions that hepatocytic nodularity, while the remaining ten
percent is represented by other palpable or US detected nodules in various sites, including lymph nodes.
Most of cases consist of outpatients from private or agreement
specialistic clinics, except for breast pathology, that are mainly posted from radiologists after screening mammography.
In every US guided-fine needle aspirated nodule, we have setted up two smeared slides coloured with hematoxylin-eosin immediately to value if cellular material is sufficient and reliable
for diagnosis, while the remaining available material is stored
in an alcoholic fixative to obtained thin prep slides, sometimes
useful to determine the immunophenotype of cellular elements.
Results. As regards breast and thyroid nodular lesion, we
have had twenty percent of inadequate cases, while all the
pathologies regarding hepatocytic, lymph nodes and salivary
gland nodularity have resulted satisfactory for a definitive and
attendable cytological diagnosis.
Cyto-histological correlation in all cases in which histological
sample has been obtained corresponds to one hundred percent,
with none cytological misdiagnosis.
Conclusions. The aim of this report is to determine if USguided FNA cytology is an appropriate test for diagnostic
purpose and if it can replace the frozen section during surgical
intervention.
Hepatocytic, salivary gland nodular pathologies and lymph
node enlargements have provided good results with the practise of cyto-assistance.
As regards breast and thyroid cases, we have had a percentage
of inadequate similar to FNA cytology without US guide and
cyto-assistance. From a detailed analysis of the reasons of the
reported results, we think that it would be necessary a better
selection of patient, excluding cases with no clear nodular
lesion, above all in breast pathology. In thyroid pathology, indequate would be some colloid-cystic goiter, where cellularity
is poor and characterized by macrophages and colloid.
In conclusion, the practise of cyto-assistance could give more
satisfactory results when equipment discussion of the single
case will be performed during the patient’s management, for
example in the periodical GIC meetings.
Lymph node fine needle aspiration in a case
of blastic plasmacytoid dendritic cell neoplasm
L. Lorenzi, M. Ungari, A. Guerini, M. Chiudinelli, S. Fisogni,
F. Facchetti
Department of Pathology 1, Spedali Civili di Brescia, Brescia, Italy
Background. Blastic plasmacytoid dendritic cell neoplasm
(BPDCN) is a rare, aggressive, haematologic malignancy, derived from precursor of plasmacytoid dendritic cells. BPDCN
may present with cutaneous lesions or as acute leukemia with
systemic involvement since the beginning. In both cases the
course is aggressive and the median survival is approximately
12 to 14 months.
Patient, materials and methods. A 73 years old woman with
history of breast cancer, presented with contralateral sovraclavear lymphadenopathy. Ultrasound-guided fine needle aspiration (FNA) was performed and in addition to routine stains,
immunocytochemistry (ICC) for CD3, CD20, CD123, TdT,
CD34 and CD68R/PG-M1 was performed.
154
Results. FNA showed abundant monotonous medium sized
cells, with nuclei showing slightly irregular contour, finely
dispersed chromatin, one or several small nucleoli, and
scant cytoplasm, sometimes eccentrically located. On ICC
neoplastic cells were negative for CD3, CD20, and CD34;
TdT was expressed by 10% of the cells. A diagnosis of
hematolymphoid neoplasia not otherwise specified was
provided and the lymph node was excised. On histology a
proliferation of blast cells immynophenotypically consistent
with BPDCN (CD4+, CD56+, CD123+, TCL1+, BDCA2/
CD303+, CD2AP+, Tdt+ (10-20%); CD3-, CD20-, CD34-,
CD68R-) was diagnosed. Immunocytochemistry was retrospectively performed on additional FNA samples and
confirmed the expression of CD123, while CD68R was
negative. Chemotherapy was started, but the patients developed peripheral blood and bone marrow monocytosis, with
subsequent cerebral fluid involvement leading to patient’s
death 8 months from diagnosis.
Conclusions. BPDCN early diagnosis can be crucial for appropriate treatment; this rare neoplasm should be included in
the differential diagnosis on nodal FNA showing blastic features and lacking most common lineage-associated markers.
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
had nuclear atypia (enlargement, dispersed chromatin, membrane irregularity and large nucleoli), having other 5 cases a
more bland appearance. One case showed a proliferation of
plasma-cells which included binucleated and immature cells.
In seven cases, including a case subsequently diagnosed as
reactive hyperplasia, the cytological diagnoses were suspect
NHL. NHL was diagnosed in other 4 cases and myeloma in
1 case. All the lesions were then histologically diagnosed as
NHL in 10 cases (one case arisen in a salivary gland), myeloma (1) and florid lymphoid reactive hyperplasia (1).
Conclusions. The cytological diagnosis of OCL is hampered
by the rarity, the specific anatomical context and the difficulties in obtaining a sufficient cellular sample and, up to date,
a final histological diagnosis is necessary. Nonetheless a preoperative cytological diagnosis may be useful to differentiate
tumoral from non-tumoral processes, to avoid unnecessary
extensive surgery and to speed up the therapeutic procedures.
Unusual presentation of adenoid cystic
carcinoma: a pitfall in aspiration cytology
of the thyroid
B.J. Rocca, M.R. Ambrosio, A. Barone, A. Disanto
Fine needle aspiration cytology of
lymphoproliferative processes of the oral cavity
I. Cozzolino1, P. Todaro3, A.M. Bonanno3, G. Troncone1,
G. Giuffrè3, M. Mignogna2, P. Zeppa 4, G. Tuccari3, A. Vetrani1,
L. Palombini1
1 Dipartimento di Scienze Biomorfologiche e Funzionali e 2 Servizio
di Patologia Specialistica Odontostomatologica, Università di Napoli
“Federico II”; 3 Dipartimento di Patologia Umana, Università di Messina e 4 Servizio di Anatomia Patologica, Università di Salerno, Italy
Introduction. The pre-surgical diagnosis of the oral cavity
masses (OCM) is difficult because of the peculiar anatomical environment and the variety of possible corresponding
lesions. Primary non-Hodgkin lymphomas (NHL) of the
oral cavity represent less than 3% of all NHL; excluding the
tonsils and Waldayer’s ring only a small percentage may be
considered primary lymphomas of the oral cavity (OCL).
Fine needle aspiration biopsy (FNAB) has been successfully
utilized in the preoperative diagnosis of both OCM and NHL.
The aim of this study is to review the FNAB cytopathological
features of a series of OCL.
Materials and methods. Ten cytological cases of NHL of the
oral cavity were retrieved; one case of myeloma and one of
florid lymphoid reactive hyperplasia were also added to the
series that accounts for 10 women and 2 men with a median
age of 69 yrs. Corresponding lesions were from palatal soft
tissue (2); palatal bone (4); the floor of the mouth (1), the vestibules (2) and the gums (3). In all cases the first FNAB one
was air dried, Diff-Quik stained and evaluated for adequacy;
the second one was alcohol fixed and Papanicolaou stained.
In 7 cases a second FNAB was performed and used either to
prepare additional smears for immunocytochemistry (ICC)
(2) or suspended in PBS and used for flow cytometry (FC)
(5). Cytological diagnoses were rendered on the basis of cytological features alone (5), or combined with ICC (2) and/or
FC (5). All the diagnoses underwent subsequent histological
confirmation.
Results. Clinical presentation was represented by different
sized sub-mucosal masses which bulged in the oral cavity.
In all the cases cytological smears showed a monomorphous
lymphoid cell population; in 6 cases, corresponding cells also
Department of Human Pathology and Oncology, Anatomic Pathology
Section, University of Siena, Italy
Background. Adenoid cystic carcinoma (ACC) is a malignant neoplasm most commonly originating in salivary glands
where it accounts for approximately 10% of all tumors. Its
occurrence elsewhere is rare and extension into thyroid gland
is even rarer and described only once. However, this possibility should be taken into account if cytologic features are
suggestive of ACC on thyroid fine needle aspiration cytology
(FNAC).
Materials and methods. A 66-year-old man presented for the
onset of a swelling in the midline neck of six months duration.
The swelling was slowly progressive and was not associated
with pain or compressive symptoms. The patient was clinically euthyroid. On physical examination, a solitary palpable
nodule was identified in the isthmic region of the thyroid.
Ultrasonography revealed a 16x14-mm mixed echoic lesion
in the thyroid parenchima, involving trachea and soft tissues, without lymphoadenopathies. FNAC of the nodule was
done; wet fixed and air dried smears were made and stained
with May-Grunwald Giemsa (MGG) and Papanicolaou. Immunocytochemistry for TTF1 and thyreoglobulin were also
performed.
Results. FNAC revealed highly cellular smears with a monomorphic population of basaloid cells in tight clusters. There
was a partial microfollicle-like pattern. The individual cells
were round, oval or slightly angulated with fine, granular
chromatin and indistinct nucleoli. Cytoplasm was scant,
and the cell borders were indistinct. There was no definite
grooves or intranuclear pseudoinclusions. Small hyaline
globules of basement membrane material were found. The
globules stained magenta with MGG, were roughly spherical
and showed marked variability in size. “Ropy colloid” was
observed. Differential diagnoses between papillary thyroid
carcinoma, follicular variant, poorly differentiated thyroid
carcinoma and primitive or metastatic ACC were considered.
Thyroid primitivity was excluded on the basis of the negativity for TTF1 and thyreoglobulin. The final diagnosis was
ACC of salivary glands infiltrating the thyroid. The diagnosis
was then confirmed by the histological examination of the
surgical sample.
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Conclusions. In the evaluation of a thyroid nodule, history,
physical examination, and ultrasonography are essential but are
of limited usefulness, and the treatment decision mostly rely on
the findings of FNAC. Although rare, metastatic tumors, including ACC, should be considered in the differential diagnosis.
Foamy gland pattern of pancreatic ductal
adenocarcinoma as a pitfall in EUS-FNA
pancreatic cytology
C. Bellevicine1, U. Malapelle1, A. Iaccarino1, P. Schettino2,
V. Napolitano2, P. Zeppa3, G. Troncone 1
1 Department of Biomorfologic and Functional Sciences, University
of Naples Federico II, Naples, Italy; 2 Endoscopic Surgery, Second
University of Naples, Italy; 3 San Giovanni di Dio e Ruggi d’Aragona,
University of Salerno, Salerno, Italy
Background/introduction. The cytological diagnosis of
pancreatic ductal adenocarcinoma (PDA) can sometimes be
challenging. In our experience most PDA fine needle aspirates (FNA), expecially when performed during endoscopic
ultrasound (EUS), do not offer difficulties to the experienced
cytopathologist, as consistent diagnostic criteria are well
established. However, in some instances microscopy may
overlap between benign and reactive processes. In particular,
the foamy gland pattern (FGP) may impart to PDA a deceptively benign appearance, thus that a false negative diagnosis
of benign or reactive process may be rendered on FNAs. This
issue is here addressed by reporting two cases of PDA with
FGP correctly diagnoses by FNA. We argue that cell-block
preparation, ancillary stains and molecular techniques are
important to give a correct diagnosis.
Materials and methods. A retrospective evaluation on a
series of 9 EUS FNA of PDA was carried out and two PDA
cases with FGP were reviewed. In our Institution on site
evaluation is carried out and material from additional passes
are formalin fixed and dedicated to cell block preparation. To
refine on-site diagnosis a wide range of ancillary techniques
is performed, including mucicarmine and Alcian Blue stain
for acid mucins, panel immunostainings and molecular mutational analysis for exon 2 KRAS mutations.
Results. The first case, from a 57 years-old female showed
only a few scattered medium-sized cells, arranged in loosely
cohesive small groups. Their cytoplasm was abundant and
finely microvacuolated; nuclei were compressed and eccentrically dislocated with irregular membrane outline conferring a
wrinkled appearance to the nuclei. Evidences obtained from
the H&E stained cell block slides were more informative. The
neoplastic cells were arranged in several strips of aligned columnar cells with basally located nuclei; their cytoplasm was
foamy displaying an apical eosinophilic condensation. The
nuclei were irregular, with raisinoid appearance and evident
nucleolation. The cytoplasm thickening observed at the cell’s
apex was strongly stained by the mucicarmine and Alcian Blue
stain for acid mucins; a strong CEA granular cytoplasmatic
positivity were also observed. For KRAS mutational analysis,
cancer cells from H&E stained cell block sections were selectively laser microdissected. The G12D mutation was detected,
confirming the microscopic diagnosis. The microscopic features together with ancillary stainings and KRAS status are
consistent with those of pancreatic adenocarcinoma with FGP.
The second case, from 64 years old woman had previously
been diagnosed as a reactive process on a six month earlier
FNAs performed in other Institution. A subsequent aspiration
performed at our endoscopic service, was on-site evaluated
and processed as above described. The microscopic and phenotypic completely overlapped with those above described.
Microscopic review of the earlier FNAs also showed that FGP
features were indeed present, having originally been misinterpreted as reactive benign process.
Conclusions. In conclusion, although FGP does not have any
additional biological and clinical significance, the practicing
cytopathologist should be aware of its occurrence in order to
avoid a false negative diagnosis.
Technical options for inclusion in needle
aspiration pathology and in selected serosal fluid
S. Erra, S. Modena, E. Mazzoni, M. Butera
SOC Anatomia Patologica, ASL AL, Casale Monferrato, Italy
Introduction. Different techniques have been adopted in surgical pathology laboratories to optimize the diagnostic yeald
of cytological material obtained from fine needle agoaspirative cytology and from serosal spontaneous fluid. In our
department,serosal fluid are prepared on thin prep slide and
paraffin cytoinclusion,while fine needle agoaspirative material is set on double thin prep slides and standard smears. In
last six months,we adopted agar to obtain cytoinclusion from
serosal fluid and agoaspirative material,compairing these agar
cytological inclusions with the classic cytoinclusions.
Materials and methods. For our purpose,we have selected
cases from agoaspirative material fixed in cytolit preparation
and some serosal fluid. We have considered only champions
with high cellularity after a previous valutation on a smear or
on a thin prep slide. We have included the remaining cytological material using agar according with the following protocol:
Place a tube of Bio-Agar to liquefy in the microwave for 1’
low power or in a water bath at 65°C for 10’; centrifuge test
sample for 10’ at 2500 RPM; discard the supernatant with
disposable pipette aspiration of avoiding any suspended fragments; add 10 drops of melted Bio-Agar; vortex for 5’’; leave
the sample to chill in the freezer until it solidifies (3’); remove
the pellets sample solidified from the bottom of the tube and
put it into a biocassetta; include as routine histologic.
Results. Agar inclusion of fine needle agoaspirative material
has obtained satisfactory results in 70% of considered cases.
The cells included in agar and derived from parenchymal tumors reproduce the structure of the original lesion,so it results
easier to interpret the morphology observed. Agar inclusions
allow to determine the immunophenotype of the neoplastic elements with better results compared to immunocytochemistry
on thin prep slides.
In pleural and peritoneal fluid,agar cytoinclusions have made
it possible make the differential diagnosis between mesothelioma and carcinoma,not only on morphological aspects of
the neoplastic cells,but on the immunophenotype of the elements observed. We have had satisfactory results in 80% of
examined cases.
Conclusions. In selected cytological cases derived from fine
needle aspiration and from serosal fluid, agar inclusion technique gives better results in differential diagnosis compared
to classic cytoinclusion or thin prep slide. We noticed that
material obtained shows the maintenance of morphology not
only of the single elements,but of the original parenchymal
structure,as well as the absence of immunophenotype alterations. This technique is useful in cytological material derived
from hepatic,lymph node and breast fine needle aspiration, with
the possibility to determine immunoprofile of breast tumors in
order to establish a neoadjuvant therapy in selected patients.
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I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Sabato, 30 giugno 2012
ore 11.00-12.30
Comunicazioni Libere
Chairmen: Guido Collina (Bologna), Gaetano Bulfamante (Milano)
Immunocytochemistry in effusion diagnosis can
reduce pitfalls
A. Di Lorito, G. Melatti*, M. De Laurentiis*, D. Gatta, S. Rosini,
D. Caraceni*
Unità Operativa di Citodiagnostica, Dipartimento di Scienze Biomediche, Università G. d’Annunzio di Chieti- Pescara; * Unità Operativa
Complessa di Citodiagnostica, Ospedale “F. Renzetti” Lanciano, Italy
Introduction. Cytology, in some situation, represents the
only type of material available in advanced cancer patients.
However morphology alone could cause problems of differential diagnosis and pitfalls, especially in effusions. Today
ancillary techniques represent the key to give a correct diagnosis. In particular immunocytochemistry (ICC) could help
morphological diagnosis in difficult conditions. We present
two group of cases of effusions for which ICC was important
to avoid pitfalls.
Material and methods. In the first group we showed pleural
effusions from a women of 57 years old (y) and a man of
65y. In both there were neoplastic cells in which we detected
a mucus- containing vacuoles with a central inclusion, PAS
positive, colored in purple in May Grunwald Giemsa- stained
aspiration smears. These findings were named such as bull’s
eye (target) inclusions, previously described in adenocarcinomas of breast, stomach, colon, lung, ovary, pancreas and
urinary bladder. For these reasons, we thought of metastases
from adenocarcinoma.
In the second group we compared pleural effusions from a
man of 64y and a peritoneal effusion from a man of 71y. Cells
showed a weak intercellular coesion in both cases.
A panel of ICC was performed in the two group of patients.
Results. In the first group, CKAE1-AE3 and EMA were
positive in malignant cells of both cases. CEA and B72.3
were positive in the first patient, negative in the second, whilst
calretinin, PAS and DPAS were positive only in the second
one. The first case was diagnosed as ductal invasive breast
carcinoma while the second as epithelioid mesothelioma, both
confirmed by histology.
In the second group, the first patient showed PAS, Ca 19,9 and
CEA positivity and HBME1 negativity. In the second case,
PAS, Ca 19-9 and CEA were negative, HBME1 was positive.
The first case was diagnosed as metastases from signet-ring
gastric adenocarcinoma, The second had a clinical diagnosis
of ascites in congestive heart failure.
Conclusion. We showed that a panel of ICC markers is helpful in effusion diagnosis especially when morphology alone
could not give a certain response.
Can KI67 cytological index distinguish low grade
from high grade GEP-nets?
G. Carlinfante1, P. Baccarini2, L. Di Tommaso3, A. Fornelli4,
L. Losi5, L. Maccio5, G. Gardini1
Department of Pathology IRCCS/ASMN of Reggio Emilia, 2 Section
of Pathology “M. Malpighi” University of Bologna, 3 Department of
1
Pathology IRCCS/Humanitas Hospital Rozzano, 4 Institute of Pathology Department of Oncology Maggiore Hospital Bologna, 5 Department of Pathology Policlinico of Modena, Italy
Introduction. Gastroenteropancreatic NETs (GEP-NETs) are
predominantly indolent low-grade neoplasm although some
are histologically and clinically aggressive. Distinguishing
characteristics of low-grade versus high grade tumour have
been described in the literature. The Ki67 labelling index was
found to bear prognostic significance in pancreatic and in
gastrointestinal NETs, in particular last 2010 WHO classification of GEP-NETs followed the ENETs proposal for a role of
ki67 immunostaining in GEP-NETs grading. Three tiers (G1,
G2, G3) have been defined based on mitotic count and Ki67
index. The grading require mitotic count in at least 50 HPF
(1HPF = 2mm2) and Ki67 index as a percentage of 500-2000
cells counted in areas of strongest nuclear labelling. In small
biopsy or cytological material, the number of tumour nuclei
may be lower and the proliferative fraction may not reflect
that of the whole tumour.
Aims. The aim of this study was to assess reliability of
tumour grading based on Ki67 cytological index in order to
distinguish low grade from high grade GEP-NETs. We compared the measurements of the cytological Ki67 expression
obtained on EUS-FNAC and the histological Ki67 expression obtained on histological sections after surgical removal
of the tumour.
Material and methods. We studied 36 lesions of GEPNETs from 32 patients retrieved from files of Department
of Pathology of IRCCS/Arcispedale Santa Maria Nuova of
Reggio Emilia, Bellaria and Maggiore Hospital of Bologna,
Policlinico of Modena and IRCCS/Humanitas Hospital of
Rozzano.
All patients underwent EUS-FNAC examination before surgery for removal of the mass. Ki67 expression was measured
by immunocytochemistry on cytological material and on the
corresponding histological sections. Cytological evaluation
of Ki67 expression has been done counting at least 500-2000
tumour cells before considering adequate the sample. The pathologist measuring Ki67 expression on histological sections
was blinded to the cytological results. Defined 2010 WHO
criteria were used for NET classification.
Results. We found a good concordance of Ki67 labelling
index (85%) in 30 of 36 lesions (from 26 of 32 patients).
In particular 24, 10 and 2 lesions graded respectively as
G1, G2 and G3 on cytological material were confirmed in
21, 7 and 2 histological specimens. In six lesions different
Ki67 cyto-histological expression we found, in particular
3 lesions graded as G1 on cytological material resulted be
G2 on histological sections, whereas, 3 cases graded G2 on
cytology resulted G1 on histology. Both cases graded as
G3 on cytological smears were confirmed on histological
evaluation.
Conclusions. Our results showed a good concordance of Ki67
index between cytological and histological specimens in GEPNETs. In particular if we consider NETs G1 and NETs G2 as
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low grade NETs whereas NECs G3 as high grade neoplasms,
we found a total reliability of cytological tumour grading with
respect to histological grading. Other studies on larger series
of patients will be necessary, however, this distinction may
represent an important information for therapeutic decisions
in inoperable cases.
UroVysion test in the follow-up of patients with
history of urothelial bladder cancer: our 1-year
experience
E. Pegolo, F. Riosa, L. Peronio, G. Raiti, C. Di Loreto
Istituto di Anatomia Patologica, Università di Udine, Italy
Introduction. The aim of the present prospective study was
to assess the diagnostic benefit of UroVysion (Vysis-Abbott
Laboratories, Downers Grove, IL) in the follow-up of patients
with a history of bladder urothelial carcinoma.
Materials and methods. An unselected cohort of 28 patients
with a history of urothelial carcinoma of the bladder was prospectively followed up by office-based cystoscopy, cytology,
and UroVysion for 1 year. Urine samples both for cytology
and UroVysion test were placed and stored in Cytolyt solution and then processed by the ThinPrep 2000 method (Cytyc
Corp., Boxborough, Mass) according to the manufacturer’s
instructions. Final cytologic results were classified into 1 of 4
categories: inadequate, normal-benign, atypical-suspicious or
malignant. The results of the UroVysion test, performed according to the package insert, were signed out as inadequate,
negative or positive. During cystoscopy, any abnormal urothelial lesions were biopsied and frank tumors were resected.
In patients with a malignant or atypical-suspicious cytology
or positive Urovysion and no obvious tumor, random bladder
biopsies were obtained. Biopsy specimens were diagnosed according to WHO 2004 classification of tumours of the urinary
system.
Sensitivity, specificity, positive predictive value (PPV), and
negative predictive value (NPV)
were calculated being the biopsy the gold standard.
Results. The median age was 72 years (range: 53-83 years).
The male to female ratio was 2.5:1. We had a total of 42 cytology tests (0 inadequate, 17 normal-benign, 15 atypical-suspicious and 10 positive), 42 UroVysion analysis (5 inadequate,
25 negative and 12 positive), 38 cystoscopic examinations
(23 negative and 15 positive for urothelial lesions), and 20
biopsy specimens (8 low grade and 8 high grade urothelial
carcinoma and 4 inflammatory lesions). Cytology showed an
overall sensitivity of 81.2% and specificity of 50%, PPV of
86.7%, and NPV of 40%. Cystoscopy yielded a sensitivity of
75%, specificity of 75%, PPV of 92.3%, and NPV of 42.8%.
UroVysion showed a sensitivity of 50%, specificity of 50%,
PPV of 77.8%, and NPV of 22.2%. Sensitivity, specificity,
PPV, and NPV for the standard follow-up scheme consisting of combined cystoscopy and cytology were 93.7%, 50%,
88.2%, and 66.7%, respectively. On the other hand, the noninvasive follow-up scheme consisting of combined cytology
and UroVysion showed a sensitivity of 93.5%, specificity of
50%, PPV of 88.2%, and NPV of 66.6%.
Conclusions. UroVysion is a worthwhile approach for the
follow-up of patients with a history of urothelial carcinoma.
The test increases the sensitivity of the urinary cytology alone
(from 81.2% to 93.5%) and shows a high specificity (100%)
in the detection of high grade urothelial lesions, which comprises carcinoma in situ that are frequently missed at the cystoscopic examination.
The cytological sample is a suitable
biospecimen for KRAS mutation analysis
C. Bellevicine, U. Malapelle, G. Troncone
Department of Biomorfologic and Functional Sciences, University of
Naples Federico II, Naples, Italy
Background. Anti-EGFR monoclonal antibodies, cetuximab,
and panitumumab, are administrated under the condition
that advanced colo-rectal cancer (CRC) carries a wild-type
KRAS gene. Thus, clinicians request pathologists to genotype
KRAS before treatment. Typically, the specimens available
for KRAS mutational analysis are formalin-fixed paraffinembedded (FFPE) primary tumor tissue blocks. However, in
patients with rectal tumours undergoing neoadjuvant therapy,
the source of FFPE material is limited. In this setting, CRC
cytological samples taken from the metastatic site may be exploited. However, these specimens show at least some degree
of necrosis; thus, their suitability for the KRAS assay needs
to be tested.
Aims. To explore the suitability of the smears aspirated from
metastatic CRC for KRAS mutational analysis by direct gene
sequencing.
Material and methods. We retrospectively recollected from
our archives a series of 19 aspirates from metastatic CRC. For
13 out of 19 patients was also available an histological FFPE
sample. On both specimen, KRAS status was assessed.
Results. Here, we show that 18/19 (94.7%) metastatic CRC
smears were perfectly adequate for codon 12 and 13 KRAS
mutational analysis by direct gene sequencing. Only one
case (5.3%) showing abundant necrotic debris and poor
cellular preservation, was not informative for KRAS status.
Codon 12 gene mutations were found in 4/18 (22.2%) of the
adequate cases (c35G>T n = 2; c34G>T n = 1; c35G>A n
= 1). Concordance between cytological and FFPE samples,
both available in 13 patients, occurred in 92.3% (12/13) of
the cases.
Conclusion. Whenever histological specimens of CRC are
not available, KRAS testing may be reliably performed on
cytological specimens.
EGFR and KRAS mutations detection on lung
cancer liquid-based cytology
U. Malapelle, C. De Luca, M. Salatiello, C. Bellevicine, G.
Troncone
Department of Biomorfologic and Functional Sciences, University of
Naples Federico II, Naples, Italy
Introduction. In advanced non-small-cell lung carcinomas
epidermal growth factor receptor (EGFR) and KRAS testing
is often performed on cytology. Currently, there is a trend
towards an increasing use of liquid-based cytology (LBC) to
diagnose non-small cell lung cancer. In fact, liquid-based cytology (LBC), which eliminates the need for slide preparation
by clinicians, may be very useful.
Aim. To detect epidermal growth factor receptor mutations
applying different molecular techniques to LBC samples with
and without laser capture microdissection (LCM).
Matherials and methods. We have retrospectively retrieved
58 LBCs and DNA was extracted twice. One sample was
obtained directly from CytoLyt solution, whereas the other
DNA sample was derived after slide preparation and LCM
of Papanicolaou-stained cells. EGFR and KRAS mutational
analyses were performed matching direct sequencing and
more sensitive molecular assays.
158
Results. The rate of mutant cases obtained by direct sequencing was discordant between CytoLyt-derived (10.3%)
and LCM-derived (17.2%) DNA. However, the same mutant rate (17.2%) was achieved on the matched samples by
high-resolution melting analysis, fragment and TaqMan
assays.
Conclusion. LCM and direct sequencing may be replaced by
more sensitive non-sequencing methods directly performed
on CytoLyt-derived DNA, an easier and faster approach to
improve epidermal growth factor receptor testing standardisation on LBCs.
Clinical utility of adding braf mutational
analysis to immunocytochemical algorithm in
thyroid carcinoma. A preliminary study
G. De Maglio, A. De Pellegrin, E. D’Alessandro, E. Masiero
Azienda Ospedaliero-Universitaria S.Maria della Misericordia, SOC
Anatomia Patologica, Udine, Italy
Background. The worldwide incidence rate of thyroid cancer
is steadily increasing.
Morphologic approach alone is not often able to solve the
aim of differential diagnosis between benign and malignant
lesions. Paraffin embedded cytologic material (cell-block)
is a useful method allowing both immunocytochemical and
molecular analysis.
Galectin-3, associated to HBME-1 and CK19, demonstrate
all together a high sensitivity and specificity in immunocytochemistry detection of malignant papillary lesions.
BRAF V600E mutation is reported in about 45% of papillary
thyroid carcinoma and it is associated with aggressive disease.
RAS mutations are described in about 15% of papillary carcinoma and 40% of follicular carcinoma.
Materials and methods. We selected from archive 57 cytologic cell-block samples, previously diagnosed by a immunocytochemistry panel (Galectin-3, HBME-1, CK19 and TPO).
We defined carcinoma those cases positive for Galectin-3,
HBME-1, CK19 and negative for TPO. Were defined negative samples expressing TPO and negative for Galectin-3,
HBME-1 and CK19. All cases with only one or two positive
markers and/or TPO negativity or reduced expression were
defined as suspicious.
Morphologic and immunocytochemical diagnosis were blinded reviewed by two pathologists.
Cases were diagnosed as follows: 16/57 (28.1%) carcinoma,
27/57 (47.4%) suspicious for carcinoma and 14/57 (24.5%)
negative.
All cases were analysed for gene status of BRAF (exon 15) by
pyrosequencing. All BRAF wild-type morphologically suspicious cases were screened for NRAS (codon 61) gene status.
Results. BRAF gene was wild-type in 35/57 (61.4%) patients
and 22/57 (38.6%) revealed a V600E mutation.
All negative cases were BRAF wild-type.
Among positive cases 15/16 (93.8%) carried a V600E mutation. The only wild-type case showed a microfollicolar appearance; histologic correlation was not available.
Among suspicious cases 7/27 (25.9%) showed a mutant status
for BRAF (V600E), while 2/20 (10%) showed a Q61R mutant
status for NRAS.
Conclusions. BRAF mutation demonstrated high sensitivity
(93.3%) and specificity (100%).
In our cohort of patients we observed a higher mutation rate
(93.8%) than expected, probably related to low number of
patients.
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Further studies are needed to correlate cytologic suspicious
cases with histology.
Regardless of prognostic value of BRAF mutations, by our
experience we suggest the immunocytochemical panel for all
samples. A much expensive and time-consuming molecular
approach could be addressed only for suspicious cases with
this algorithm: BRAF first and then NRAS and KRAS for BRAF wild-type samples.
The management of thyroid patient on
indeterminate FNAC specimen using a predictor
model and molecular test of point mutation
V600 BRAF
G. Di Benedetto, A. Fabozzi, C.R. Rinaldi, C. Rinaldi
Service of Cytopathology, AslCe, Division of Medical Oncology, F.
Magrassi-A. Lanzara. Department of Clinical and Experimental
Medicine, Second University of Naples School of Medicine, Doctor
of Medicine Consultant of Department of Haematology United Lincolnshire Hospital (NHS), Trust, Boston, United Kingdom, Service of
Molecular Biology
Background/introduction. Fine needle aspiration cytology
(FNAC) is considered the gold standard diagnostic test in
the evaluation of a thyroid nodule 1 2.The management of
patients with indeterminate or suspicious FNAC specimens
still remains problematic and the main topic is the identification, between the cytological indeterminate lesions, of nodules
requiring surgical treatment and the benignant ones that can
be only clinically observed 1 2.The use of a predictor model
considering different features of patient or of disease 3, could
further decrease the overall thyroidectomy rate in patients
with benignant disease even if it does not eliminate all unnecessary surgery.
A further contribution to the question is given by molecular
testing to thyroid FNAC 4 5. In particular, the V600E BRAF
activating point mutation, is highly specific for papillary carcinoma 4 5.
Our study was started to evaluate the thyroid FNAC diagnostic accuracy and true positive rate of malignant lesions, using
a clinical and cytological predictor model 3 on cytologic indeterminate examinations in addition to BRAF mutation test 3-5.
Material and methods. From September 2008 to December
2009, in the endocrinology department of Clinical Hospital of
Marcianise – Italy, we performed a neck ultrasonographic examination on 1296 patients with clinical evidence of a thyroid
nodule. 124 patients subsequently underwent an ultrasoundguided fine needle aspiration cytology (FNAC); the aspirated
specimen was smeared onto glass slides, air dried and stained
with Diff-Quick colouration.
Diagnostic categories. The results obtained, were classified
according to the Italian Society of Pathology and Cytology
(SIAPEC) Consensus Conference morphological criteria, and
put in five different categories:
Tir 1-not diagnostic; Tir2-negative for malignant cells; Tir3follicular neoplasia/atypia of indeterminate significance; Tir4suggestive for malignant neoplasm; Tir5-positive for malignancy.
Predictor Model. To predict thyroid malignancy on indeterminate cases, we employed a score risk proposed by Banks et al.
in 2008 3. This predictor model implies the presence of three
different risk categories, such as the nodule size, the age at
diagnosis and Cytopathology features. Every category is characterized by a definite score, such as reported in the Table 1;
therefore, in order to sub-classify indeterminate diagnoses
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Tab. I. Risk score analysis (3).
Risk score
Nodule size (cm)
1.5–2.5
<1.5
2.5–3 1.
>3 1.
0
3
0
2
Age at diagnosis
(year)
50–60 1.00 (Reference) 0
<30
30–40
40–50
60–70
>70
0
2
2
2
3
2
Cytopathology
Hu¨ rthle cell
Atypia
0
3
Follicular feature
Suspicious for PTC
3
12
(Tir3), we considered risk score as low (0-5) and moderate
(> 5). Applying this model score to our reported cases, we
obtained two subcategories:
• little suspicion for malignancy lesion (Tir3a)
• moderately suspicion for malignancy lesion (Tir3b)
Molecular biology. In patients with a diagnosis of Tir3a,
Tir3b, Tir4 and Tir5 we performed the molecular biology test
for V600E - BRAF gene mutation, deemed to be specific for
the papillary carcinoma.
We performed DNA Extraction on cells obtained by FNAB
and resuspended in a 0.9% NaCl solution. DNA was
achieved with a salting-out method 4, modified in our laboratory. Purity assay and evaluation was assessed by spectrophotometry (BiophotomakerEppendorf), while the degree of
integrity was evaluated with electrophoresis. Mutation study
was performed by PCR – ARMS and for the confirmation we
employed the gene sequencing (Biosystem kit) and scanning
on automatic analyzer ABI PRISM 310 (Genetic Applied
Biosystem).
Histology and follow-up. The patients with diagnosis of Tir3b,
Tir4 and Tir5, subsequently underwent a surgical resection
with histological assessment of the pathology. Patients with
a diagnosis of Tir2 and Tir3a were studied every six months
with follow-up in the subsequent years, while Tir3b, Tir4 and
Tir5 ones underwent thyroidectomy.
Cyto-Histologic and follow-up correlation. The thyroid nodules were histologically classified and compared with the
cytology (Tab. I).
All the cases not related with the histological diagnosis were
considered false negative (Fn) and False Positive (Fp). True
positive (Tp) and true negative (Tn) represent the cytologic
diagnosis according to histology examination and clinical
follow-up.
No late resections or re-aspiration were performed.
Statistical analysis. Sensitivity, Specificity and Positive
Predictive Value of a positive cytological examination, were
calculated according to Galen and Gambino (Tab. I).
Results. Following ultrasound guided FNAC, 124 patients
were classified into five diagnostic categories: Tir1 = 18
The probability of thyroid malignancy as a function of risk score.
Scores <5 are low risk
Scores between 5 and 15moderate risk
Scores greater than 15, high risk. () .
(14,5%); Tir2 = 86 (69,4%); Tir3 = 14 (11,3%); Tir4 = 2
(1.6%); Tir5 = 4 (3,2%).
Patients classified as Tir1 repeated the FNAC and were reclassified as Tir2 in 17 cases and as Tir5 in 1 case.
On Tir3 diagnosis, according to the malignancy predictive
model, previously described, we assigned a risk score for each
feature considered (Tab. I): two points are assigned to nodules
> 3.0 cm and in patients with less than 50 years or more than
70 years, three points in patients with nodules smaller than
1.5 cm, in the age group between 60 and 70 year, as well as
cellular atypia and available in follicles of cells. No point is
awarded to nodules of variable dimension between 1.5 and
2.5 cm, patients between 50 and 60 years, and the presence
of Hurthle cells.
Summing the scores obtained, we calculated the score of
the 14 Tir3 samples (Tab. I): the cases 1,2,3,4,5,6 had a
score lower than 6 and we considered them at low risk
of malignancy (Tab. I) and classified as Tir3a. The cases
7,8,9,10,11,12,13,14 obtained a score variable between 6 and
9 and were considered at moderate risk of malignancy (Tab. I)
and classified as Tir3b. After the repetition of FNAC on not
diagnostic specimen (Tir1) and the subdivision of inadeguate
specimen (Tir3) in two sub-categories, we obtained this cytological classification: Tir2 = 103, Tir3a = 6, Tir3b = 8, Tir4
= 2; Tir5 = 5 (Tab. I). The V600E - BRAF mutation test was
performed on patients with a Tir3, Tir4 and Tir5 ctology (n =
21). BRAF mutation was found in one case of Tir3-b, one case
of Tir4 and 2 Tir5. The Tir3b and the Tir4 resulted positive to
BRAF mutation has been re-classified Tir5.
Table I shows the final cytologic classification: Tir2 = 103,
Tir3a = 5; Tir3b = 8; Tir4 = 1, Tir5 = 7. In the light of these
new results, surgery was performed on 16 patients classified
as Tir3b, Tir4 and Tir5. Histological examination of these
patients was typed and compared with cytological diagnosis. Histological examination results were: 2 nodular goiters
(False positive), 5 follicular adenoma, atypic adenoma, follicular carcinoma, 6 papillary carcinomaand1metastatic adenocarcinoma (True positive).
Clinical follow-up was performed on Tir2 and Tir3a diagnoses and no late resections or re-aspiration was necessary;
therefore, we potentially avoided unnecessary surgery.
Statistical analysis
Diagnostic accuracy was assessed as following: Tp=14; Fp=2;
Tn=108; Fn=0, Sensitivity= 14 Tp /14 Tp + 0 Fn = 00 %;
Specificity= 108 Tn/ (108 Tn +2Fp) = 98.2 %: Predictive
Positive Rate = 14 Tp/( 14 Tp +2 Fp) =9 7.5 %.
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I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Conclusions. Cytological diagnosis is a fundamental element for the surgical indication of thyroid nodules and, at the
same time, is the only useful method to reduce unnecessary
surgery. In this way, it can restrict the surgical indications
to the cytological diagnosis of suspect and/or positive for
malignancy. Notwithstanding the use of FNAC techniques,
many surgery still result excessive or useless. To select with
more accuracy the patients candidate to surgery, in this study,
we have applied, on indeterminate lesions , the predictive
model sec Banks 3 using a score risk of malignancy. We
divided the follicular neoplasms in two subcategories, giving
points to individual cases diagnostic,.Tir3-a: little suspicion
for malignancy;Tir3-b: moderately suspicion for malignancy.
Finally, we have sought with the molecular biology the mutation
V600-B-Raf . In our study we found 1 BRAF V600 positive on 1
Tir3-b that in histology was diagnosed follicular variant of papillary carcinoma 5, as well as 1 positive on tir4 and two positivity
of Tir5 already classified as papillary carcinomas, confirming the
usefulness of Braf as help diagnostic cytology carcinoma.
Using model predictor and researching V600 BRAF we have
improved the sensitivity (98.2%) and the True Positive Rate
(97.5%).
Our experience, even if performed in a small number of patients, leaves us some interesting topics to think about.
References
1
Dean DS, Gharib H. Epidemiology of thyroid nodules. Best Pract Res
Clin Endocrinol Metab 2008;22:901-11.
2
Koss’ Diagnostic Cytology and its histopathologic bases. Volume II,
5th edition, Lippincott Williams & Wilkins.
3
Banks ND, Kowalski J, Tsai HL, et al. A diagnostic predictor
model for indeterminate or suspicious thyroid FNA samples. Thyroid
2008;18:933-4.
4
Kucukodaci Z, Akar E, Haholu A, et al. A valuable adjunct to FNA
diagnosis of papillary thyroid carcinoma: In-house PCR assay for
BRAF T1799A (V600E). Diagn Cytopathol 2010 Jul 6.
5
Troncone G, Cozzolino I, Fedele M, et al. Preparation of thyroid FNA
material for routine cytology and BRAF testing: a validation study.
Diagn Cytopathol 2010;38:172-6.
RISK SCORE ACCORDING TO BANK’S PREDICTOR MODEL
CASE
SIZE (cm)
AGE
CYTOLOGY
SCORE
1
2.7
55
Follicular
4
2
2.9
58
Follicular
4
3
3.5
30
0
4
4
1.3
65
Hurtle cell
5
5
1.7
28
Follicular
5
6
1.5
37
Atypia
5
7
1.2
39
Follicular
8
8
2
55
Follicular + Atypia
6
9
1.7
56
Follicular + Atypia
6
10
1.3
1
Follicular + Atypia
8
11
1.2
63
Follicular
9
12
3.5
57
Follicular + Atypia
7
13
3.8
33
Follicular
6
14
3.7
32
Follicular
6
CYTOLOGIC DIAGNOSES
CYTOLOGY
CLASSIFICATION
RESULTS
Tir2
103 (83.1%)
Not neoplastic
Tir3-a
Tir3-b
5
8(
(4%)
6.5%)
Suggestive for malignancy
Tir4
1
(0.8%)
Positive for malignancy
Tir5
7
Follicular suspicious
Total
(5.6%)
124
DIAGNOSTIC ACCURACY
CYTOLOGIC-HYSTOLOGIC DIAGNOSIS CORRELATION
CYTOLOGY
NOT
FOLLICULAR FOLLICULAR
NEOPLASTIC ADENOMA CARCINOMA
Tir2
Tir3-a
108
follow-up
Tir3-b
2 FP
Tir4
5 TP
PAPILLARY
CARCINOMA
1TP
1 TP
Tir5
6 TP+ 1TP
(Metastatic
Adenocarcinoma)
Total
True Positive: 14
False Positive: 2
True Negative:108
False Negative: 0
Sensitivity= Tp/(Tp+Fn)= 14/14=100%
Specificity=Tn/(Tn+Fp)=108/110=98.2%
True Positive Rate= Tp/(Tp*Fp)= 14/16=97.5%
The role of BRAF (V600E) mutational analysis on
LBC-processed aspiration biopsies. a prognostic
factor for multifocality and nodal involvement
in thyroid microcarcinoma
E.D. Rossi, M. Martini, P. Straccia, M. Raffaelli1, S. Capodimonti, C.P. Lombardi1, A. Pontecorvi2, E. Stigliano, L.M.
Larocca, G.Fadda
Division of Anatomic Pathology and Histology, 1 Division of Endocrine Surgery, 2 Division of Endocrinology- Università Cattolica del
Sacro Cuore, “Agostino Gemelli” School of Medicine, Rome
Background. Activating mutations in the V600E locus of
BRAF-1 gene are frequently detected in papillary thyroid carcinoma (PTC). These mutations have been identified in about
29-69% of PTC and more than 80% of the tall cell variant
(TCV) whereas they have not been detected in benign lesions
and in the majority (80%) of the follicular variant of PTC
(FVPC). The aim of this study is the evaluation of the role
of the BRAF mutation in predicting the outcome of thyroid
PTC up to 1 cm diagnosed on liquid based cytology (LBC),
and, hence, in guiding the clinical and surgical management
of these patients.
Materials and methods. From October 2010 through June
2011 230 cases were diagnosed as PTC on FNA processed
by LBC. Out of them, 80 cases of microcarcinoma underwent
BRAF mutational analysis. The aspirated material was processed with the ThinPrep 2000 technique (Hologic Co, Marl-
161
comunicazioni libere
borough MA). After DNA extraction on the residual material
the BRAF mutation analysis using direct sequencing method
was carried out.
Results. Sixty cases out of 80 (75%) underwent surgery. Out
of them 48 (83.3%) expressed a BRAF mutation (34 PTC, 11
TCV and 3 FVPC). Unlike TCV and PTC cases, in which the
BRAF mutation occurred with high frequency (in all TCV
and 34 out of 36, 94.5% PTC), only 3 out of 13 patients with
FVPC had this mutation (23%). Furthermore a significant association between BRAF mutation and multifocality of cancer
was found (p = 0.0003, Odd Ratio, OR 0.071 CI from 0.014
to 0.353). The presence of BRAF mutation was significantly
associated with nodal involvement (p = 0.0001, OR 0.042, CI
from 0.005 to 0.346) but not with extra-capsular infiltration
even if its value tends to significance (p = 0.0653, OR 0.21 CI
from 0.044 to 1.070).
Conclusions. The BRAF gene mutation can be successfully
identified on LBC processed material as well as other cytological method with high reproducibility, feasibility and
100% informative results. It may predict preoperatively the
behaviour of micro PTC and may suggest a more aggressive
surgical approach involving the central neck dissection.
Role of lifestyles in breast cancer risk:
a retrospective analysis of Trieste’s female
population
F. Giudici1 2, S. De Martino1, C. Bottin1, A. Bianco3, L. Di
Bonito1 4, F. Martellani4, E. Ober4, A. Romano4, A. Zacchi4, F.
Zanconati1 4, M.A. Cova1 5, M. Tonutti5, C. Gasparini5, M. Assante5, F. Frezza5, M.P. Bortolotto5, C. Cressa5, R. Perrone5, E.
Makuc5, G. Petz6, P.L. de Morpurgo7, G. Pellis7, N. Lizza7, M.
Bortul1 8, S. Scommersi8, A. Dell’Antonio8, C. Convertino8, B.
Borea8, N. Renzi8, Z.M. Arnez1 8, C. Dellach9, G. Mustacchi1 9,
A. Franzo 10, L. Zanier10, L. Torelli2
Dep of Medical, Surgery and Health Sciences, University of Trieste,
2
Dep of Mathematics and Geoscience, University of Trieste, 3 Dep
of Life Sciences, University of Trieste, 4 Pathology Unit, University
Hospital of Trieste; 5 Radiology Unit, University Hospital of Trieste;
6
Radiology Unit of Salus Trieste, 7 Radiology and Surgery Units of
Sanatorio Triestino, 8 Dep of Surgery. University Hospital Trieste,
10
Oncologic Unit ASS1 Triestina, 11 Epidemiological Service; Direzione Centrale della Salute Friuli Venezia Giulia
1
Background. Friuli Venezia Giulia holds the highest raw
incidence rate for breast carcinoma in Italy (218%oo) and
Trieste, where live 125.000 women (105.000 women of age
below 75), has the highest incidence rate among the other
provinces of the region (220%oo). The risk of developing
breast carcinoma seems to be correlated with many different
factors. The goal of this study is to focus on some specific
breast carcinoma risk factors in the Trieste population.
Methods and materials. This is a case-control study in
which groups are made up as follows: 1113 cases of breast
carcinoma (period 2006-2009) and 1821 controls (biennium
2009-2010). All women of both groups, contextually to the
exam, were asked to answer a questionnaire about anamnestic information regarding breast carcinoma risk factors. The
questionnaire includes information about the woman’s age,
Body Mass Index (BMI), smoking habits, breast carcinoma
familiarity, hormonal background. The analysis of risk factors was limited to women aged below 75 years for lack of
an adequate number of controls in women above 75. A first
univariate statistical analysis was focused on those risk factors
which information, obtained through the interview, proved to
be complete and reliable. Only factors with O.R. (Odds Ratio)
statistically significant to the univariate analysis were chosen
for a multivariate analysis. Smoking was analyzed on the two
aspects of duration in years and intensity. For the statistical
analysis we used R and STATA software.
Results. The univariate analysis of smoking factor shows that
women below 50 years who smoke for more than 15 years
have a strong risk (OR 1.95; C.I. 1.25-3.00; p-value = 0.0007)
that decreases for women between 50 and 75 years of age,
(OR 1.30; C.I. 0.99-1.71). As for smoking intensity (independently from years of smoking), in women below 50 the risk
increases among those smoking more than 10 cigarettes a day
(OR 1.85; C.I. 1.10-3.05; p-value = 0.002), versus non smokers. The same applies for women aged between 50 and 75
(OR 1.41; C.I. 1.03-1.93; p-value = 0.002). The multivariate
analysis involved the following risk factors: BMI (Body Mass
Index) higher or equal to 25; age of menarche before 12; nulliparity; first and second grade breast carcinoma familiarity.
From our study we confirm as risk factors: BMI in women
above 50 (OR 1.87; C.I. 1.52-2.29; p-value < 0.001), nulliparity (OR 1.20; C.I. 0.90-1.60; p-value < 0.001) and familiarity
(OR 1.34; C.I. 1.09-1.64; p-value < 0.001) while for the menarche age there are no statistically significant results.
Conclusions. From the analysis of risk factors relating to
women living in Trieste, we have observed that the modifiable
factors, like BMI and smoking, result to be more effective in
the onset of breast carcinoma versus genetic factors as familiarity. Our results support the important role of a healthy life
style in reducing the risk of breast cancer
Statistics and histopathology: a mixed-effects
model approach to digital image analysis
M. Borelli1, F. Zanconati2, L. Bortolussi1, F. Giudici2,
G.Barbati2, L.Torelli1
Department of Mathematics and Geoscience, University of Trieste,
Italy; 2 Universitary Clinical Department of Medical, Surgical and
Health Sciences, University of Trieste, Italy
1 Background/introduction. In quantitative histology several
image analysis software are available with good results in detecting objects both on morphometrical side (perimeter, diameter, area, shape factor) and densitometrical features (RGB or
gray densities). Classification usually is performed by extracting overall information by low-level features (i.e. pixel values),
mid-level features (relationships between pixels and objects)
and high-level features (histological textures / structures). In
this communication we focus on the possibility to exploit random effects approach in histopathological image analysis.
Materials and methods. By means of free softwares (ImageMagick, http://www.imagemagick.org) we developed a
semi-automated code to import a pnm-format histological
image into the open source statistical package R (http://
www.r-project.org). We exploited the capabilities of the pixmap CRAN (http://cran.r-project.org) library to manipulate
pixel information while the lme4 CRAN package was used
to provide mixed effect modelling. Bootstrapping techniques
were applied to perform Likelihood Ratio test between models
obtained by different images.
Results. Our procedure is able to extract morphometrical
features (e.g. objects area) and to evaluate their reference
values in terms of a centrality parameter (i.e. the mean), the
within-object variability, and the residual variability. Our estimates benefit of the statistical literature well-known feature
called shrinking phenomenon, allowing us to compare images
sourced from different histological regions.
162
Conclusions. Mixed-effects models statistical approach reveals
to be a reliable technique to assess variability in histopathological digital image analysis. Our result offers the possibility to
develope an automated classifier (or even a learning machine)
able to identify morphometrical and densitometrical features
evaluated in terms of sample/population estimated variability.
Diagnostic accuracy and malignancy risk for
thyroid fine-needle aspiration according to
the Bethesda System for Reporting Thyroid
Cytopathology
S. Rossi
Dipartimento Immagini e Medicina di Laboratorio., Anatomia, Istologia e Citologia patologica, Azienda Ospedaliero-Universitaria di
Ferrara, Ferrara
Background. The Bethesda System for Reporting Thyroid
Cytopathology (BSRTC) is the offspring of the 2007 National
Cancer Institute State of Science Conference on thyroid fineneedle aspiration (FNA) and provides a new standardized
6-tiered diagnostic terminology for pathologist to communicate with clinicians. Each category has an implied risk of malignancy which is linked to the management. We applied the
BSRTC in the routine diagnostic since the 1th January 2010.
Aims. To test the BSRTC categories in the routine diagnostic after one year experience (between 01.01.2010 and
31.01.2011) and to define the risk of malignancy for each
diagnostic category.
Materials and methods. The casuistic included 3710 ultrasound-guided FNA performed by endocrinologists or radiologists. Each case consisted of MGG-stained conventional
smears with or without a Papanicolau-stained thin-layer slide
(Thin Prep®, Cytyc Inc.). The diagnostic categories included
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
nondiagnostic (ND), benign (BFN), atypia of undetermined
significance (AUS), follicular lesion of undetermined significance (FLUS), suspicious for follicular neoplasm (SFN), suspicious for malignancy (SFM), malignant (M). A check-list
with the diagnostic categories and subcategories was adopted
to facilitate reporting.
Results. Of a total of 3710 thyroid FNA performed, there were
507 ND (13,67%), 2835 BFN (76,41%), 113 FLUS (3,04%),
67 AUS (1,81%), 62 SFN (1,67%), 64 SFM (1,73%) and 62
M (1,67%). In 146 (28,7%) ND cases, reaspiration within 3
Months resulted in adequate samples. Follow-up cytology and
histology were pursued. The statistic parameters, expressed
in percentages, were: complete sensitivity 90,78, complete
specificity 83,07, positive predictive value (PPV) of M 100,
PPV SFM 81,25, PPV FLUS 9,73, PPV AUS 26,86, PPV SFN
12,90. The negative predictive value was 99,54%. The false
negative rate was 8,55%. There were no false positive. Correlation between cytology and histology: 8,8% (10 cases) of
FLUS were confirmed as papillary carcinoma (PCT) on histology, half of these represented by the follicular variant. 27%
(18 cases) of AUS, 72% of which subclassified as “atypia with
focal features suggestive of PCT”, proved to be PCT. The risk
of malignancy was: ND 0%, BFN 0,45%, FLUS 22%, AUS
48,64%, SFN 18,18%, SFM 81,25% and M 100%.
Conclusions. The incidence of each categories in our casuistic
fell within the ranges expressed by BSRTC and the literature.
Sensitivity, specificity and predictive values were good. AUS
proved to be a PCT in a significant proportion of cases and
carried out in our casuistic a higher risk of malignancy than
in BSRTC. Based on the risk of malignancy, we confirmed
in most categories the recommendations for management
suggested by BSRTC and proposed a triage with molecular
testing (BRAF) in AUS: if negative reaspiration, if positive
intraoperative frozen section and subsequent thyroidectomy
after confirmation of carcinoma.
163
Poster
Sabato, 30 giugno 2012
Discussione Poster
Chairmen: Attilio Leotta (Catanzaro), Ferdinando Quarto (Castellamare di Stabia)
Report of ASC-US and ASC-H in cervical cancer
screening: what role in detecting high-grade
lesions?
G. Accinelli, F. Maletta, P. Luparia, G. Alfonso, M.T. Benenti,
V.Buratti, D. Maso, M. Verga, L. De Marco, A. Gillio Tos, E.
Allia, A. Sapino, B. Ghiringhello
Centro Unificato di Screening, San Giovanni Battista Hospital, Turin, Department of Biomedical Sciences and Human Oncology, University of Turin, Italy
Background. The terms ASC-US and ASC-H were introduced
to designate equivocal changes that may reflect a squamous
intraepithelial lesion (ASC-US) or changes suggestive, but not diagnostic, of HSIL (ASC-H). Early detection of high-grade lesions
is critical to cancer prevention and our study aims at describing
the detection rate of high-grade lesions in ASC-US and ASC-H.
Materials and methods. At the “Centro Unificato di Screening” (Turin), 87264 1° level PAP-smears were examined from
6/6/2009 to 31/12/2011; of these 1104 were derived from previous positive high risk HR-HPV DNA test. The following diagnoses were made: inadequate (2,1%), negative (95,6%), ASCUS (0,6%), LSIL (1,3%), ASC-H (0,1%), HSIL (0,3%), others
(AGC, carcinoma) (< 1%).
Results. The 497 cases diagnosed as ASC-US represented the
0,5% (448/86160) of all cases of the conventional group, and the
4,4% (49/1104) of cases positive for HR-HPV testing.
Within the conventional group, 96 cases were lost at follow-up (FU);
179 had a cytological FU (negative in 113 cases, ASC-US in 4, LSIL
in 58 and HSIL in 4) and 173 had a histological FU (negative in 94
cases, CIN1 in 51 and CIN2 or higher in 28). In the subgroup of
HR-HPV positive cases, 11 patients had a cytological FU (negative
in 6 cases, LSIL in 5) and 38 had a histological FU (negative in 17
cases, CIN1 in 17 and CIN2 or higher in 4). By combining cytology
and histology ASC-US diagnoses from the conventional group had a
negative FU in 207/352 cases (59%), low-grade lesions in 113 cases
(32%) and high-grade lesions in 32 (9%); ASC-US derived from the
HR-HPV positive group had a negative FU in 23/49 (47%), lowgrade lesions in 22 (45%) and high-grade lesions in 4 (8%).
The 115 ASC-H represented the 0,1% (99/86160) of all cases of
the conventional group, and the 1,4% (16/1104) of cases positive
for HR-HPV testing.
Within the conventional group, 13 cases were lost at FU, while in
the remaining 86, histological FU was negative in 5 cases (6%),
CIN1 in 13 (15%) and CIN2 or more in 68 (79%). Within the
HR-HPV positive group, histological FU was negative in 2 cases
(12,5%), CIN1 in 2 (12,5%) and CIN2 or higher in 12 (75%).
No statistical differences were found in the detection of highgrade lesions between conventional and HR-HPV positive groups
both in ASC-US and ASC-H.
The positive predictive value (PPV) in detecting low and highgrade lesions during FU was 43% (CI 95% 38-48) for ASC-US
and 93% (CI 95% 86-97) for ASC-H, while it decreased to 9%
(CI 95% 6-12) for ASC-US and 78% (CI 95% 69-86) for ASC-H
in detecting high-grade lesions only.
Conclusions. Our results showed that ASC-H had a high PPV
and was more strongly associated with underlying high-grade
lesions than ASC-US. Anyway, in spite of the low percentage
of ASC-US cases (0,5%), obtained thanks to constant quality
controls, ASC-US was associated to a relevant amount of highgrade lesions (9%), thus strengthening the importance of efforts
to standardize the current diagnostic criteria.
Diffuse large B-cell extranodal lymphoma of the
uterine cervix: an incidental pap smear finding
with histological and immunohistochemical
correlates
A. Zabatta1, R. Boschi1, G. Marino1, C. Bellevicine1, U. Malapelle1, P. Zeppa2, G. Troncone1, A. Vetrani1
Department of Biomorfologic and Functional Sciences, University of
Naples Federico II, Naples, Italy; 2 San Giovanni di Dio e Ruggi d’Aragona, University of Salerno, Salerno
1
Background. Hematologic malignancies rarely affect the female genital tract, representing an uncommon finding on a
Papanicolaou (Pap) smear. In fact, less than 1% of extranodal
lymphomas arise from the gynecological tract with the uterine
cervix most commonly being affected. The majority of primary
cervical lymphomas are classified as non-Hodgkin lymphomas
(NHL) of B-cell origin. Here we present a case of diffuse Large
B-cell extranodal lymphoma of the uterine cervix diagnosed on
Pap-smear and subsequently confirmed by histological examination.
Case. A 79 years-old woman with no significant past medical
history, presented with abnormal vaginal bleeding after a perineal trauma. On colposcopy, cervical carcinoma was suspected
and therefore a Pap smear was taken. Microscopically, scattered
among benign atrophic epithelial cervical cells, a highly atypical cell population was evident. The atypical cells were mostly
dispersed as single isolated elements, three to four time larger
than a mature lymphocyte, with scant eosinophilic cytoplasm,
round to oval nuclei and a central evident eosinophilic nucleolus. Several multinucleated cells with striking Reed-Sternberglike feature and occasional mitotic figures were also evident.
These cytological features suggested the occurrence of a high
grade lymphoma. Since, other malignancies (e.g. metastatic
melanoma) could not be ruled out, the patient underwent histological biopsy.
Results. The tissue fragment from bioptic specimen showed a
diffuse infiltration of medium and large cells with morphological features that perfectly recapitulated the Pap smear features.
In fact, the nuclei were irregularly rounded with a characteristic
central nucleolus; several multinucleated Reed-Sternberg-like
cells were also present. An high mitotic index and a focal
“starry sky” appearance were readily evident. The immunohistochemistry showed a B cell phenotype (CD20+) and a positive
signal for bcl2 and CD5. Converesely, CD10, bcl6 and CD30
were negative. As suggested by mitotic index, a nearly all neoplastic cells displayed Ki67 labelling. Thus, a final diagnosis of
diffuse Large B-cell extranodal lymphoma, immunoblastic type
was rendered.
Conclusions. The diagnosis of hematologic malignancies on a
Pap smear is challenging because its rarity and the scarcity of
neoplastic cells, usually scattered among non neoplastic cervical
epithelial cells. Correlation with clinical history and imaging features are essential to correctly diagnose these rare malignancies
of the gynecologic tract.
164
Correlating HPV DNA test to pap-test for the
detection of cervical cancer and its precursors
M. Onorati, C. L. Bianchi, L. Ruggero1, G. Petracco, P. Uboldi,
F. Di Nuovo
Pathology Unit, Garbagnate Milanese, AO “G. Salvini” Garbagnate Milanese, Italy; 1 Pathology Unit, Rho, AO “G. Salvini” Garbagnate Milanese, Italy
Background. Human Papillomavirus infection is the most
common sexually transmitted disease and the most important
pathogenetic factor of cervical cancer. The question is how to
distinguish lesions which progress from lesions which regress
spontaneously. On the basis of Pap-test, cytological abnormalities
ranging from low-grade (L-SIL) to high-grade (H-SIL) squamous
intraepithelial lesions can be diagnosed. HPV-DNA test has been
introduced in cervical screening programs to increase the sensitivity of detection of lesions related to HPV and to select the
category of progressive lesions. The aims of the present study is
to evaluate the sensitivity and specificity of a combined use of
Pap-test and HPV-DNA test on a series of cases selected from the
data of a screening program.
Methods. A total of 380 women were retrieved from the archive
files of the Anatomic Pathology of “G. Salvini” Hospital in Garbagnate from 2007 to 2011. The mean age of the patients was
45,5 years (range 15-76 years). Among these patients only 142
were chosen on the basis of the use of both HPV-DNA test and
Pap-test for the first diagnosis and the follow-up. Cytological
diagnoses were confirmed by reviewing the original smears. A
comparison has been made between the results of Pap-test and
those of HPV-DNA test.
Results. The results of Pap-test were the following: 63 positive
(L-SIL or H-SIL), 26 negative, 32 ASC-US. Among the positive
cases, 52 (82%) were positive at HPV DNA test, while, among
the negative cases, 21 (81%) were HPV DNA negative and 5
(19%) HPV DNA positive. Among the ASC-US cases, 22 (69%)
were HPV DNA negative and 10 (31%) positive. In the concordant cases between Pap-test and HPV-DNA test, the biopsy
confirmed the presence of H-SIL and/or carcinomatous lesions,
of which the 69% were treated (negative at follow-up). In the
ASC-US cases, at biopsy the lesions were L-SIL or H-SIL.
Discussion. Several studies have demonstrated that the combined
use of HPV DNA test and Pap-test consents to select the women
to be submitted to colposcopy among the ASC-US cases, while
the only use of HPV-DNA test allows to increase the screening
intervals. Our data confirm that the screening of cervical lesions
is firstly committed to the Pap-test, while the association with
HPV-DNA test represents a good tool in the triage of ASC-US.
The introduction of polyvalent HPV vaccination can induce a
reduction of abnormal smears in the target population of approximately 40% and a potential reduction of cancer risk of 70%. The
reduction of HPV infection due to vaccine use in young females
may consent re-evaluate the Pap-test as the most important diagnostic tool in screening programs.
Detection of high-risk HPV genotypes in cervical
samples: a comparison study of a novel real time
pcr/reverse line blot-based technique and the
digene HC2 assay
S. Mason1, A. Gani1, M. Vettorato1, G. Negri2, C. Mian2, F. Brusauro1, K. Bortolozzo1
1 AB ANALITICA srl, Padova, Italy; 2 Department of Pathology, Central
Hospital, Bolzano, Italy
Background. High-risk genotypes of Human Papillomavirus
(hrHPV) are etiologically linked to cervical carcinomas and their
precursors. HPV DNA testing has therefore become an important part of cervical carcinoma screening and management in
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
several countries. Many different techniques have been proposed
to detect the presence of hrHPV in cervical samples. Digene
HC2 High-Risk HPV DNA Test (HC2; QIAGEN, Germany) is
one of the most widely used. Recently a new Real Time PCR/
Reverse Line Blot-based commercial assay has been introduced:
it allows for the detection of all genital HPV and the typing of
29 genotypes.
Aims. To compare the performance of two commercially available assays, the HC2 and the REALQUALITY RI-HPV STAR/
AMPLIQUALITY HPV-TYPE (AB ANALITICA, Italy), to
detect the presence of hrHPV in cervical samples.
Materials and methods. In 2010, 224 PreservCyt samples were
collected from female patients aged 16-74 years and analysed using both HPV detection techniques. Using the AB ANALITICA
device, DNA was extracted with EZ1 Advanced XL DNA extraction system (QIAGEN) and evaluated for HPV by Real Time
PCR with REALQUALITY RI-HPV STAR kit. Positive samples
were selected and their amplicons were directly genotyped by
Reverse Line Blot, using AMPLIQUALITY HPV-TYPE kit. The
HC2 test was performed according to the manufacturer’s instructions. Non concordant samples were verified using MY09/MY11
primer PCR and direct sequencing.
Results. Comparison of the two methods showed an overall concordance of 91.4%. The HC2 assay gave positive results for nontargeted HPV types in 10 samples in which HPV 81 (3 samples),
73, 70, 61, 54, 43, 42 or 11 were detected by Real Time PCR/
Reverse Line Blot and confirmed by direct sequencing. Diagnostic sensitivity and specificity of the Real Time PCR/Reverse Line
Blot test were 95.8% and 98.4% respectively.
Conclusions. This study shows that HC2 and the novel Real
Time PCR/Reverse Line Blot-based commercial assay give
comparable results, both being suitable for routine use. Despite a
similar overall and manual working time, REALQUALITY RIHPV STAR/AMPLIQUALITY HPV-TYPE provides complete
information about the specific genotype of all carcinogenic and
probably/possibly carcinogenic HPV types.
Cervical vaginal screening. PCR multiplex-nested
and western-blot: interaction between E6-p53 and
E7-pRb
G. Di Benedetto, M. Gravina, P. Nuzzo, A. Di Benedetto,
M. Gentile, C. Rinaldi
Cytopathology Service, Asl (Caserta), Department of Molecular Biology,
Clinical Hospital, Marcianise Ce
Background/introdution (aims). Patients with Low and High
Squamous Intraepithelial Lesions (LSIL-HSIL), Hp-Virus positive, demonstrate, in most cases, the integration of the viral genome into the chromosomes of the infected cells. This integration
is relative only to the genes E6 and E7 which are stably expressed
in the infected cell, suggesting a role for the E6 and E7 proteins
in the induction of malignant transformation.
These oncoproteins are known to play several roles critical to
the infected epithelial cells, primarily concerning the balance between cell proliferation and differentiation with the inactivation
of onco-suppressors p53 and pRb. The expression of two viral
oncoproteins is the event necessary for the initiation of a wide
range of complex events that develop cancer.
The aim of our work was to establish a biomolecular protocol to
identify the m-RNA of E6 and E7 genes and their protein products with PCR technique. We demonstrated successful activation
between proteins E6 and E7 respectively with the proteins p53
and retinoblastoma.
Material and methods. The study was conducted on 1085
patients of Asl Ce. The search for virus DNA was carried out
on cell samples obtained by sampling for cytobrush, using the
Polymerase Chain Reaction (PCR) and the PCR multiplex nested.
The analysis of messenger RNA was performed using RT-PCR
165
Poster
and PCR multiplex nested. The study of viral proteins used the
Western blot method.The colposcopy, histology and cytology
(Pap test) was performed on every patients.
Results. Of the 1085 samples examined 240 (22,1%) cases tested
positive for HPV infection.
The results showed the absence of mRNA in 24 (10%) of 240
samples examined. This means no integration into the cellular
genome and the virus is only in the episomic form.
In 211 (87.9%) of the remaining 216 samples a perfect correlation was observed between the results of the analysis of viral
DNA and those of the analysis of the cDNA was found.
Analysis by Western blot allowed us to detect the protein products of the 2 genes and especially the interaction occurred E6-p53
and E7-pRb.
Colposcopic, hystologic and cytologic examination of 130/211
(61.6%) cases, including episomic form, were diagnosed, in
histology as normal, in cytology as negative for intraepithelial
lesions.
52/211 (24.6%) cases were diagnosed, in histology as reactive
inflammatory, incytology as 32 Abnormal Squamous Cell Unsignificance (ASCUS) and 20 negative for intraepitelial lesions.
On 29/211 (13.8%) cases the Cyto-Histologic diagnosis was 20
LSIL, 8 HSIL and 1 Cancer.
Conclusion. Our protocol allows a selection of some patients at
risk of carcinogenesis by addressing an appropriate follow-up.
We are suggesting a new screening protocol that will enable the
selection of at risk
carcinogenic patients. The procedure screening + follow-up
should then help avoid lesions and improve patient cure rate.
Can the asbestos occupational exposure
be revealed by induced sputum (IS) and
bronchoalveolar lavage fluid (BALF) investigation?
D. Bellis1 3, S. Capella2 3, E. Belluso2 3, D. Antonini1, L. Viberti1
ASLTO1, Ospedale Martini, Dipartimento dei Servizi, Servizio di Anatomia Patologica; 2 Dipartimento di Scienze della Terra, Università di
Torino; 3 Centro Interdipartimentale per lo Studio degli Amianti e di altri
Particolati Nocivi ‘‘Giovanni Scansetti”; 4 Istituto di Geoscienze e Georisorse, CNR, Unità di Torino
1
Diagnosis of asbestos-related diseases requires information on
past asbestos exposure, which can be often obtained through the
occupational history that in some cases is limited or unknown.
Asbestos bodies (ABs) are a hallmark of asbestos exposure in
human lung. When the precise nature of the fibrous core is not
known it has been suggested to use the noncommittal term of ferruginous bodies (FBs). They are present in few amount in lungs
of the great majority of general population, but higher quantities
are found in lungs of occupationally asbestos-exposed subjects.
Detection of their presence in living people required surgical lung
biopsy. In order to avoid this invasive procedure attention has
turned to induced sputum (IS) and bronchoalveolar lavage fluid
(BALF) analysis.
Cytological samples from 62 subjects were investigated. They
were divided into three groups:16 BALF samples from general
population with radiological pulmonary fibrosis with clinical possible pneumoconiosis (group A), 16 IS samples from healthy
general population (group B) and 40 IS samples from healthy
people with indirect asbestos exposure (electricians, hydraulics
and maintenance workers which have worked in presence of old
asbestos containing pipes in the work place: group C).
Cytological samples, prepared using two different techniques,
were investigated to detect the FBs presence. A portion of each
sample was fixed, embedded, sectioned and treated with H-H
routine stain for cytopathologic screening. The other portion was
digested and collected on a mixed cellulose ester membrane for
optical microscope (OM) examination. One BALF sample was
investigated also by scanning electron microscope (SEM).
FBs were found in 11 out of 16 (69 %) BALF samples (group A).
The only sample investigated by SEM was positive too.
In IS samples FBs were found only in group C and in 4 out of 40
(10 %), while no FBs were found in samples of group B.
Previous studies of IS analysis for FBs in heavy occupationally
exposed subjects report low percentages of positive cases. Therefore when IS sample is positive for FB this is suggestive of a high
lung asbestos burden.
Our results confirm that IS investigation is less sensitive than
BALF to reveal hidden or doubtful asbestos exposure although the
collection of IS is more simple, less invasive, and less expensive
than BALF. In fact IS sample were negative for FBs in the majority of subjects with occupational asbestos exposure (group C).
The absence of FBs in IS samples from subjects of healthy general population (group B) confirms that they didn’t undergo to
heavy occupational asbestos exposure in the past.
The high percentage (69 %) of BALF samples from subjects with
diagnosis of lung diseases (group A) in which FBs were detected
indicates a possible past asbestos exposure.
Lymphomatoid granulomatosis of the lung: fine
needle aspiration cytology diagnosis and cytohistological correlations
M. Onorati, G. Petracco, P. Uboldi, F. Di Nuovo
Pathology Unit, Garbagnate Milanese, AO “G. Salvini” Garbagnate Milanese, Italy
Background. Lymphomatoid granulomatosis (LG) is a rare
extranodal B-cell lymphoproliferative disorder on a background
of reactive T lymphocytes. It is characterized by the association
with Epstein-Barr virus (EBV). It has an angiocentric and angiodestructive behaviour, and grade 3 LG is considered a diffuse
large B cell lymphoma (DLBCL). It can originate at any age, but
80% of the cases occur between the 4th and 6th decade, with a
male prevalence. As other EBV-associated lymphoproliferative
disorders, lymphomatoid granulomatosis occurs with increased
frequency in immunosuppressed patients and the lung is the most
common involved organ.
Methods and results. A 77 year-old male affected by MGUS
(monoclonal gammopathy of undetermined significance) presented with severe dyspnea. A thoracic computed tomography
was made and it showed a diffuse interstitial lung disease. A
fine-needle aspiration cytology (FNAC) of selected TC-guided
suspicious areas, was performed by means of a 22G needle.
Smears were air-dried and stained with Papanicolaou and MayGrunwald Giemsa. The cell population was constituted by small
lymphocytes admixed with scattered cells with blastic appearance
and with rare, large, pleomorphic. Multinucleated “Hodgkin-like”
cells were also observed. On the basis of the smear a pulmonary
lymphomatoid granulomatosis was hypothesized and a pulmonary biopsy was performed. Pulmonary parenchyma showed
nodular infiltrates of T cell lymphocytes (CD4+, CD3+, CD8+),
intermingled with rare, large, pleomorphic, multinucleated B
cells, actively proliferating (CD20+, LMP +, EBER+/-). Blood
vessels showed obliteration of the lumen and transmural infiltration of tumor cells. Necrotic foci were absent. The diagnosis of
pulmonary lymphomatoid granulomatosis grade 1 was made.
Conclusions. Although the evolution of LG from grade 1 to grade
3 has not been unequivocally demonstrated, several studies showed
that an early diagnosis followed by a specific treatment with
corticosteroid, interferon, monoclonal antibodies and sometimes
chemotherapy, may avoid the development of a DLBCL. Fineneedle aspiration cytology is an important tool for an early correct
diagnosis and the correlation between cytopathological and histopathological findings favour a more accurate and rapid diagnosis.
166
Papillary serous carcinoma of peritoneum in pleural
effusions: a case report
A. Di Lorito1, R. Streppa2, M. De Laurentiis2, S. Capanna1,
S. Rosini1, D. Caraceni2
Unità Operativa di Citodiagnostica, Dipartimento di Scienze Biomediche, Università G. d’Annunzio di Chieti-Pescara; 2 Unità Operativa Complessa di Citodiagnostica, Ospedale “F. Renzetti”, Lanciano
1
Introduction. Primary peritoneal serous papillary carcinoma
(PSPC) is a rare primary peritoneal tumor but it has to be distinguished from mesothelioma and from papillary serous carcinoma
of the ovary. In the first case, differential diagnosis can be made
with a panel of immunocytochemistry (ICC). The extraovarian
PSPC is morphologically identical to ovarian serous carcinoma,
however, it can spare or minimally involve the ovaries. In that
condition, clinical and pathological examination of the ovaries
is needed.
Materials and methods. We presented a case of a women of
76 years old with initial manifestation of abdominal swelling,
dyspnea and ascites, with elevated serum CA125. She presented
also left pleural effusions. Abdominopelvic chest computed tomography revealed nodular thickening of the parietal peritoneum,
mesenteric and omental nodules. Exploratory laparotomy findings of carcinomatosis were not able to reveale the primary site
and PSPC was strongly considered. Cytological examination of
pleural fluids was carried out.
Results. Cytology pleural effusions revealed malignant cells in
papillary structure, psammoma bodies and high grade nuclear
atypia.
The tumor’s differential diagnosis from malignant mesothelioma
was established with PAS and DPAS positivity and with a panel
of ICC markers.
Tumor cells were positive for CKAE1-AE3, Ca125, PLAP, Ca
15.3, BerEp4, B72.3, and EMA while they were negative for
CEA, ER, PR, Ca19.9 and Calretinin.
The last diagnosis was PSPC, confirmed also by surgical biopsies
of the omental nodule. Patient was treated with platin- based
chemotherapy.
Conclusion. The use of a panel of markers, in our patient, was
useful to distinguish in cytology effusions PSPC from malignant
mesothelioma, to establish the best type of treatment.
Intraoperative cytological evaluation for guiding
surgical treatment of Tir3/Tir 4 thyroid fine needle
aspiration cases
S. Crippa, J. Barizzi, C. Cannizzaro, E. Merlo, B. Flores,
M. Bongiovanni
Institute of Pathology, Locarno, Switzerland
Background/introduction. Thyroid Fine Needle Aspiration is
the most effective tool for guiding the initial management of
patients with thyroid nodules. Thyroid Fine Needle Aspiration is
accurate, safe, efficient, and cost-effective. Unfortunately, a few
cases are suspicious for, but non-diagnostic of malignancy (Tir 4
in the 5-tiered SIAPEC-IAP reporting system), most of these are
papillary carcinomas. Confirmation of malignancy is desirable
for guiding the best surgical treatment is desirable.
Aims. To verify the impact of intraoperative cytological evaluation, with or without histological frozen section, on diagnosis of
malignancy in selected cases.
Materials and methods. We reviewed our intraoperative diagnosis of thyroid nodules in the last year. All cases were papillary
carcinoma at definitive histological diagnosis. We performed the
intraoperative cytological evaluation of 1 “indeterminate” case
(Tir 3) and 5 “suspicious for papillary carcinoma” cases (Tir 4).
Touch or scrape preps were made of the cut surface of the nodule.
In 5 out of 6 cases we also performed intraoperative histological
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frozen section also. Fast Hematoxilin-Eosin stain was used for all
of the specimens.
Results. At intraoperative citological evaluation, classic nuclei
of papillary thyroid carcinoma were seen in 5 of 6 cases; nuclear
atypia was not enough to suggest papillary carcinoma in 1 case,
previously classified as Tir 3. At intraoperative histological evaluation, features of papillary carcinoma was seen in 5 of 6 cases;
nuclear atypia was not enough to suggest papillary carcinoma in
1 case, previously classified as Tir 4.
Conclusions. Intraoperative citological evaluation of selected
cases is a useful, simple and very cost-effective method for directing surgical treatment.
TROP-2 expression in undetermined thyroid
lesions a preliminary cyto-histological study
G. Simone, T. Addati, G. Achille, S. Petroni, M. Centrone,
G. Giannone, F. Palma, S. Russo, L. Grammatica
Istituto Tumori “Giovanni Paolo II” IRCSS Bari
Background. The ThyFNCs has reduced the incidence of unnecessary thyroid surgery. However its usefulness is still limited
by the variable number of undetermined diagnoses. Immunohistochemical (IHC) markers have been considered an usefull tool to
reduce the incidence of undetermined FNCs but, the NCI Thyroid
FNA Conference still stated that there is not sufficient evidence
at this time to use IHC or molecular techniques in Undetermined/
souspicious FNAs.
Aims. To study expression of glycoprotein TROP2 in thin layer
cytological smears and in corresponding histological specimens
and compare the results with cytological and histological results
and with the HBME1, considered as referral marker.
TROP2 is a cell surface glicoprotein, codified by a gene located
on p32 of Chr1, highly expressed in trophoblasts cells, forming
the outer layer of the blastocyst, an ‘invasive’ normal tissue.
TROP2 is present also in choriocarcinomas and in a wide variety
of epithelial cancers but there is little or no expression in adult
normal tissue.
Materials and methods. Entered the study 121 US guided
ThyFNCs occurred in 120 patients (male 26 and 94 female,
from 18 to 73 years old, mean age 53 years). The nodule size
ranged between 6 to 44 mm (mean 21 mm). After reclassification according to Bethesda Conference- 2007- most of them (69,
58.2%) received a cytological diagnosis of Atypia/Follicular
lesion of Undetermined Significance. Other 5 cases (4.3%) as
Follicular/Oncocytic Neoplasm, 6 (5.1%) as souspicious-PTC,
10 as PTC (8.5%) and 25 (21.4%) as Benign nodules diagnosed,
were included.
In all cases HBME1 was performed on cytology and 82 of them
underwent to lobectomy/ thyroidectomy.
Results. Related to HBME1 expression in ThyFNCs and in corresponding histological samples we found, at histology, 3 HBME1
negative resulted PTC and 10 HBME1+/Benign. Moreover, 3
Oncocytic Tumor, 2 UPM-Follicular neoplasm and 1 Follicular
carcinoma were HBME1, whereas 10 cases (2 Souspicious and 8
malignant in cytology) HBME1+ were confirmed as PTC.
Agreement, in terms of absence of HBME1-IR in Benign nodules
or its presence in PTC, was reached in 66/79 evaluable cases
(82%). Sensitivity of HBME1 was 86% and specificity 81%.
Incidence of TROP2 in ThyFNCs evidenced 32 negative and 7
Positive in benign nodules; 3 Oncocytic Tumor, 1 UPM/Follicular neoplasm and 1 Follicular carcinoma were TROP2 negative,
whereas 16 cases (6 Souspicious and 10 malignant in cytology)
TROP2 were confirmed as PTC. Agreement with HBME1 determination was detected in 55/60 FNCs. We evaluated also 75 surgical samples in which both the two markers were tested. Twelve
cases were positive and 50 negative for both the markers whereas
10 showed positivity only for HBME1 (4 malignant) and 3 only
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Poster
for TROP2 in 3 cases (1 malignant).Agreement was reached in
62/75 (82.7%).
Conclusion. In our experience, TROP2 shwed to be un usefull
markers, such us HBME1 but with an highe specificity in predicting malignancy in TIR 3 FNCs.
Diagnostic dilemma in fna of salivary gland:
warthin tumor with signet-ring cell features
C. Bellevicine, A. Iaccarino, G. Troncone
Biomorphological and Functional Science, University Federico II, University Federico II, Naples, Italy
Background. Warthin tumor (WT) is a common parotid lesion
frequently encountered in FNA practice. Several reports described either metaplastic or rare malignant trasformation in WT.
Here we want to underscore the possibility of unusual signet- ring
metaplasia in a case of an otherwise classic WT.
Case. A 61 years-old man presented with a multinodular 3.6 cm
mass of right parotid gland, with US features suggestive of WT.
A FNA was performed and one smear was air-dried, DiffQuik
stained and on-site evaluated: the cytomorphology was readily
compatible with the clinical suspect of WT. Other passes were
performed and the smears were alcohol-fixed for Papanicolaou
staining. This latter smears revealed, scattered among the WT
classical cytological features consisting of lymphocytes, oncocytes sheets and granular debris, small groups and single cells
with evident signet-ring features. Interestingly, some groups featured oncocytes with secretory aspects similar to those observed
in the signet-ring cells. Considering the clinical context together
with the predominant cytological aspects, a diagnosis of Warthin
tumor with signet-ring cell features was rendered. However, in
the final report, special care was taken to note
that, rarely, mucoepidermoid carcinoma can be associated to WT
and that the surgical specimen should be thoroughly sampled.
Conclusion. Warthin tumor with signet-ring cell features can
cause diagnostic dilemma in FNA from parotid gland. However,
clinical and classical cytological features of WT can be useful to
assess this additional features as metaplastic.
Salivary gland fine-needle aspiration cytology:
a case of oncocytic carcinoma with mucinous cells
of the parotid gland
S. Fisogni, M. Chiudinelli, L. Lorenzi, M. Ungari.
Department of Pathology 1, Spedali Civili-University of Brescia, Italy
Background. Fine needle aspiration (FNA) is a safe diagnostic
technique that is widely employed for lesions of the head and
neck. Among head and neck sites, the parotid gland is unique in
number, diversity and peculiarity of its pathological processes.
FNA cytology is useful in avoiding surgery (inflammatory lesions), limiting surgical procedures (benign tumors) and for
planning the extent of surgery of malignant tumors. The presence
of oncocytes is a common finding in both normal and neoplastic
salivary gland (SG). Many frequent benign lesions can show
oncocytic features such as oncocytosis, oncocytoma, Warthin’s
tumor, pleomorphic adenoma and myoepithelioma. Malignant
SG neoplasms may have more or less focal oncocytic features
and among these the most frequent are mucoepidermoid carcinoma and acinic cell carcinoma. However, malignant SG tumors
comprised entirely oncocytes (oncocytic carcinoma) are very
uncommon.
Materials and methods. A 59 years female with history of papillary carcinoma of the thyroid and colorectal adenocarcinoma,
presented with a 2 cm irregular mass of the right parotid gland.
Ultra-sound guided FNA was performed and, in addition to routine stains (Papanicolau), immunocytochemistry (ICC) for TTF1
was performed.
Results. FNA showed abundant epithelioid cells in cohesive
mono-multilayered aggregates. The cells demonstrated abundant
finely granular cytoplasm of oncocytic type. The nuclei were
round to oval and centrally located with fine granular chromatin
and single, often prominent nucleoli. No nuclear grooves or
pseudo-inclusions were identified. Most cells appeared regular
with little or no pleomorphism. Mitosis were not seen. No other
cells type were identified. On ICC, neoplastic cells resulted negative for TTF1. A diagnosis of oncocytic neoplasm not otherwise
specified, more probably benign, was provided and the parotid
gland was excised. On histology, the lesion was larger (about 5
cm) and resulted completely constituted by oncocytic cells. The
lesion was partially pseudo-capsulated with a solid, lobular and
cordonal architecture, with evident infiltrative pattern of growth
into glandular parenchyma and vascular neoplastic embolization.
Rare mitosis and glandular neoplastic structures outnumbered by
mucous cells were focally recognized.
Conclusions. Cytologically, the recognition of the oncocytic
nature of the lesion is usually straightforward, but in SG FNA
the differential diagnosis should contemplate different primary
benign or malignant neoplasms and metastatic disease. SG neoplasms composed entirely of oncocytic cells are very rare. The
lack of significant nuclear atypia did not necessarily indicate
benignancy.
FNA features of a case of pleomorphic lipoma
of the breast
P. Grassi, B. Flores Pereira, J. Barizzi, V. Martin, M. Bongiovanni
Institute of Pathology, Locarno, Switzerland
Background. Pleomorphic lipoma is a rare and benign lipocytic
neoplasm that most commonly occurs in the shoulder and in the
head and neck region. It has also been documented in the tongue,
orbit, bulbar conjunctiva, parotid gland, oral cavity, dermis, scalp
and breast. Few cases have been reported to be diagnosed by
cytology and only one was documented in the breast.
Materials and methods. A 72-year-old female presented with a
lump in the upper inner quadrant of the left breast. Fine-needle aspiration (FNA) was performed, and the smears were stained with
the Papanicolaou staining. Following the cytological diagnosis,
the nodule was excised.
Results. Microscopic examination of the smears revealed a fragment of mature adipose tissue with interspersed atypical cells presenting hyperchromatic and pleomorphic nuclei and vacuolated
cytoplasm. Intranuclear inclusions and mitotic figures were also
observed. Multinucleated giant cells with nuclei arranged in a
crown configuration (so called “floret cells”) were found occasionally in the smear. Due to the presence of highly atypical
cells, a cytological diagnosis of suspicious for malignancy was
done and surgical excision followed. On histology, besides “floret
cells”, the presence of important atypical cells suggested the diagnosis of liposarcoma. The negativity for the immuonohistochemical markers MDM2 and CDK4 and absence of amplification of
the gene MDM2 (12q14-15) by FISH were finally consisted with
the diagnosis of pleomorphic lipoma.
Conclusions. Subcutaneous lump in the breast and atypical cells
in lipocytic neoplasm may create apprehension in patients and
diagnostic confusion in cytopathologists. Knowledge of the existence of pleomorphic lipoma, the recognition of “floret cells”, the
possible presence of atypical features and adequate immunocito/
histochemical and FISH investigations can assist in the correct
diagnosis.
168
Fine-needle aspiration cytology of Kikuchi-Fujimoto
lymphadenitis: a report of six cases
M. Chiudinelli, S. Fisogni, L. Lorenzi, F. Facchetti, L. Lucini,
M. Ungari
Department of Pathology 1, Spedali Civili-University of Brescia, Italy
Background. Kikuchi-Fujimoto disease (KFD), also known as
histiocytic necrotizing lymphadenitis, is an uncommon, generally
self-limited disease of unknown etiology, usually occurring in
young females, presenting as an acute onset febrile illness, associated with firm, tender, unilateral cervical lymphadenopathy.
Despite the histological changes in KFD are rather characteristic
(patchy paracortical areas of necrosis with a variable mixture of
karyorrhectic nuclear debris, plasmacytoid dendritic cells, immunoblasts, signet ring and foamy histiocytes), the diagnosis might
be challenging and KFD is often confused with lymphomas. The
accuracy of fine-needle aspiration (FNA) in the diagnosis of KFD
has not been very satisfactory and cytological findings can be
indistinguishable from other nonspecific reactive lymphadenitis
or lymphomas. We report the FNA findings in six cases of KFD.
Materials and methods. Six patients (4 men and 2 women) aged
between 12 and 54 years (mean 29.6), presented with localized
lymphadenopathy (4 cases laterocervical, 1 case sovraclavear,
1 case axillary) ranging from 1.1 to 2.3 cm in size. Clinical
information was limited, with the exception of one patient, who
presented fever and suspected hemophagocytic syndrome in Still
disease or tuberculosis.
Results. In all cases FNA cytology showed similar features,
with highly cellularity, polymorphous lymphoid cell population, including immunoblasts, abundant karyorrhectic debris,
macrophages and histiocytes; the latter showed eccentrically
placed round to oval nucleus and abundant cytoplasm, with
ingested nuclear debris (“crescentic histiocytes”). Granulomas,
a significant number of epithelioid histiocytes, eosinophils, or
neutrophils were not observed. In three cases a diagnosis of
reactive lymphadenopathy with features suggestive of KFD was
provided, while in the other 3 cases KFD was diagnosed. In the
first 3 cases the lymph node was excised and KFD was proved
histologically.
Conclusions. The accurate diagnosis of KFD on fine-needle
aspiration is possible given correct clinical data, an adequately
sampled and well-prepared specimen in which the characteristic
intra- and extracellular apoptotic nuclear debris with admixed
crescentic macrophages are identified on a reactive lymphoid
background, devoid of epithelioid and multinucleated giant cells
and neutrophils. Some Authors retain that FNA alone will suffice
the diagnosis of KFD, with the approach of close clinical followup, but the diagnosis should probably be confirmed on a lymph
node biopsy in all circumstances.
HHV8-related and HHV8-unrelated primary
effusions lymphomas: similarities and differences
V. Ascoli1, G. Marangi1, I. Cozzi1, V. Giannelli2, M. Merli2, F.
Petrachi3, C. Lorusso3, G. Della Grotta3, I.C. Danese3, I. Della
Starza4, R. Guarini4
1 Dipartimento di Scienze Radiologiche, Oncologiche e Anatomo-Patologiche; 2 Dipartimento di Medicina Clinica, Divisione di Gastroenterologia; 3 Dipartimento di Medicina Interna e Specialità Mediche; 4 Dipartimento di Biotecnologie Cellulari ed Ematologia, Università Sapienza,
Roma
Background. Primary effusion lymphoma (PEL) is a rare liquidphase non-Hodgkin lymphoma localized in the body cavities. The
PEL tumor clone is infected by human herpesvirus-8 (HHV8),
the etiologic agent of Kaposi’s sarcoma. More rarely, primary
lymphomatous effusions unrelated to HHV8 may also occur.
We describe two paradigmatic examples of HHV8-related and
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HHV8-unrelated primary effusions lymphomas. The aims are: (1)
to discuss the main similarities and differences between these two
entities, and (2) to highlight the pivotal diagnostic role of cytology, with emphasis on the workup of these cases.
Materials and methods. Case 1. A 73-year-old man (HIV-neg,
HBV-neg, HCV-neg) with type II diabetes, recent herpes-zoster
and recurrent peritoneal effusions (Mar/Jun-2011).
Case 2. A 80-year-old man (HIV-neg, HBV-posHCV-neg) with
hypertension, atrial fibrillation, HBV-related cirrhosis and hepatocellular carcinoma, and recurrent right pleural effusions (Oct/
Dec-2011).
In both patients total body CT was negative for lymphoadenopaties. Fluid removal was followed by morphologic plus
immunophenotypic studies and molecular analysis of tumor
cells by PCR.
Results. Case 1. Fluid cytology revealed granulocytes, macrophages and medium-to-large-size lymphoid cells with abundant basophilic cytoplasm. Most cells had a single nucleus;
there were also multinucleated cells. Other features were high
mitotic activity and apoptotic bodies. Lymphoid cells were
CD3-neg, CD20-neg, CD45-pos, BCL2-pos, HHV8/LANApos, and Ki-67-pos (> 40%). Ascites LDH was 2500 U/ml;
EBV-DNA 1.4x107; HHV8-DNA 3.6x107. Clonal rearrangements of both heavy and light chains (kappa) of the immunoglobulin (Ig) gene were demonstrated by PCR (VH-DH-JH
and VK-K). Case 2. Fluid cytology revealed small lymphocytes and medium-size lymphoid cells with irregular, indented
nuclei and light-blue cytoplasm. Lymphoid cells were CD3neg, CD20-pos, EMA-neg, CD45-pos, HHV8/LANA-neg, and
Ki-67-pos (> 80%). Serum beta-2 microglobulin was high as
well as LDH (in serum and pleural fluid). Clonal rearrangement of the heavy chain of the Ig gene was demonstrated by
PCR (FR2/JH).
A diagnosis of HHV-8-related PEL was rendered in Case 1 and of
HHV8-unrelated B-cell effusion lymphoma in Case 2.
Conclusions. The first suspicion of PEL is by fluid cytology.
When a primary solid NHL is excluded, HHV-8 detection is first
required to distinguish between HHV8-related (PEL) and HHV8unrelated effusions lymphoma. The final diagnosis of PEL
requires the molecular proof of clonality. Also, on a cytologic
basis alone, it is impossible to differentiate primary lymphomatous effusions from the more frequent secondary lymphomatous
effusions.
Ufo in urinary cytology
M. Schiavo Lena, A. Cornacchiari, M. Gattamelata, M. Bonardi,
R. Tardanico
II Anatomia Patologica, Spedali Civili, Brescia
Background/introduction. We found in the urine of 4 patients
carriers of ileal neobladder with skin stoma and outer bag, oval
(pseudo?)-cellular formations difficult to interpret. We thought at
first they were eggs of Enterobius Vermicularis (comparing the
images of histological sections of Enterobius in appendices), however, the microbiological tests were repeatedly negative. A specific
antiparasitic therapy was performed but later cytologic samples remained unchanged even in a period of almost 4 years (2009-2012).
Materials/methods. We have studied the bag-kit setting up cytological preparations by washing the bag, scrape the gum adhering
to the bag and the modeling paste that is used as filler of skin
irregularities.
Results. From this sample we found the same structures that we
have seen in the urine. Feulgen stain was negative.
Conclusion. This is not a side effect of the paste. It may be a
mimic of parasite eggs and cause a “false positive” misdiagnosis. The patients did not require treatment. It should just be
aware that although rarely pasta in some patients may release
thismaterial.
169
Poster
Don’t forget the possible detection of HPV infection
in urinary cytology!
Immunoistochimica in citologia urinaria: P16 e Ck20,
due in uno
G. Petracco, C. L. Bianchi, P. Uboldi, M. Onorati, F. Di Nuovo
R. Rapezzi, B. Selmi, S. Negri, A. Bondi
Pathology Unit, Garbagnate Milanese, AO “G. Salvini” Garbagnate Milanese, Italy
U.O. Anatomia, Istologia Patologica e Citodiagnostica, Ospedale Maggiore, Azienda USL di Bologna
Background. Urinary cytology is widely used as a diagnostic
tool for the detection of urothelial lesions because of its simplicity, safety and the possibility of repetition. It is also a very costeffective, helpful, accurate diagnostic test to select patients with
urothelial carcinoma. We don’t forget that many lesions can be
detected by urinary cytology: lesions due to human papillomavirus (HPV) infection may be detected in squamous cervical cells
intermingled with urothelial cells in urinary cytology. Due to this
unexpected finding, women further undergo Pap-test to confirm
HPV infection and discover possible cervical lesions.
Case report. A 50- year- old woman was admitted to “G. Salvini” hospital of Garbagnate Milanese for recurrent haemorragic
cystitis. Three urine samples were collected and smears were
performed, routinely fixed and stained. The smears showed a
background of acute inflammation, red blood cells, reactive
urothelial cells. There were also epithelial squamous cells with
nuclear enlargement and perinuclear clear “koilocytosis-like”
halo that resulted in an increased nuclear to cytoplasmic ratio.
Rarely, binucleation and multinucleation were present. These
particular cellular alterations were suspicious for low-grade squamous intraepithelial lesion (LG-SIL) due to HPV-infection. For
this reason the patient was submitted to a gynecologic examination and a Pap-test was performed. The uterine cervical smear
confirmed HPV infection.
Discussion. The discovery of HPV related lesions in urinary
cytology is not a rare event. It is well known that HPV LG-SIL
may remain hidden, in particular in middle aged women, who are
likely to avoid screening programs for cervical cancer. Urinary
cytology, a cheap diagnostic tool, does not imply any contact
with physicians, and, mainly for this reason, may allow to recruit
a number of women who would otherwise escape clinical control.
The observation of this case allowed us to re-evaluate urinary
cytology not only as a tool for the detection of urothelial lesions
but also for HPV infection related lesions in women. We must
to bear in mind and not miss this feasible and easy-to perform
opportunity.
Ogni anno oltre 7000 pazienti si rivolgono al’Unità Operativa
(UO) di Anatomia Patologica dell’Ospedale Maggiore di Bologna per effettuare un esame citologico di urina.
Dal 2009 l’accesso all’UO per questa prestazione è stato progressivamente implementato grazie all’introduzione di una nuova
procedura di raccolta dei campioni che prevede l’utilizzo di un
fissativo alcoolico in grado di conservare in modo ottimale e stabile per almeno 7 giorni gli elementi cellulari presenti.
Nel laboratorio del’UO i campioni vengono allestiti filtrando contemporaneamente e completamente i 3 campioni utilizzando una
rampa da filtrazione in aspirazione e membrane di policarbonato
a porimetria ø 5 micron, ad una depressione media di circa 25
mmHg esercitata con una pompa vuoto; quindi per ogni paziente viene allestito un unico vetro, colorato con Papanicolaou, e
prodotta un’unica diagnosi.
La citologia oncologica urinaria ha un ruolo importante soprattutto nel follow up dei pazienti trattati per neoplasia uroteliale,
con un’alta specificità (95-100%) ma una sensibilità (38-60%)
non adeguata per l’incapacità di poter discriminare morfologicamente le lesioni benigne floride dai carcinomi ben differenziati.
Soprattutto in esiti di cistectomie o lesioni iperplastiche possono
essere utili indagini immunocitochimiche.
L’espressione di proteina P16 rappresenta una stima indiretta
della più frequente alterazione genetica riscontrata nel carcinoma della vescica ed una indicazione sulle sue capacità di replicazione. P16INK4a non si riscontra nell’urotelio non neoplastico,
mentre è espressa nel 50% dei carcinoma uroteliali, ed aumenta
all’80% nei carcinomi di alto grado. La più frequente alterazione
genetica nel carcinoma uroteliale è costituita dalla parziale perdita del cromosoma 9: su questo cromosoma è presente la regolazione della sintesi di P16.
Ck20 è una citocheratina ad alto peso molecolare che poco o
nulla espressa nell’urotelio normale mentre è decisamente presente nei carcinomi transizionali.
Disponendo di un solo vetrino, è stata messa a punto una doppia
colorazione immunocitochimica: P16 è stata evidenziata in bruno
con DiamminoBenzidina (DAB) mentre la Ck20 è stata colorata
in rosso, dopo un’adeguata diminuzione dei tempi e della temperatura di incubazione dell’anticorpo primario.
Sviluppando P16 con DAB, aumentandone i tempi di incubazione e utilizzando un sistema di amplificazione si è ottenuto un
miglioramento della performance. CK20 invece è stata sviluppata
in RED; è stato ridotto il segnale positivo diminuendo tempi di
incubazione e temperatura dell’anticorpo primario.
La colorazione risulta ben differenziata, facilmente interpretabile
ed utile nella pratica clinico-diagnostica.
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SCHEMI DI REFERTAZIONE CITOLOGICA PROPOSTI DA SIAPEC-IAP
Citologia mammaria agoaspirativa
Esame n. ……..
Tipo di accesso:
SCREENING (organizzato / spontaneo) SINTOMI (si/no) Prelievo del ………..
(TAT)………….gg
Pervenuto il………..
ALTRO……….
Cognome ……………………………………..
Nome………………… Data di nascita…………...
C.F………………………………………….....
Struttura inviante …………………………….................
Prelevatore: Patologo, Radiologo , Clinico Valutazione estemporanea adeguatezza: sì / no
Sede del Prelievo: Mammella Dx / Sn
Quadrante ……
Linfonodo ascellare ………….
Tipo prelievo: Agoaspirato Capillarità
Tipo di guida per centratura:
Calibro dell’ago….. N. passaggi …..
Palpazione
Mammografica
Ecografia
N. vetrini ….
Altro (RM…)
Tipo Preparato: Convenzionale/ Fase Liquida / Cell Block Colorazione: PAP H&E Giemsa.
Anamnesi: Sintomi: No Si ( Secrezione; dolore; retrazione; arrossamento; ulcerazione)
Familiarità: No Sì (Grado parentela ……….. Sede neoplasia …………. …Età insorgenza …)
Data ultima mestruazione ……. Età menopausa: ………… Pregresse terapie (ormonali,
chemio, Radio, altre ……...................)
Caratteristiche cliniche lesione: Palpabile
NON Palpabile Dimensioni in mm …………
Caratteristiche Eco-Mammografiche: Microcalcificazioni, Distorsione, Asimmetria densità,
Lesione solida/ cistica / complessa/ altro……………………..
Classe Radiologica di rischio (BiRADs 1-5) ……
oppure (R1-5 / U1-5) ………………..
Immunocitochimica: ER: ______ PR:________ Biologia Molecolare (FISH) ___________ Tipo di campione…………
Materiale conservato Si/No Modalità …………………
Caratteristiche Microscopiche: epiteli duttali: Presenti Assenti; quantità (Scarsa Moderata
Abbondante); presenza di
metaplasma apocrina, strutture tipo papillare, nuclei nudi,
nucleoli; atipia si no; grado atipia nucleare ( lieve, moderata, severa); fondo: Sangue,
infiammatorie, macrofagi, cellule stromali adipociti muco …………
Categoria Diagnostica: C1
C2
C3
C4
C5
(Inadeguato) causa: ematico; assenza cellule duttali;
(Benigno)
(atipia, verosimilmente benigno)
(sospetto per malignità)
(maligno)
necrosi,
cellule
artefatti
Compatibilità con diagnosi Istologica di ……………………………………….
Raccomandazioni: 1) Ripetizione 2) Controllo clinico/radiologico entro 6-12 mesi 3) Core Biopsy 4) Esame intraoperatorio 5) Chirurgia
Data referto …………… Il Patologo
171
appendice
NOTE ESPLICATIVE per la compilazione della scheda citologica mammaria
La scheda di refertazione si riferisce alla singola lesione e rappresenta il documento finale, sottoscritto dal Patologo,
relativo all’atto diagnostico, rilasciato al Medico richiedente e/o alla Paziente, (in unico esemplare).
Le lesioni non palpabili e i linfonodi ascellari, dovrebbero essere sottoposti a prelievo esclusivamente sotto guida radio/
ecografia.
Per quanto riguarda la citologia su secreto e scraping del capezzolo e preparati per apposizione non si ritiene obbligatorio
di utilizzare la codifica in categoria secondo le linee guida europee, preferendo una diagnosi descritta, con l’indicazione
ad eventuali approfondimenti.
TAT ( Turn Around Time): Inserimento suggerito ma facoltativo
Calibro dell’ago espresso in gauge (G), n. di passaggi e n. di vetrini allestiti : Inserimento suggerito ma facoltativo
Descrizione microscopica/ Commento: Deve contenere, in sintesi, la descrizione di quanto osservato al M.O.; può essere utile, soprattutto per i grandi numeri (p.e. > 200 campioni/anno), organizzare tutte queste informazioni in una scheda
sostitutiva del Commento.
C2 (precisare nella descrizione la presenza/assenza di cellule duttali/aciniche normali)
C5: Indicare un valore di “ grading citologico” del carcinoma sulla base dei seguenti parametri: Grado di dissociazione,
Dimensioni e forma del nucleo, presenza di nucleoli e di necrosi oppure si può utilizzare una gradazione in 3 classi (come
per l’istologia) assegnando un valore numerico ai singoli parametri e stabilendo dei valori cut off.
Immunocitochimica/Biologia molecolare:
Deve essere indicato:
- su quale tipo di campione viene eseguita ( FNC convenzionale, LBC, Cell Block)
- se si tratta di una recidiva o di una lesione neoplastica sincrona / metacrona.
- se eseguita per trattamenti di tipo “Neoadiuvante”
- il confronto, ove possibile, con i risultati ottenuti sul materiale chirurgico post-operatorio
Standard DI QUALITA’ minimi in diagnostica citologica mammaria
SENSIBILITà ASSOLUTA
SENSIBILITà COMPLETA
SPECIFICITà (solo casi biopsiati) SPECIFICITà (inclusi i casi non biopsiati ed assumendo che siano benigni)
VALORE PREDITTIVO POSITIVO C5 VALORE PREDITTIVO POSITIVO C4 VALORE PREDITTIVO POSITIVO C3 VALORE PREDITTIVO NEGATIVO C2 TASSO DI FALSI NEGATIVI
TASSO DI FALSI POSITIVI TASSO DI INADEGUATI
TASSO DI INADEGUATI (diagnosi finale carcinoma) TASSO DI C3 TASSO DI C4 TASSO DI SOSPETTI ( C3 + C4)
> 60%
> 80%
> 60%
> 60%
> 98%
> 80%
< 20%
> 60%
< 5%
> 1%
< 25%
< 10%
< 20%
< 20%
< 20%
(< 5%)
(<1%)
172
I CONGRESSO NAZIONALE DI CITOPATOLOGIA SIAPEC-IAP
Citologia urinaria
A) NOTIZIE CLINICHE (da organizzare come checklist)
Motivo del prelievo (esempio: ematuria, sintomatologia clinica, sospetto ecografico, screening in lavoratori esposti, FU
in pregresso tumore uroteliale, altro)
Pregresse terapie endovescicali e quando (es.: BCG, chemioterapia – specificare-, radioterapia)
Fumo (es.: pregresso o attuale)
Pregresso carcinoma uroteliale (es. sede e periodo)
Pregressi interventi chirurgici all’app. urinario (es. sede e periodo)
Precedenti cistoscopie (es. data ed esito)
Nefrolitiasi
Altro
B) PRELIEVO
Spontanee [ ]
da catetere [ ]
lavaggio vescicale [ ]
ileali [ ]
altro [ ]
C) ALLESTIMENTO
Tipo di preparazione: striscio normale [ ], citocentrifugato [ ], strato sottile [ ], altro [ ]
D) DIAGNOSI MORFOLOGICA (a fianco la corrispondenza con le conclusioni)
Non atipie uroteliali di rilievo [1]
Aggregati papillari di cellule uroteliali iperplastiche [1]
Alterazioni regressive delle cellule uroteliali [1]
Atipie uroteliali lievi [1]
Modificazioni cellulari indotte da terapia [1]
Alterazioni associate
[ ] Flora Batterica
[ ] Miceti
[ ] Cilindri
[ ] Virus
[ ] Cellule intestinali
[ ] Cristalli [ ] Altro
Atipie uroteliali severe (sospetta neoplasia uroteliale) [2]
Cellule uroteliali neoplastiche maligne (CTM) [3]
Cellule neoplastiche non uroteliali (ca. squamoso, adenoca., ca. renale, metastasi, altro) [4]
E) CONCLUSIONI DIAGNOSTICHE (vedere la corrispondenza con la diagnosi morfologica)
[1] NEGATIVA LA RICERCA DI C.T.M [3] C.T.M. UROTELIALI *
[2] SOSPETTO PER C.T.M. *
[4] C.T.M. DI ALTRA ORIGINE
* Si consiglia di indirizzare il paziente allo specialista urologo
F) ESAMI SPECIALI (specificare)
LETTORE:
173
indice degli autori
A
Accinelli G. 132, 163
Achille G. 166
Addati T. 166
Alfonso G. 132, 163
Alì G. 112
Allia E. 163
Al-Omoush T. 147
Ambrosio M.R. 154
Antonini D. 151, 165
Arnaud S. 132
Arnez Z.M. 161
Ascoli V. 119, 168
Assante A. 147
Assante M. 161
Assi A. 144
B
Baccarini P. 156
Baccigalupo B. 121
Barbati G. 118, 161
Barbero S. 153
Barizzi J. 166, 167
Barone A. 154
Basolo F. 141
Battolla E. 121
Beccati M.D. 134
Bellevicine C. 155, 157,
163, 167
Bellis D. 165
Bellomi A. 152
Belluso E. 165
Benenti M.T. 132, 163
Bergeron C. 131
Bianchi C.L. 164, 169
Bianco A. 147, 161
Boldrini L. 112
Bollito E. 115
Bonanno A.M. 154
Bonardi M. 168
Bondi A. 169
Bongiovanni M. 166, 167
Bonifacio D. 128, 147
Borea B. 161
Borelli M. 118, 161
Bortolotto M.P. 147, 161
Bortolozzo K. 164
Bortolussi L. 161
Bortul M. 147, 161
Boschi R. 163
Bottin C. 128, 147, 161
Brollo A. 128
Brusauro F. 164
Bulzoni O. 134
Buratti V. 132, 163
Buriani C. 134
Burlo P. 132
Butera M. 155
C
Campagnone G. 152
Canessa P.A. 121
Cannizzaro C. 166
Capanna S. 166
Capella S. 165
Capitanio A. 124, 137
Capodimonti S. 160
Caraceni D. 156, 166
Carantoni A. 134
Carbone A. 127
Carlinfante G. 156
Castellano I. 146
Cavicchi C. 134
Centrone M. 166
Cesari V. 149
Chiudinelli M. 153, 168
Coccia A. 132
Coda R. 146
Convertino C. 161
Cornacchiari A. 168
Corponi L. 129
Corrado R. 129
Cova M.A. 143, 147, 161
Cozzi I. 168
Cozzolino I. 154
Cressa C. 147, 161
Crippa S. 140, 166
D
D’Alessandro E. 158
Dalla Palma P. 115
Danese I.C. 168
Davidson B. 119, 122
de Biase D. 149
De Laurentiis M. 156, 166
De Luca C. 157
De Maglio G. 158
De Manna M. 152
De Marco L. 163
De Martino S. 161
De Morpurgo P.L. 147,
161
De Pellegrin A. 158
Delazer A.L. 134
Dell’Antonio A. 161
Della Grotta G. 168
Della Starza I. 168
Dellach C. 161
Dessanti P. 121
Di Benedetto A. 164
Di Benedetto G. 158, 164
Di Bonito L. 128, 147,
161
Di Loreto C. 113, 157
Di Lorito A. 156, 166
Di Napoli M. 128
Di Nuovo F. 164, 165,
169
Di Tommaso L. 156
Dina R. 132
Disanto A. 154
Dudine S. 147
E
Erra S. 152, 153, 155
F
Fabozzi A. 158
Facchetti F. 153, 168
Fadda G. 140, 160
Fedeli F. 121
Ferro P. 121
Fiaccavento S. 147
Fiandrino G. 123
Fiorito C. 132
Fisogni S. 153, 168
Flores Pereira B. 166, 167
Fontana V. 121
Fontanini G. 112
Fornari A. 115
Fornelli A. 156
Franceschini M.C. 121
Franzo A. 161
Frezza F. 147, 161
G
Gani A. 164
Gardini G. 156
Gasparini C. 147, 161
Gatta D. 156
Gattamelata M. 168
Gentile M. 164
Gentili C. 135
Ghiringhello B. 132, 163
Giannelli V. 168
Giannone G. 166
Gillio Tos A. 163
Giovagnoli M.R. 133
Giudici F. 118, 128, 147,
161
Giuffrè G. 154
Grammatica L. 166
Grassi P. 167
Gravina M. 164
Graziani B. 129
Guarini R. 168
Guerini A. 153
I
Iaccarino A. 155, 167
Immovilli S. 134
Isidoro E. 128
L
Larocca L.M. 160
Lizza N. 161
Loche D. 132
Lombardi C.P. 160
Lorenzi L. 153, 168
Lorusso C. 168
Losi L. 156
Lucini L. 168
Lucioni M. 123
Luparia P. 132, 163
Lupi C. 112
M
Maccio L. 156
Macrì L. 146
Magnani C. 129, 151
Maioli P. 115
Makuc E. 147, 161
Malapelle U. 155, 157,
163
Maletta F. 163
Manoiero N. 153
Marangi G. 168
Marino G. 163
Martellani F. 147, 161
Martin V. 167
Martini M. 160
Masiero E. 158
Maso D. 132, 163
Mason S. 164
Mazzoni E. 153, 155
Melatti G. 156
Merli M. 168
Merlo E. 166
Mian C. 164
Mignogna M. 154
Modena S. 153, 155
Monica V. 136
Montarolo F. 146
Mustacchi G. 161
N
Napolitano V. 155
Navone R. 151
Negri G. 164
Negri S. 152, 169
Nicastro E. 128
Nicastro E.M. 130
Nicola M. 123
Nuzzo P. 164
O
Ober E. 147, 161
Onorati M. 164, 165, 169
P
Palma F. 166
Palombini L. 139, 154
Papotti M. 136
Parolini R. 134
Pascale M.G. 134
Pasqualetti P. 117
Pastormerlo M. 152
Paulli M. 123
Pegolo E. 113, 157
Pellis G. 161
Pelosi G. 112
Peronio L. 157
Perrone R. 147, 161
Pession A.L. 149
Petracco G. 164, 165, 169
Petrachi F. 168
Petroni S. 166
Petz G. 147, 161
Pietribiasi F. 152
Pinamonti M. 147
Pistillo M.P. 121
Pizzolato R. 143
Pontecorvi A. 160
Privitera S. 132
Proietti A. 112
R
Raffaelli M. 160
Raiti G. 113, 157
Rapezzi R. 169
Renzi N. 161
Righi L. 136, 146
Rinaldi C. 158, 164
Rinaldi C.R. 158
Riosa F. 113, 157
Rocca B.J. 154
Romano A. 128, 147, 161
Roncella S. 121
Roncoroni L. 144
Rosini S. 156, 166
Rossi E.D. 140, 160
Rossi S. 162
Rosso S. 152
Rostan I. 151
Ruggero L. 164
Russo M. 125
Russo S. 166
Ruzza G. 129
S
Salatiello M. 157
Salmaso G.V. 153
Santeusanio G. 151
Sanzone A. 152
Sapino A. 132, 146, 163
Schettino P. 155
Schiavo Lena M. 168
Schmitt F.C. 142
Scommersi S. 161
Selmi B. 169
Sensi E. 112
Servadio A. 112
Simone G. 166
Sosa Fernandez L.V. 125
Spagnolo L. 153
Stigliano E. 160
Straccia P. 160
Streppa R. 166
T
Tallarigo F. 138
Tallini G. 149
Tardanico R. 168
Todaro P. 154
Tonutti M. 147, 161
Torelli L. 118, 147, 161,
161
Troncone G. 154, 155,
157, 163, 167
Tuccari G. 154
U
Uboldi P. 164, 165, 169
Ungari M. 126, 153, 168
V
Verga M. 163
Vetrani A. 154, 163
Vettorato M. 164
Viberti L. 133, 165
Vielh P. 146
Vigliar E. 125
Visani M. 149
Z
Zabatta A. 163
Zacchi A. 147, 161
Zamboni G. 144
Zanconati F. 118, 128,
147, 161
Zandonà L. 147
Zanier L. 161
Zeppa P. 125, 154, 155,
163
CellPrep è un sistema
che appone cellule esfoliative
su vetrino in un’area circolare di 20 mm,
disponendole in monostrato
e mantenendo integri gli aggregati cellulari
Sistema automatico a
passaggio singolo
Trasporto e caricamento
automatico della membrana
filtrante
“Surely Speedy Solution”
Sistema dotato di schermo
LCD per un facile utilizzo