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

IMMUNE SYSTEM thymus bone marrow spleen lymph nodes

Cause piu frequenti di linfoadenopatia infezionitumori linfatici EBV linfoma di Hodgkin Toxoplasmosilinfoma non-Hodgkin Cytomegalovirus LLC Febbre da graffio di gattoLLA FaringiteLMA Tubercolosi Scarlattina tumori metastatici Varicella Zoster Virus melanoma HBVcarcinoma mammario HIVcarcinoma polmonare Rosoliacarcinoma gastrico parotitecarcinoma prostatico carcinoma renale malattie del collagene e vasculiti tumori del collo RA LES

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

(^) incremento del 150% dal 1950, con netto incremento per NHL rispetto ad HD (*) notevole variabilità a seconda dell'età: 70 casi/ adulti di oltre 80 anni Patologia No/ / per anno Linfomi (^) Mielomi 3-5 Leucosi Acute 8-10 Leucosi Croniche (*) 5-10 Incidenza delle principali neoplasie ematologiche

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

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 dorgano (fegato, cuore, polmone, rene e midollo emopoietico) terapie con immunosoppressori AIDS

Cronica stimolazione antigenica Malattie autoimmuni Sindrome di Sjögren Tiroidite di Hashimoto Artrite reumatoide Lupus eritematoso sistemico Morbo celiaco Gastrite da Helicobacter pylori Flogosi croniche intestinali Dermatite erpetiforme Sostanze tossiche e farmaci Utilizzate per lo più in ambiente lavorativo Erbicidi Pesticidi Solventi Tinture per capelli Fumo Difenilidantoina Virus EBV HTLV-I HTLV-II HHV-8 HHV-6 HIV HCV Fattori associati ad aumentato rischio di insorgenza di linfoma non-Hodgkin (II) 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 , 2002

B-cell precursor Naive B- lymphocy te Mantle- zone Germinal Centre Marginal zone Mature B- lymphocytes plasmacells ALL CLL without somatic mutations FCL DLCL MM MZL Bone marrow and peripheral blood MCL BL Lymph node CLL with somatic mutations LPL

The 1990 idea of mature linfoproliferative disorders: GC derivedNon-GC derived FCL, DLCL GC derived, Burkitt CLL, MALT, MCL, LPC, MM, DLCL activated CURABILITY no yes

APPROCCIO DIAGNOSTICO: biopsia linfonodale biopsia osteo-midollare tipizzazione immunofenotipica tipizzazione citogenetica tipizzazione molecolare

ANALISI AL FACS DI LINFOCITI OTTENUTI DA UN LINFONODO DI LINFOMA FOLLICOLARE

CD20

CD79

CD3

CD30

CD15

Consequences of chromosomal translocations regulatory coding regulatory coding regulatory coding regulatory coding regulatory coding regulatory coding translocation fusion protein (leukemia)deregulated transcription (lymphoma)

t(8;14)(q24;q32)BurkittcMYCIgH t(2:8)(q12;q24)BurkittcMYC Ig-kappa t(8;22)(q32;q11)BurkittcMYCIglambda t(1;14)(q21;q32)ALL BCL9 IgH t(14;18)(q32;q21)FCLBCL2IgH t(11:14)(q13;q32)MCLBCL1IgH t(14;19)(q32;q13)B-CLLBCL3IgH t(11;14)(p13;q11)T-ALLTCL2TCR t(14;16)(q32;q23)MMec-MAFIgH t(4;14)(p16;q32)MMFGFR3IgH t(11;14)(q13;q32)MMBCL1??IgH t(6;14)(p25;q32)MM IRF4IgH t(9;14)(p13;q32)LPLPAX5 IgH inv(14)(q11;q32)PTCLTCR IgH Chromosomal translocations in lymphoid malignancies

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)

* 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%) NON-HODGKIN LYMPHOMAS:

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

FCL non follicular low grade NHL B-cell DLCL T-cell NHL Other lymphomas 30% 6% 16% 28% 20% INCIDENZA DEI VARI TIPI DI LINFOMA NON-HODGKIN

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 Nodular sclerosis Bands of fibrosis, Most frequent type, Good Lacunar cells more common in womenmost are stage I-II Mixed cellular Composed of many Most frequent Fair different cells in older persons, most are stage III second most frequent overall Lymphocyte-rich classical Hodgkin's lymphoma Mostly B-cells and few Uncommon Good Reed-Sternberg variant cellsmost are stage I or II Lymphocyte depletion Many Reed-Sternberg Uncommon Poor cells and variantsmost 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)

Overall Survival in DLCL according to risk group defined by Age-Adjusted IPI (PS, stage, LDH) Risk groupScore C R Rate (%) 5-yr survival (%) Low09283 Low-intermediate17869 High-intermediate25746 High34632

Age ( 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) IIL prognostic system

Federico M et al., Blood 2000, 95: 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

Prognostic classificationFactor Factors with independent prognostic valuefor survival in lymphomas of both high and low grade histology I P I (New Engl J Med 1993) age >60 performance status serum LDH level Ann Arbor stage extranodal involvement aa I P I (New Engl J Med 1993) Performance status serum LDH level Ann Arbor stage I I L (Blood 2000) 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 Corradini P et al, Blood 1997 Jan 15;89: Tarella C et al, Leukemia 2000 Apr 14:740-7 a high-dose approach aimed to obtain maximal tumor cytoreduction and to exploit the in vivo-purging effect operated by chemotherapy

VP-16 2 g/sqm APO x 2 DHAP x 2 G - C S F CTX 7 g/sqm PBPC/BM harvest MITOX + L-PAM + PBPC autograft I-HDS SCHEME FOR HIGH-RISK FCL PATIENTS G - C S F MTX 8 g/sqm DEX 40

I-HDS REGIMEN IN FCL: results of the Torino group experience Leukemia 2000, 14: CR RATE OF 79% ACCEPTABLE RATE OF EARLY AND LATE TOXICITIES A PROJECTED EFS AT 9 YEARS OF 62% AND A PROJECTED OS OF 78% years % surviving Event-free survival years % surviving Overall survival

Gianni AM et al; NEJM 1997; 336: HDS vs MACOP-B in aggressive B-cell NHL

MOUSE CHIMERICAL HUMANIZED HUMAN DEVELOPMENT OF MONOCLONAL ANTIBODIES

UNLABELED CHIMERIC ANTIBODY IMMUNOTOXIN RADIOCONJUGATE

Meccanismo dazione mAbs effetto diretto signaling apoptosi citossico (tossine o radiomarcati) effetto indiretto complemento ADCC (NK, GN) immunosensibilizzazione

Principali anticorpi monoclonali unlabelled ANTIGENNAME INDICAZIONI CD20 CD25 CD52 CD22 STRUTTURA Rituximab Basiliximab Daclizumab Campath 1H Epratuzumab Chimerico umanizzato FCL,MCL,HCL, DLCL Trapianto LLC, Trapianto FCL

Radiation-induced cytolysis Effector mechanisms TARGET ANTIGENS: NOT SHED NOT INTERNALIZED ? RADIOIMMUNOCONJUGATE NB. Properties of each immunoconiugate depend on which isotope is chosen

hd-CY VP16 + CDDP hd- Ara-C PBSC autografting A P O PBSC harvest G-CSF PBSC harvest G-CSF C-HDS + Rituximab schedule RITUXIMAB

C-HDS + Rituximab in high-risk DLCL patients: a multicenter italian study R- HDS historical 82% 46-57% 71 % (3yr.) CROS 32-46% (5yr.)

identification of residual disease The role of 67 Ga 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

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: , 1999

week Id/KLH (0.5 mg mg) GM-CSF (150 µg/sqm) VACCINATION SCHEDULE 15 MM in first remission after HDS and PBPC infusion; Protein-based vaccine

tumor burden remission threshold time diagnosisrelapse remission phase

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

% III-IV (n=259) I-II (n=153) Years after relapse SURVIVAL by Ann Arbor stage at relapse I. I. L p<0.0001

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 - micosi (aspergillo) - virus (CMV) - protozoi (pneumocistis carinii) più marcata immunodepressione, con prolungati deficit T e B linfocitari, e incremento dei rischi infettivi per:

le complicanze gravi, potenzialmente fatali dei trattamenti per linfoma 2. Le neoplasie secondarie 1. Complicanze infettive

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