• linfomi: diagnosi e classificazione
LINFOADENOPATIA
definizione:
linfonodi di dimensioni anormali (> 1,0-1,5 cm)
linfonodi di consistenza anormale
epidemiologia:
eta’ > 40 anni: 4% rischio di tumore
eta’ < 40 anni: 0.4% rischio di tumore
Studio olandese 1% delle linfoadenopatie senza causa nota sono
dovute a tumore
linfoadenopatia
localizzata:
1 sola area interessata
generalizzata: 2 o piu’ aree interessate non contigue
thymus
IMMUNE
SYSTEM
bone marrow
lymph nodes
spleen
Cause piu’ frequenti di linfoadenopatia
infezioni
EBV
Toxoplasmosi
Cytomegalovirus
Febbre da graffio di gatto
Faringite
Tubercolosi
Scarlattina
Varicella Zoster Virus
HBV
HIV
Rosolia
parotite
tumori linfatici
linfoma di Hodgkin
linfoma non-Hodgkin
LLC
LLA
LMA
tumori metastatici
malattie del collagene e vasculiti
RA
LES
melanoma
carcinoma mammario
carcinoma polmonare
carcinoma gastrico
carcinoma prostatico
carcinoma renale
tumori del collo
Anamnesi
graffio da gatto (Toxoplasmosi)
ingestione di carne cruda (Toxoplasmosi)
puntura di zecca (Lyme Disease)
esposizione alla TBC
tossicodipendenze (IV)
trasfusioni
viaggi in Africa, Asia, Australia
Leishmania
febbre tifoide
Diagnosi:
emocromo ed ematochimici completi
sierologia per HIV, EBV, toxo, CMV, HBV, HCV
Rx torace
Eco addome
Biopsia linfonodale (FNA, core biopsy or excision biopsy)
linfonodo iperplastico
linfoma diffuso a piccole cellule
Incidenza delle principali neoplasie ematologiche
Patologia
No/100.000/
per anno
Linfomi (^)
15-20
Mielomi
3-5
Leucosi Acute
8-10
Leucosi Croniche (*)
5-10
(^) incremento del 150% dal 1950, con netto incremento per NHL rispetto ad HD
(*) notevole variabilità a seconda dell'età: 70 casi/100.000 adulti di oltre 80 anni
LINFOMI NON-HODGKIN
LINFOMI NON-HODGKIN
Lymphoid malignancies: the last 50 years
Incidence +150%
Mortality + 90%
Several factors may explain the lower increase in
mortality compared to incidence, in particular:
BETTER DIAGNOSIS
MORE EFFECTIVE THERAPY
Tabella 1. Dati complessivi di incidenza di linfomi in diversi stati Europei nel periodo 1985-1992
e negli Stati Uniti d’America nel periodo 1990-1996.
Nazione
§
No. di casi/anno/100.000 abitanti
Maschi
Femmine
Totale
Danimarca
19,5
14,5
16,8
Finlandia
20,9
15,8
18
Francia
16,2
10,7
13,2
Gran Bretagna
15,0
9,8
12,2
Italia*
17,2
12,6
14,7
Olanda
21,8
12,2
16,4
Spagna
10,4
8,7
9,5
Stati Uniti d’America
19,4
12,1
15,5
Media
17,5
12,0
14,5
§
i valori sono stati ottenuti dal lavoro citato in referenza 18 e dai dati del SEER Cancer Statistics
Review, 1973-1996, accesso a: http://www-seer.ims.nci.nih.gov
*i dati si riferiscono al registro di Firenze, ottenuti su una popolazione di 1,2 milioni
Fattori associati ad aumentato rischio di insorgenza di linfoma non-Hodgkin (I)
Fattori generali di rischio aumentato




Età avanzata
Sesso maschile
Razza bianca
Stato socioeconomico avanzato
Stato di immunodepressione
 Congenita
 sindrome di Wiskott-Aldrich (WAS)
 common variable immunodeficiency (CVID)
 ataxia teleangectasia (AT)
 severe combined immunodeficiency (SCID)
 X-linked lymphoproliferative disorder (XLP)
 Acquisita
 trapianto d’organo (fegato, cuore, polmone, rene
e midollo emopoietico)
 terapie con immunosoppressori
 AIDS
Fattori associati ad aumentato rischio di insorgenza di linfoma non-Hodgkin (II)
Cronica stimolazione antigenica
Virus
 Malattie autoimmuni
 Sindrome di Sjögren
 Tiroidite di Hashimoto
 Artrite reumatoide
 Lupus eritematoso sistemico
 Morbo celiaco
 Gastrite da Helicobacter pylori
 Flogosi croniche intestinali
 Dermatite erpetiforme
 EBV
 HTLV-I
 HTLV-II
 HHV-8
 HHV-6
 HIV
 HCV
Sostanze tossiche e farmaci
 Utilizzate per lo più in ambiente lavorativo
 Erbicidi
 Pesticidi
 Solventi
 Tinture per capelli
 Fumo
 Difenilidantoina
Allergie?
APPROCCIO DIAGNOSTICO:
biopsia linfonodale
biopsia osteo-midollare
iperplasia linfonodale
Linfoma di Hodgkin
Linfoma follicolare
a piccole cellule clivate
Linfoma diffuso a piccoli linfociti
Linfoma diffuso
a grandi cellule
PTLD
APPROCCIO DIAGNOSTICO:
biopsia linfonodale
biopsia osteo-midollare
APPROCCIO DIAGNOSTICO:
biopsia linfonodale
biopsia osteo-midollare
tipizzazione immunofenotipica
tipizzazione citogenetica
tipizzazione molecolare
diagnosis of hematological malignancies:
1950: morfologia
1960: citochimica
1970: citogenetica
1980: mAbs
1990: biologia molecolare
2000: genomics and proteomics
Nature Reviews Cancer 2. 177-189, 2002
Lymph node
Bone marrow and peripheral blood
B-cell
precursor
ALL
MM
Naive Blymphocy
te
CLL without somatic
mutations
MCL
Mantlezone
BL
Marginal
zone
Germinal
Centre
CLL with
somatic
mutations
DLCL
LPL
LPL
plasmacells
FCL
Mature Blymphocytes
MZL
ANALISI AL FACS DI LINFOCITI OTTENUTI DA UN LINFONODO DI
LINFOMA FOLLICOLARE
CD20
CD3
CD30
CD15
Consequences of chromosomal translocations
regulatory
coding
regulatory
coding
regulatory
coding
regulatory
coding
translocation
regulatory
coding
fusion protein (leukemia)
translocation
regulatory
coding
deregulated transcription (lymphoma)
Chromosomal translocations in lymphoid malignancies
t(8;14)(q24;q32)
t(2:8)(q12;q24)
t(8;22)(q32;q11)
t(1;14)(q21;q32)
t(14;18)(q32;q21)
t(11:14)(q13;q32)MCL
t(14;19)(q32;q13)
t(11;14)(p13;q11) T-ALL
t(14;16)(q32;q23)
t(4;14)(p16;q32)
t(11;14)(q13;q32)MM
t(6;14)(p25;q32)
t(9;14)(p13;q32)
inv(14)(q11;q32)
Burkitt
Burkitt
Burkitt
ALL
FCL
cMYC
cMYC
cMYC
BCL9
BCL2
BCL1
B-CLL
IgH
BCL3
TCL2
MMe
MM
IgH
TCRd
c-MAF
FGFR3
BCL1??
MM
LPL
PTCL
IgH
Ig-kappa
Iglambda
IgH
IgH
IgH
IgH
IgH
IRF4
PAX5
TCRa
IgH
IgH
IgH
normal bone marrow and karyotype
Fluorescence In Situ Hybridisation (FISH)
In situ hybridisation is a technique to directly identify a specific region of DNA
or RNA in a cell. Complementary probes linked to molecules such as biotin or
fluorescein can be used to visualise the target, which can be chromosomes,
interphase nuclei or tissue biopsy.
Advantages and Disadvantages of FISH Techniques
Direct in situ technique is relatively rapid and sensitive.
No cell culture needed.
Result easier to interpret than karyotype.
Can combine FISH with immunostaining ie FICTION
FISH will only provide information about the probe being tested, other aberrations
will not be detected.
Illustration of FISH technique in metaphase and interphase cells.
Normal interphase cells show 2 green spots (BCL-1) and 2 red spots (IgH).
Abnormal interphase cells show 3 green spots and 2 red spots, but there
must be co-localisation of 1 red and 1 green spot.
FISH
CD5+ B-cell lymphoproliferative disorders
Trisomy 12, t (11;14), deletion 13q14, deletion of 17p(p53), deletion of 11q
Diffuse large B-cell lymphoma
3q27 deletion, t (14;18).
Suspected Burkitt lymphoma
t (8;14)
Suspected follicular lymphoma
or CD5- B-cell lymphoproliferative disorder in bone marrow
t (14;18)
NON-HODGKIN LYMPHOMAS:
*1) non-Hodkin lymphomas are a diverse collection of approximately
*40 entities, with different immunopathologic and cytogenetic characteristics
*2) the most frequent entities are the:
*
- Follicle Centre lymphoma (FCL)
*
- Diffuse Large Cell lymphoma (DLCL)
*3) B-cell derived are by far more frequent compared to T-cell derived
*(90% vs. 10%)
B-Cell Neoplasms
I. Precursor B-cell neoplasm:
a. Precursor B-lymphoblastic leukemia/lymphoma
II. Mature (peripheral) B-cell neoplasms
a. B-cell chronic lymphocytic leukemia / small lymphocytic lymphoma
b. B-cell prolymphocytic leukemia
c. Lymphoplasmacytic lymphoma
d. Splenic marginal zone B-cell lymphoma (+/- villous lymphocytes)
e. Hairy cell leuekmia
f. Plasma cell myeloma/plasmacytoma
g. Extranodal marginal zone B-cell lymphoma of mucosa-associated
lymphoid tissue type
h. Nodal marginal zone lymphoma (+/- monocytoid B-cells)
i. Follicle center lymphoma, follicular,
j. Mantle cell lymphoma
k. Diffuse large cell B-cell lymphoma
• Mediastinal large B-cell lymphoma
• Primary effusion lymphoma
l. Burkitt's lymphoma/Burkitt's cell leukemia
T-Cell and Natural Killer Cell Neoplasms
I. Precursor T cell neoplasm:
a. Precursor T-lymphoblastic lymphoma/leukemia
II. Mature (peripheral) T cell and NK-cell neoplasms
a. T cell prolymphocytic leukemia
b. T-cell granular lymphocytic leukemia
c. Aggressive NK-Cell leukemia
d. Adult T cell lymphoma/leukemia (HTLV1+)
e. Extranodal NK/T-cell lymphoma, nasal type
f. Enteropathy-type T-cell lymphoma
g. Hepatosplenic gamma-delta T-cell lymphoma
h. Subcutaneous panniculitis-like T-cell lymphoma
i. Mycosis fungoides/Sézary's syndrome
j. Anaplastic large cell lymphoma, T/null cell, primary cutaneous type
k. Peripheral T cell lymphoma, not otherwise characterized
l. Angioimmunoblastic T cell lymphoma
m. Anaplastic large cell lymphoma, T/null cell, primary systemic type
INCIDENZA DEI VARI TIPI DI LINFOMA NON-HODGKIN
28%
6%
16%
30%
20%
FCL
B-cell DLCL
non follicular low grade
NHL
T-cell NHL
Other lymphomas
Hodgkin's lymphoma (Hodgkin's Disease)
a.Nodular lymphocyte predominance Hodgkin's lymphoma
b.Classical Hodgkin's lymphoma
• Nodular sclerosis Hodgkin's lymphoma
• Lymphocyte-rich classical Hodgkin's lymphoma
• Mixed cellularity Hodgkin's lymphoma
• Lymphocyte depletion Hodgkin's lymphoma
Hodgkin's Disease - Classification
Type
Histologic Features
Frequency
Prognosis
Bands of fibrosis,
Lacunar cells
Most frequent type,
more common in women
Good
most are stage I-II
Composed of many
different cells
Most frequent
in older persons,
second most frequent overall
Fair
most are stage III
Nodular sclerosis
Mixed cellular
Lymphocyte-rich classical Hodgkin's lymphoma
Mostly B-cells and few
Uncommon
Reed-Sternberg variant cells
Good
most are stage I or II
Lymphocyte depletion
Many Reed-Sternberg
cells and variants
Uncommon
Poor
most are stage III or IV
CARATTERIZZAZIONE RISCHIO PROGNOSTICO:
biopsia linfonodale
biopsia osteo-midollare
tipizzazione immunofenotipica
tipizzazione molecolare
stadiazione della malattia
DEFINIZIONE RISCHIO PROGNOSTICO:
biopsia linfonodale
biopsia osteo-midollare
tipizzazione immunofenotipica
tipizzazione molecolare
stadiazione della malattia
fattori di rischio
Fattori con valore prognostico sfavorevole indipendente:
 performance status > 2
 LDH > normale
 siti extranodali  2
 stadio III o IV
 età > 60 anni
No. di fattori presenti
Tipo di rischio prognostico
0-1 basso (L)
2
intermedio-basso (LI)
3
intermedio-alto (HI)
4-5 alto (H)
A World Health Organisation classification indicating a
PERSON's status relating to activity / disability.
0 Able to carry out all normal activity without restriction
1 Restricted in physically strenuous activity, but able to walk
and do light work
2 Able to walk and capable of all self care, but unable to
carry out any work. Up and about more than 50% of waking
hours
3 Capable of only limited self care, confined to bed or chair
more than 50% of waking hours
4 Completely disabled. Cannot carry on any self care. Totally
confined to bed or chair
Tab 4 – Correlazioni tra gruppi di rischio definiti dall'IPI e possibilità di remissione completa,
sopravvivenza libera da ricadute di malattia e sopravvivenza globale nei linfomi aggressivi
Rischio
prognostico
secondo IPI
Basso
Intermedio-basso
Intermedio-alto
Alto
No. Fattori
sfavorevoli
presenti
0-1
2
3
4-5
% Remissione
Completa
87
67
55
44
% sopravvivenza a
%
5 anni senza
sopravvivenza
ricadute di malattia globale a 5 anni
70
73
50
51
49
43
40
26
Overall Survival in DLCL according
to risk group defined by Age-Adjusted IPI
(PS, stage, LDH)
Score
CR
Rate
(%)
5-yr
survival
(%)
Low
0
92
83
Low-intermediate
1
78
69
High-intermediate
2
57
46
High
3
46
32
Risk group
IIL prognostic system
• Age (< vs. > 60 vs)
• Sex (F vs M)
• Extranodal sites (0-1 vs  2)
• Serum LDH (normal vs elevated)
• B symptoms (absent vs present)
• ESR (less than 30 vs at least 30)
Federico M et al., Blood 2000, 95: 783-789
Prognosis of follicular lymphoma: a predictive model based on a retrospective analysis of 987 cases
Hodgkin's lymphoma (Hodgkin's Disease)
a.Nodular lymphocyte predominance Hodgkin's lymphoma
b.Classical Hodgkin's lymphoma
• Nodular sclerosis Hodgkin's lymphoma
• Lymphocyte-rich classical Hodgkin's lymphoma
• Mixed cellularity Hodgkin's lymphoma
• Lymphocyte depletion Hodgkin's lymphoma
Factors with independent prognostic value
for survival in lymphomas of both
high and low grade histology
Prognostic
classification
IPI
(New Engl J Med 1993)
aa I P I
(New Engl J Med 1993)
IIL
(Blood 2000)
Factor
age >60
performance status
serum LDH level
Ann Arbor stage
extranodal involvement
Performance status
serum LDH level
Ann Arbor stage
Age (>60)
Sex (male)
ESR ()
Serum LDH level ()
Systemic symptoms
extranodal involvement
13-gene predictor:
cured gene-espression
signature
fatal/refractory gene-espression
signature
13-gene outcome
predictor:
IPI-outcome
predictor:
13-gene predictor:
cured gene-espression
signature
fatal/refractory gene-espression
signature
Overall Survival of advanced-stage DLCL
with 3rd generation chemotherapy regimens
Overall Survival of FCL patients
Turin-group experience
with the i-HDS scheme
a “high-dose” approach aimed to obtain
maximal tumor cytoreduction and to exploit
the in vivo-purging effect operated by
chemotherapy
Corradini P et al, Blood 1997 Jan 15;89:724-31
Tarella C et al, Leukemia 2000 Apr 14:740-7
APO x 2
VP-16
DHAP x 2 2 g/sqm
MTX
8 g/sqm
CTX
7 g/sqm
DEX
40
G-CSF
G-CSF
PBPC/BM
harvest
MITOX + L-PAM
+ PBPC autograft
I-HDS SCHEME FOR HIGH-RISK
FCL PATIENTS
I-HDS REGIMEN IN FCL:
results of the Torino group experience
Leukemia 2000, 14: 740-747
•CR RATE OF 79%
•ACCEPTABLE RATE OF EARLY AND LATE TOXICITIES
100
90
80
70
60
50
40
30
20
10
0
% surviving
% surviving
•A PROJECTED EFS AT 9 YEARS OF 62% AND A PROJECTED OS
OF 78%
Event-free survival
0
1
2
3
4
5
years
6
7
8
9
100
90
80
70
60
50
40
30
20
10
0
Overall survival
0
1
2
3
4
5
years
6
7
8
9
Gianni AM et al; NEJM 1997; 336: 1290-97
“HDS vs MACOP-B in aggressive B-cell NHL“
DEVELOPMENT OF MONOCLONAL
ANTIBODIES
HUMAN
MOUSE
CHIMERICAL
HUMANIZED
UNLABELED CHIMERIC
ANTIBODY
IMMUNOTOXIN
RADIOCONJUGATE
Meccanismo d’azione mAbs
• effetto diretto
• signaling  apoptosi
• citossico (tossine o radiomarcati)
• effetto indiretto
• complemento
• ADCC (NK, GN)
• immunosensibilizzazione
Principali anticorpi monoclonali “unlabelled”
STRUTTURA
INDICAZIONI
ANTIGEN
NAME
CD20
Rituximab
Chimerico
FCL,MCL,HCL, DLCL
CD25
Basiliximab
Daclizumab
Chimerico
umanizzato
Trapianto
CD52
Campath 1H
umanizzato
LLC, Trapianto
CD22
Epratuzumab umanizzato
FCL
RADIOIMMUNOCONJUGATE
Effector mechanisms
Radiation-induced cytolysis
TARGET ANTIGENS:
•NOT SHED
•NOT INTERNALIZED ?
NB. Properties of each immunoconiugate depend on which isotope is
chosen
hdAra-C
hd-CY
A P O
VP16 +
CDDP
PBSC
harvest
G-CSF
PBSC
harvest
G-CSF
RITUXIMAB
G-CSF
PBSC autografting
C-HDS + Rituximab schedule
C-HDS + Rituximab in high-risk DLCL patients: a
multicenter italian study
R- HDS
historical
CR
OS
82%
71 % (3yr.)
46-57%
32-46% (5yr.)
identification of residual disease
The role of 67Ga scanning or FDG-PET
in discriminating between active or fibrotic
residual masses is well established
identification of residual disease
The value of molecular biology techniques (PCR)
in evaluating the minimal residual disease in patients
with Bone Marrow involvement at presentation
DFS comparison between PCR-positive
and PCR-negative patients
100
PCR negative
% surviving
80
60
40
PCR positive
P<0.005
20
0
0
2
4
6
years
8
10
12
IMMUNOTERAPIA NEI DISORDINI LINFOPROLIFERATIVI
• percentuale di guarigione ancora insufficiente
• crescita abbastanza lenta
• markers tumore-specifici o lineage-specifici
• chemioterapia efficace ma non eradicante
• monitoraggio della malattia minima residua (MMR)
• MMR spesso MDR+
• immunosensibilita’ della MMR
• modelli animali disponibili
Bendandi et al., Nat Med 5: 1171-1177, 1999
Protein-based vaccine
VACCINATION SCHEDULE
week
0
2
6
10
14
24
28
Id/KLH
(0.5 mg + 0.5 mg)
GM-CSF
(150 µg/sqm)
• 15 MM in first remission after HDS and PBPC infusion;
tumor burden
remission
threshold
diagnosis
time
remission phase
relapse
Early detection of recurrent disease
1.
The efficacy of “salvage treatment” is well known in
both high and low-grade lymphomas
Early detection of recurrent disease
2.
“salvage treatments” are more effective if the
recurrent disease is not extensively spread
SURVIVAL by
Ann Arbor stage at relapse
100
75
%
p<0.0001
50
I-II (n=153)
25
III-IV (n=259)
0
I.I.L.
1999
0
5
10
Years after relapse
le complicanze gravi,
potenzialmente fatali
dei trattamenti per linfoma
1. Complicanze infettive
sviluppo di nuovi programmi di
chemioterapia intensiva e
chemioimmunoterapia
per cercare di migliorare la risposta
più marcata immunodepressione,
con prolungati deficit T e B linfocitari,
e incremento dei rischi infettivi per:
- micosi (aspergillo)
- virus (CMV)
- protozoi (pneumocistis carinii)
le complicanze gravi,
potenzialmente fatali
dei trattamenti per linfoma
1. Complicanze infettive
2. Le neoplasie secondarie
La gestione di un paziente con
linfoma è complessa in tutte le fasi
della storia clinica, dalla diagnosi al
trattamento sino alle fasi a lungo
termine
In questa gestione è indispensabile una
stretta collaborazione tra tutti i medici,
specialisti in vari settori ed internisti