CLL: definition and nosography Chronic lymphoproliferative disorder Clonal Expansion of B lymphocytes expressing CD5 Localization in the PB, BM, lymph nodes and spleen Extranodal localizations rare at diagnosis, more frequent in advanced phases Heterogeneous clinical course

CLL belongs to a wide group of leukemic CLD B-derived CLL (typical, mixed cell type, CLL/PL) PLL HCL SLVL/SMZL NHL in leukemic phase (LPL, FCL, MCL, others) T-derived LDGL T-PLL Sezary S. ATLL PTCL in leukemic phase The distinction of these disorders is based on morphology, histology and immunopenotyping

Epidemiological data: age at diagnosis show that CLL is a disease predominantly of the elderly populatuion 100% Incidence 4 / 100.000 Stable incidence M/F 2:1 White > black > asians Risk factor Family history of lymphoid neoplasia Genetic predisposition > 75 aa 65-75 aa < 65 aa 80% 44 60% 40% 31 9% 45-54 y.o. 2% < 45 y.o. 20% 25 0% SEER

Pathogenesis of CLL Cell of origin Mechanisms of cell growth and clonal expansion Mechanisms of autoimmune phenomena

It is possible to define the cell origin of B-lymphoid neoplasia (pre GC vs post GC) based on the presence of IGHV mutations Mantle cell Lymphoma Follicular lymphoma Large cell lymphoma (part) Burkitt’s lymphoma Multiple myeloma B-ALL Lymphoplasmacytic lymphoma Pre-plasma cell Plasma cell BM B-precursor cell Naive B-cell (Not Encountered Ag) Marginal zone Lymphoma CLL Germinal Centre cells Memory B-cell IGHV unmutated (30% of CLL) IGHV “mutated” (70% of CLL)

Cell of origin of CLL “MUTATED” “UNMUTATED” Bone Marrow CLL CLL Genetic insult “MUTATED” CLL “UNMUTATED” CLL Germinal centre MEMORY B-CELL IGVH mutations Genetic insult No IGVH mutations Stimulation by T-independent Ag or autoantigen Somatic hypermutation Activation- induced Cytidine deaminase Marginal zone Circulating naive B-lymph Germinal centre cell precursor Bone Marrow Pre-B cell VDJ rearrangement

Four pathogenetic moments in CLL Antigenic stimulation of lymphocytes with specific BCR 1 CD5+/B lymphs in the mantle or marginal zone Autoantigens (e.g anti RA, myosin) Other Ag (CMV? EBV?) Continuous Ag stimulation Active BCR signalling Antigen driven oligoclonal expansion Telomere shortening Genetic Instability 2 Transgenic mouse Primary anomaly (miR15-16, TCL1?) 13q-; +12, 6q-, 14q32, 11q-; 17p- Chromosome translocations Secondary events Late appearance Genetic features 3 Interaction with microenvironment Clinical phenotype CLINICOPATHOLOGIC MANIFESTATIONS 4 7 7

Pathogenetic moments Unfavourable prognostic markers 1 2 3 4 Antigenic stimulation of lymphocytes with specific BCR Specific BCR subsets e.g. stereotyped VH3-21 1 Continuous Ag stimulation Antigen driven oligoclonal expansion Activation markers e.g. CD38, ZAP70, TCL1, LPL 2 Markers of genetic instability Telomere shortening 11q-, 17p-, TP53, IGHV unmutated Chromosome translocations Complex karyotype Genetic features 3 Markers of tumor expansion e.g. stage ß2 micro, sCD23, sTK Bulky disease Clinical phenotype 4 8 8

The frequency of chromosome lesion varies according to the clinical condition in CLL. CLL stage A no indication for treatment CLL stage B/C indication for treatment CLL fluda refractory

Survival from diagnosis in 217 CLL Ferrara’s series 1.0 ,8 ,6 ,4 ,2 0,0 unfavourable abnormal favourable 6q- 92 pts with 13q- single, normal Cumulative Survival 69 pts with + 12, 1-2 aberrations 13 pts with 6q- 43 pts with 17p-, 11q-, complex karyotype 0 50 100 150 200 250 300 350 400 Months Cuneo et al, Leukemia, 2004

DEATH CLL: single disease with variable clinicobiologic features lymphocytosis Early stage Advanced stage Tumor mass adenopathies splenomegaly BM infiltrate Progression Secondary effects of treatment Cutis Digestive tract Liver CNS Extra nodal organs Autoimmune phenomena DEATH Infections / Cachexia 5 10 15 20 YEARS

Mechanisms of autoimmune hemolytic anemia in CLL Galletti J et al, J immonul 2008: 181: 3674-83 Recognition of erythrocyte protein band 3 (N-terminal portion) by an unknown receptor by CLL cells 2. Internalization of protein band 3 by CLL cells 3. Resting B3-pulsed CLL cells are unable to induce T-cell activation 4. CD40L-activated CLL cells are able to induce T-cell activation in a HLA-DR dependent fashion CLL cells can specifically bind a RBC antigen, They are able to present it to T-cells, thus functioning as Ag-presenting cells This function require them to be in an activated status

Quadri di presentazione più frequenti della LLC: 1) Emocromo di routine o per lieve malessere generale o per astenia. riscontro di linfocitosi e, talora, anemia o piastrinopenia iniziale Neu 32% Linf 64% Mono 4% WBC 12.9X10^9/L Hb 13,9 GR 4.8X10^9/L MCV 88 MCH 28 Pst 250X10^9/L Neu 12% Linf 84% Mono 4% intermedio Neu 3% Linf 96% Mono 1% avanzato WBC 67.9X10^9/L Hb 12,9 GR 4.5X10^9/L MCV 86 MCH 27 Pst 220X10^9/L WBC 160X10^9/L Hb 9,9 GR 3,1X10^9/L MCV 83 MCH 27 Pst 95X10^9/L

Altri frequenti quadri di presentazione della LLC: 2) Adenopatia (in genere multipla, di dimensioni medio-piccole (1-3 cm), di consistenza parenchimatoso-tenera, non duro 3) Anemia emolitica autoimmune Neu 12% Linf 84% Mono 4% WBC 29.9X10^9/L Hb 7,9 GR 2,8X10^9/L MCV 98 MCH 27 Pst 220X10^9/L Sub-ittero o ittero franco Test di Coombs positivo Indici di emolisi positivi

La diagnosi differenziale è incentrata su Morfologia delle cellule linfocitarie Immunofenotipo delle cellule linfocitarie Distinzione tra linfocitosi reattive e linfocitosi monoclonali Le principali linfocitosi reattive da distinguere rispetto a quelle monoclonali sono: - EBV - CMV - Brucellosi - Toxoplasmosi Morfologia: Linfociti in varie fasi di Attivazione Immunofenotipo: Linfociti policlonali Rapporto Catene K/l normale (3/2) Non incremento linfociti B/CD5+

Le principali linfocitosi monoclonali sono: LLC Linfomi leucemizzati (follicolare, mantellare) Linfoma splenico con linfociti villosi circolanti Leucemia a cellule capellute

La diagnosi differenziale è incentrata su Morfologia delle cellule linfocitarie Immunofenotipo delle cellule linfocitarie Distinzione tra LLC e altre sindromi linfoproliferative morfologia immunofenotipo LLC Linfomi leucemizzati Piccoli linfociti alcuni prolinfociti alcuni grandi linfociti Ombre di Gumprecht Cellule clivate Irregolarità nucleo Linfociti monoclonali Evidente squilibrio K/l Netto aumento linfociti B/CD5+ Intensità espressione sIg debole Marcatori specifici per ogni tipo di linfoma

Diagnosi e stadiazione LLC Conta ematica (>5000 linfociti) ed aspirato midollare (>30% linfociti) Morfologia e immunofenotipo (espansione monoclonale B CD5+) Se adenopatia con istologia positiva per LLC, ma presenti < 5000 linfociti B nel sangue periferico la diagnosi è di linfoma linfocitico che presenta le stesse caratteristiche generali della LLC, eccetto per la localizzazione ematomidollare, che peraltro può comparire nelle fasi evolutive

Diagnosi e stadiazione LLC Anamnesi per condizioni generali, febbre, sudorazione, prurito, dimagramento, infezioni pregresse, pregresse anemizzazioni (emolisi) Visita con particolare riguardo a stazioni linfonodali, fegato e milza Rx torace ed eco addome Profilo biochimico per funzionalità renale, enzimi epatici e bilirubinemia, uricemia, LDH, beta-2-microglobulinemia. Coombs Elettroforesi: possibile ipogammaglobulinemia Valutazione dei fattori di rischio importanti per decidere il tipo di terapia nel paziente giovane (< 65 anni) - stadio clinico secondo Rai o Binet - lesioni citogenetiche - beta-2-microglobulinemia - CD38 - profilo mutazionale geni IgVH

Sintomi e segni Assenti nella > parte dei casi alla diagnosi Quadro Adenopatie (raro bulky) Splenomegalia Sistemici per neoplasie linfoidi Astenia ed ittero per anemia emolitica autoimmune Astenia per anemia Emorragie per piastrinopenia (da insuff midollare o autoimmune) Localizzazioni extra nodali (cute SNC, digerente Infezioni ricorrenti Quadro iniziale Quadro avanzato

Leucemia a prolinfociti Insufficienza midollare Anemia e piastrinopenia refrattarie Molto frequente Immunodepressione Infezioni Ricorrenti Molto frequente Quadri evolutivi e/o avanzati adenopatie massive sintomi sistemici versamenti sierosi cachessia S Richter Infrequente Sottostimata (?) 5-10% dei casi Poco frequente Leucemia a prolinfociti linfocitosi anemizzazione piastrinopenia splenomegalia

Fattori contribuenti all’immunodeficit nella LLC Ridotti livelli Ig seriche Ridotti livelli di CD4 circolanti Ridotta risposta agli antigeni (sovvertimento dell’architettura linfonodale) Terapie efficaci, ma fortemente immunosoppressive! Fludarabina +/- endoxan, Atc monoclonali, rituximab e in particolare alemtuzumab (anti CD52)

Sostituzione midollare Linfocitosi milza Sostituzione midollare con insufficienza funzionale

Sopravvivenza dei pazienti con LLC in rapporto alle anomalie citogenetiche Normale o 13q- isolato +12 6q- 17p- o 11q-

β2-microglobulin levels. Survival of MDACC previously untreated patients by β2-microglobulin levels. MANAGEMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA: A CHANGING FIELD Michael J. Keating Rev Clin Exp Hematol • vol 6.4 • December 2002

CD38 in CLL Ibrahim S et al (MDACC) Blood 2001 Kaplan-Meier survival curve comparing patients with B-CLL whose samples were positive (20% or more) or negative (less than 20%) for CD38 expression; the difference is significant at P = .00005. (B) Kaplan-Meier survival curve showing the survival difference between CD38+ and CD38    patients in the group of patients with CLL who were previously untreated.

German CLL study group Leukemia 2002 SURVIVAL IN 300 CLL German CLL study group Leukemia 2002 “mutated” “unmutated”

Indicazioni al trattamento Aumento dei GB con raddoppio < 6 mesi Stadio avanzato (III o IV Rai) Passaggio a stadio più avanzato Sviluppo di adenopatie o splenomegalia progressive Anemia emolitica autoimmune poco responsiva allo steroide Sintomi sistemici GB > 100.000 – 300.000 / ml

Opzioni terapeutiche Fluda +endoxan + anti CD20 < 65 anni > 65 anni Fluda +endoxan + anti CD20 Anti CD52 da solo o in combinazione Trapianto allogenico (sperimentale) Clorambucile (Leukeran) Fludarabina Fluda + anti CD20 Fluda + endoxan Fluda + endoxan + anti CD20 Anti CD52

Ten-year relative survival curves for patients younger than 70 years in Binet stage B/C according to whether they were diagnosed in the calendar periods 1980-1994 or 1995-2004 1980-1994 1995-2004 1.0 0.9 0.8 0.7 0.6 Relative survival 0.5 0.4 0.3 0.2 0.1 1 2 3 4 5 6 7 8 9 10 Years from diagnosis Improving survival in patients with CLL (1980-2008): the Hospital Clinic of Barcelona experience. Blood 2009 114: 2044-2050 31

Overall survival probabilty Historical controls! F (CALGB 9011) F+R (CALGB 9712) F (n=190) FCM (n=140) FCR (n=300) 1.0 1.0 0.8 0.8 0.6 0.6 Overall survival probabilty Proportion alive 0.4 0.4 F (n=190) FCM (n=140) p = 0.37 0.2 0.2 FCM (n=140) FCR (n=300) p < 0.001 20 40 60 80 100 120 140 12 24 36 48 60 72 84 96 108 Months Months Byrd et al. Blood, 2005 Tam, C. S. et al. Blood 2008;112:975-980 32 32

Binet stage does not affect overall survival with first-line MabThera-FC: Long-term MDACC data 1.0 0.9 0.8 0.7 0.6 Overall survival 0.5 0.4 Binet stage n Dead A 78 22 B 144 42 C 29 0.3 0.2 0.1 1 2 3 4 5 6 7 8 9 10 Time (years) Tam CS, et al. Blood 2008; 112:975–980. 33 33

Addition of rituximab to fluda and CTX in CLL: a randomised, open-label, phase 3 trial M Hallek et al Lancet 2010; 376: 1164–74

Overall survival in all patients Addition of rituximab to fluda and CTX in CLL: a randomised, open-label, phase 3 trial M Hallek et al Lancet 2010; 376: 1164–74 Overall survival in all patients