CD40 stimulation on CLL cells

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1 Pathways of apoptosis 0 stimulation on cells 0 stimulation on cells NC Survivin Actin ymphoid tissues in Proliferation Centres Survivin Proliferation Centres Ki7 <% Normal N p27 >9% N 0 cl-2 Aberrant immune response? he proliferating compartment Survival Proliferation CC22 (CC17) Cognitive ic elements Activation/proliferation Affinity maturation Resting Naive cells Mantle zone ight zone Dark zone 0 (I-) 1

2 cells show IGV somatic mutations () DEFINIION Accumulation of small, resting, mature + lymphocytes in peripheral blood, bone marrow, lymph nodes and spleen () omogeneous phenotype: +, CD23 +, sigm low eterogeneous clinical course Indolent course Aggressive course Survival: 12-1 years Survival: 3- years herapy: No need or late herapy: Early and frequent Small ymphocytic ymphoma (S) ow-grade, indolent ymphomas Fais et al, 1998 Gastric ymphoma Chronic. pylori infection 10-20% of cancers associated with infections Direct. pylori specific cells + Stimulation Infiltrating autoreactive cells Early. pylori MA t(11;18) dependent ymphoma t(1;1)/t(1;2) C-10 mutations? ate Aggressive. pylori MA MA independent ymphoma ymphoma ransformed? MA ymphoma epatitis C iver cancer ymphoma (splenic y villous lymphocytes) V1 Adult -cell leukaemia Campylobacter ymphoma Epstein-arr virus odgkin's disease Intest. ymphoma Nasopharyngeal cancer Kaposi's sarcoma V8 Chlamydia Psitt Primary effusion lymphoma Papilloma virus Cervical cancer ymphoma Ocul. Adn. Gastric cancer elicobacter pylori Gastric lymphoma..catastrophes ususally depend upon a combination of errors. lood count, blood film and immunophenotype omogeneous phenotype: +, CD23 +, sigm low eterogeneous clinical course (years vs decades) lood count, blood film and immunophenotype omogeneous phenotype: +, CD23 +, sigm low eterogeneous clinical course (years vs decades) lood count, blood film and immunophenotype omogeneous phenotype: +, CD23 +, sigm low eterogeneous clinical course (years vs decades) Classic Prognostic factors Classic Prognostic factors iological Prognostic factors Clinical Stage (Rai/inet) Define disease Clinical Stage (Rai/inet) Define disease β2-microglobulin Correlate with one Marrow istology extension one Marrow istology extension Serum CD23 disease burden D D Serum hymidine Kinase Staging reatment strategy New biological Prognostic factors Stage A (0) Wait and watch IGV mutational status Predict prognosis Stage (I-II) reat if progression CD38 expression at diagnosis Stage C (III-IV) reat at onset ZAP-70 expression FIS 2

3 ZAP-70, CD38 and IGV mutations Zap-70 expression is a continuum IGV genes in IGV3-21 ~ 3% of all IGV genes used in M M M M UM UM Rassenti,. Z. et al. 200 Crespo et al, 2003 IGV1-9 ~ 10% of all IGV genes used in amblin,. Z. et al Rassenti,. Z. et al ymphocytes ( + )...a distinct cell lineage? In the fetus and newborn: predominant population In the adult: in the Mantle Zone (1% of circulating cells) Natural autoantibodies production Reconstitution Experiments in the mouse Absence of somatic mutations Conventional cells produce autoantibodies hey readily appear after M Few somatic mutations = Naive Why do cells interact with? CEMOKINE P N CC22/MDC - + CC17/ARC - +/- CC/Rantes + + CC3/Mip-1! + + CC20ARC - - CC19/EC - - CC21/SC - - CC1/I CC2/eck - - CXC8/I CXC10/IP CXC9Mig - - CXC12/SDF CXC12CA CXC13/CA CXC3C1/Fractalkine - - Activated + Cells Express CCR cells: short vs long term survival CD100 and Plexin-1 in Stroma ime (days) 3

4 Scenario A: ic stimulation is an activation marker Scenario A: antigenic stimulation (auto) anti-µ Mutated IG + cell Mutated I- + cell Unmutated I-1 Unmutated IG I-2 Scenario : no CR stimulation Caligaris-Cappio et al. lood 1989; Wortis et al, PNAS 199 Scenario : auto (super) antigenic stimulation Unrelated patients carry similar CDR3 () () Case P131 IA-D2 FRA-33 IA- N13 FRA-28 CDR3 AR DAN GMDV (K)CDR3 QVWDSGSDPWV IGV3-21 DIAGNOSIS 1) Absolute cell lymphocytosis >000/ul for > weeks 2) (>30% linfociti in MO) 3) Immunophenotype - ight chain (κ/λ) restriction Characteristic phenotype: +, CD23 +, low, sigm low IA-1 P1321 FRA M Q R , +, CD ow surface Ig FRA D ---- SPA R G P32 IA-3 FRA-09 -I -R A D G FRA Q- D--- IA P D-----Y- FRA-299 N A A--- QQYNNWPPE M-G--- G obin et al, lood 2002 Ghia et al, lood 200 allek et al, lood 2008 cell clonal expansions in the elderly rasforming events Microenvironment Interactions Sequential genetic abnormalities rasforming events Microenvironment Interactions Sequential genetic abnormalities Monoclonal Gammopathy of uncertain significance (MGUS) Step 1: ransformation Step 2: Accumulation Step 3: Autonomous Growth Step 1: ransformation Step 2: Accumulation Step 3: Autonomous Growth MGUS: 1%/year MM

5 Accumulation of lymphocytes: +, CD23 +, sigm low IGV N1 IGD N2 IGJ FRA-081 C A R E Q W V R S F D Y W P3073 C A R E Q W V R V N F D Y W P23 C A R A Q W V V N F D Y W FRA-178 C A R E Q W V R F D Y W P3129 C A R E Q W D A F D Y W SPA-91 C A R A Q W V P F D Y W FRA-270 C A R E Q W V F D Y W N3088 C A R E Q W V K E F F D Y W FRA-19 C A R Q W V R D Y F D Y W N2837 C A R V Q W V R E Y F D Y W FRA-190 C A R D Q W V D Y F D Y W P39 C A R W Q W V G Y F D Y W FRA-210 C A R E Q W A K P F D Y W P1173 C A R E Q W G I K N F D Y W FRA-22 C A R I Q W G P P S F D Y W FRA-293 C A R E Q W G P F D Y W FRA-290 C A R D Q W P N N F D Y W SPA- C A R D Q W P I N Y F D Y W N17 C A R K Q W P Q Y Y F D Y W SPA- C A R A Q W S I N Y Y F D Y W omogeneous phenotype: +, CD23 +, sigm low cells in the Proliferation Centres he proliferating compartment omogeneous phenotype: +, CD23 +, sigm low eterogeneous clinical course Survival Indolent course Aggressive course CD23 CC22 (CC17) Proliferation Survival: 12-1 years herapy: No or late need New biological Prognostic factors IGV mutational status CD38 expression ZAP-70 expression Genomic abnormalities Survival: 3- years herapy: Early and frequent Predict prognosis at diagnosis CD Medium only Medium ime (days) Granziero et al, 2001; Ghia et al, 2002; Granziero et al, 2003 Ki7 0 0 (I-) CDR3 analysis EAVY CAIN V D J C V J C IG CAIN IMMUNOOGY AND MAEMAICS ' 3' ' 3 ' 3 9- x 2 3 x 3-37 x 300 POENIA RECOMINAIONS POENIA RECOMINAIONS N-DIVERSIY SOMAIC MUAIONS X x - ambda ' 3 ' ' 3' Mathematical chances that two different cell clones might share the same CR ~.3 x 10 POSSIIIIES ~ 3. x 10 POSSIIIIES 2 x DIFFEREN ANIODIES 1:10-12 FR1 FR2 FR3 IGV CDR1 CDR2 IGD CDR3 IGJ 0, %

6 1, 1, 1,2 1,0 0,8 0, 0, 0,2 0,0 ZAP70 is differentially expressed in cells ZAP70 correlates with clinical course Cell Receptor (CR) β α ε δ γ ε Rassenti,. Z. et al. 200 Crespo et al, 2003 SP-7 Itk/Rlk Zap-70 ζ ζ ck Rosenwald et al 2001 Wiestner et al, 2003 Rassenti,. Z. et al. 200 PC-γ ZAP70 is expressed in normal cells ZAP70: recent/persistent activation? Scenario A: initial CR stimulation CD3+ Memory Naive GC ZAP-70 β actin ZAP-70/β -ac tin (OD ratio) CD3+ Memory Naive GC CD1+ CD1+ CD3+ ZAP-70 β actin activation Mantle zone ight zone Dark zone cell Good prognosis ad prognosis P Cells Scelzo et al, eukemia 200 ZAP-70 expression Scenario : ongoing CR stimulation Aberrant immune response? Cognitive Activation/proliferation Resting omogeneous phenotype: +, CD23 +, sigm low omogeneous phenotype: +, CD23 +, sigm low eterogeneous clinical course ic elements Indolent course Aggressive course Survival: 12-1 years Survival: 3- years herapy: No or late need herapy: Early and frequent

7 Chromosomal abnormalities in cells in the Proliferation Centres he proliferating compartment Survival CD23? CD3 mir-1a mir-1-1 p3 100 risomy 12: 1% 90 13q deletion: 3% Medium only Medium Ki AM? Proliferation CC22 (CC17) ime (days) 0 11q deletion: 18% 17p deletion: 7% Dohner et al (I-) Granziero e t al, 2001; Ghia et al, 2002; Granziero et al, 2003 Ghia et al, Sem Cancer iol 2002 Aberrant immune response? Cognitive Activation/proliferation Resting ic elements 7