PARALLELISMO TRA PROFILO PARALLELISMO TRA PROFILO GENETICO-MOLECOLARE ED GENETICO-MOLECOLARE ED

ETEROGENEITA’ FENOTIPICA, CLINICA ETEROGENEITA’ FENOTIPICA, CLINICA E PROGNOSTICA DELLA LLCE PROGNOSTICA DELLA LLC

Robin FoàRobin FoàEmatologiaEmatologia

Università “Università “La SapienzaLa Sapienza”, Roma”, Roma

Rieti, 27 Ottobre 2006Rieti, 27 Ottobre 2006

CHRONIC LYMPHOCYTIC LEUKEMIA

• Most frequent leukemia in Western countries (30% of all leukemias vs 2-5% in Asian countries).

• Median age at diagnosis: c65 yrs • M/F ratio 1.5:1• Familial cases (4-5% first-degree relatives?)• Diagnosis usually made in asymptomatic individuals• Lymphadenopathy, splenomegaly, infections• Immune disturbances hypogammaglobulinemia

(20 - 40%), AIHA (10 - 20%), T and NK cell defects

CLL: WHY RELATIVE INTERESTCLL: WHY RELATIVE INTERESTIN THE PAST YEARS?IN THE PAST YEARS?

• A disease of the ‘elderly’; median age c65 yrs A disease of the ‘elderly’; median age c65 yrs

• A disease with a chronic, often indolent, A disease with a chronic, often indolent, clinical courseclinical course

• No grips on the heterogeneous clinical No grips on the heterogeneous clinical coursecourse

• Limited therapeutic options, namely Limited therapeutic options, namely chlorambucilchlorambucil

• A conservative, often A conservative, often ‘wait and watch’‘wait and watch’ management has been the treatment of choicemanagement has been the treatment of choice

CHANGE IN ATTITUDE TOWARDS CLLCHANGE IN ATTITUDE TOWARDS CLL Increased prevalence due to extended life expectancyIncreased prevalence due to extended life expectancy

Biologic age Biologic age who is old nowadays??who is old nowadays??

Increasingly diagnosed in younger individuals Increasingly diagnosed in younger individuals c20% of patients under 55 yrsc20% of patients under 55 yrs

Increased diagnosis due to broader use of routine blood tests

Identification of biologic features of prognostic relevanceIdentification of biologic features of prognostic relevance

New therapeutic options, including auto and New therapeutic options, including auto and allotransplantation procedures, MoAb and new drugsallotransplantation procedures, MoAb and new drugs

Concept of living (and working) with leukemiaConcept of living (and working) with leukemia quality of life and overall expectationsquality of life and overall expectations

CLL - EXTENDED BIOLOGIC CHARACTERIZATION

• For a better understanding of theFor a better understanding of the pathophysiology pathophysiology of the diseaseof the disease

• For a more accurate For a more accurate diagnostic definitiondiagnostic definition• For a biologically-based For a biologically-based prognosticprognostic stratificationstratification

of patientsof patients• For the dFor the definition of markers for MRD monitoring• For an optimal For an optimal therapeutictherapeutic (or non-therapeutic) (or non-therapeutic)

algorythmalgorythm• ? For the design of ? For the design of innovative therapeutic innovative therapeutic

strategiesstrategies

Approaches Utilized for a Modern Characterization of CLL

• Morphology dd, typical vs atypical CLL• Immunophenotype dd, typical vs atypical CLL,

CD38 expression, degree of antigen expression• Cytogenetics for prognostic stratification• Mutated or unmutated IgVH profile for

prognostic stratification• ZAP-70 expression for prognostic stratification• Further biologic properties of leukemic cells, cell-to-

cell interplay and cytokine network(s)• Host immune status T cells, NK cells, DC, etc• Gene profiling

CLLCLL PLL HCL SLVL MCL FL PLL HCL SLVL MCL FL

sssIIIggg +++++++++ ++++++ ++++++ ++++++ ++++++

CCCDDD555 +++ +++

CCCDDD222333 +++ ///+++ ///+++

FFFMMMCCC777 +++ +++ +++ +++ +++

CCCDDD222222 ///+++ +++ +++ +++ +++ +++

CCCDDD111000 (((+++))) +++

CCCDDD777999bbb +++ ///+++ +++ +++ +++

PHENOTYPE OF B-CELL CHRONIC PHENOTYPE OF B-CELL CHRONIC LYMPHOPROLIFERATIVE DISORDERSLYMPHOPROLIFERATIVE DISORDERS

(TdT-/CD19/20+)(TdT-/CD19/20+)

CLLCLL

SIgSIg++

CD5+CD5+

CD22-/+CD22-/+CD20CD20++

CD23++CD23++

FMC-7-/+FMC-7-/+

CD19+

SIg++SIg++

CD5+CD5+

FMC7+FMC7+

CD22++CD22++

CD20++CD20++

CD23-/+ CD23-/+ MCLMCL

"ATYPICAL" CLL"ATYPICAL" CLL• On morphological grounds: On morphological grounds: cases with a cases with a

typical immunophenotypic profile, but with typical immunophenotypic profile, but with more than 10% of large lymphocytes and/or more than 10% of large lymphocytes and/or prolymphocytes and/or cells with nuclear cleftsprolymphocytes and/or cells with nuclear clefts

• On immunophenotypic grounds: On immunophenotypic grounds: cases with cases with a typical morphology, but with the expression a typical morphology, but with the expression of FMC7 and/or of bright sIgof FMC7 and/or of bright sIg

‘‘Atypical’ CLL account for c20% of Atypical’ CLL account for c20% of cases, have a more advanced disease and a cases, have a more advanced disease and a

worse prognosisworse prognosis

FREQUENCY OF CHROMOSOME LESIONS IN CLLFREQUENCY OF CHROMOSOME LESIONS IN CLL(comparison of CCA and FISH)(comparison of CCA and FISH)

Aberration Cytogenetics (*) FISH (**)

Clonal 30-50% 82%

Normal 50-70% 18%

del 13q14 single 7-11% 36-45%

12q trisomy 13-19% 11-14%

del 11q22-23/ATM 6-13% 8-25%

del 17p/p53 1-5% 3-7%

del 6q21 1% 2%

(*) % total abnormal(**) Dohner et al, 2001 EHA

Survival and specific chromosome aberrations Survival and specific chromosome aberrations Dohner et al, NEJM (2000)Dohner et al, NEJM (2000)

SURVIVAL BASED on IgV MUTATION STATUS and SURVIVAL BASED on IgV MUTATION STATUS and CD38 EXPRESSION among B-CLL of RAI CD38 EXPRESSION among B-CLL of RAI

INTERMEDIATE RISKINTERMEDIATE RISK

Damle et al. Blood 1999;94:1840

B-CLL UTILIZING VB-CLL UTILIZING VHH3-21 REPRESENT 3-21 REPRESENT

an UNFAVORABLE PROGNOSTIC SUBSET an UNFAVORABLE PROGNOSTIC SUBSET

of B-CLL with MUTATED IgVof B-CLL with MUTATED IgVHH GENES GENES

Blood 99:2262, 2002

““SUPERSTABLE” CLL PATIENTSSUPERSTABLE” CLL PATIENTS

Guarini et al, Blood 102, 1035, 2003

IgVH, CD38, p53 & CD4/CD8 IN ‘IgVH, CD38, p53 & CD4/CD8 IN ‘SUPERSTABLESUPERSTABLE’ CLL’ CLLCaseCase % Mutated IgV% Mutated IgV % CD38% CD38 p53 CD4/CD8 ratiop53 CD4/CD8 ratio 11 4.7 4.7 1 1 wt wt n.d.n.d. 22 7.6 7.6 3 3 wt wt 2 2 33 3.3 3.3 48 48 wt wt 1.2 1.2 44 5.0 5.0 1 1 wt wt 2.182.18 55 3.6 3.6 2 2 wt wt 1 1 66 7.0 7.0 1 1 wt wt 4 4 77 11.4 11.4 1 1 wt wt 2.5 2.5 88 6.0 6.0 1 1 wt wt 3 3 99 6.1 6.1 1 1 wt wt 2 2 1010 3.6 3.6 1 1 wt wt 1.661.66 1111 12.7 12.7 1 1 wt wt 2.282.28 1212 7.5 7.5 1 1 wt wt 2.2 2.2 1313 8.4 8.4 1 1 wt wt 2 2 1414 1.3 1.3 1 1 wt wt 2 2 1515 10.7 10.7 1 1 wt wt 1 1 1616 7.5 7.5 9 9 wt wt 2.6 2.6 1717 3.8 3.8 4 4 wt wt 1 1 1818 4.0 4.0 6 6 wt wt 2.5 2.5 1919 4.7 4.7 2 2 wt wt 4.5 4.5 2020 10.8 10.8 1 1 wt wt 3.663.66

Guarini et al, Blood 2003

Plasma cells

Germinal Center and Lymphomagenesis

Naive B-cell

GC cells

Memory B-cells

BCL-6

Syndecan

CLL MCL FL BL

Apoptosis

IgV mutationsDLCL MM

- ++

IgV hypermutationIg isotype switch

CLL

-

MZ

Schroeder & Dighiero. Immunol Today, 1994Hamle et al. Blood, 2000Hamblin et al. Blood, 2000

OTHER DLBCL CLL

NON MUTATED

MUTATED HIGH & LOW

Rosenwald et al (J Exp Med 2001)

NON MUTATED

MUTATED HIGH & LOW

Klein et al (J Exp Med 2001)

CLL is a unique disease with two different features:

Rosenwald et al (J Exp Med 2001)

<20% ZAP70

20% ZAP70

Crespo et al., NEJM 2003

Survival probability according to ZAP70 expression (A stage)

ZAP70 expression and IgVH mutational status

Diagnosis• CLL is a heterogeneous disease, not only

from the clinical standpoint– Morphology– Immunophenotype– IgV mutated vs IgV unmutated– Zap-70+ vs Zap-70-– Cytogenetic/genetic -clinical correlates– Gene profile

How many diseases?

CHRONIC LYMPHOCYTIC LEUKEMIACHRONIC LYMPHOCYTIC LEUKEMIA PASTPAST

WAIT & SEEWAIT & SEE

CHLORAMBUCIL CHLORAMBUCIL

CONSERVATIVE CONSERVATIVE APPROACHAPPROACH

PRESENTPRESENT

* Fludurabine CR

* Combined chemotherapy (eg Fluda+Cyclo)

* MoAb (Rituximab, Campath)

* Chemo + MoAb (FCR, FluCam)

* Other purine analogs (2-CdA, DcF)

* Transplantation programs (auto, allo, ric allo)

* Allografting/minitransplant ± DLI

* New drugs and compounds

HEMATOPOIETIC TOXICITY ASSOCIATED WITH IMMUNE DEPRESSION (DEFICIT T, NK, DC)

A Few Concepts to be Considered for the Optimal Management of CLL Patients

• Age-dependent treatment (taking into account biologic age)

• Early and aggressive treatment may be curative

• Change in definition of remission

• High remission rate after first-line therapy associated with longer PFS

• Choice of first-line treatment is very important

MODERN APPROACH TO THE MODERN APPROACH TO THE MANAGEMENT OF CLLMANAGEMENT OF CLL

• An integrated biologic An integrated biologic work-up at diagnosiswork-up at diagnosis

• A biologically-based A biologically-based prognostic stratificationprognostic stratification and and possible design of a new possible design of a new scoring systemscoring system

• A biologically-based A biologically-based therapeutic (and non-therapeutic (and non-therapeutic) algorythmtherapeutic) algorythm

• Change in “therapeutic” approach for “younger” Change in “therapeutic” approach for “younger” patients with poor prognostic factors?patients with poor prognostic factors?

• Early and aggressive treatment?Early and aggressive treatment?

• Aim at Aim at disease eradication disease eradication (MRD monitoring) ??(MRD monitoring) ??

CD38 EXPRESSION FREQUENCY IN B-CD38 EXPRESSION FREQUENCY IN B-

CELL CHRONIC LYMPHOPROLIFERATIVE CELL CHRONIC LYMPHOPROLIFERATIVE

DISORDERS DISORDERS AT DIAGNOSISAT DIAGNOSIS

PATIENTSPATIENTS CD38+>10%CD38+>10% CD38+ >20%CD38+ >20%

CLL (N° 110)CLL (N° 110) 19 (17.2%)19 (17.2%) 15 (13.6%)15 (13.6%)

CD5+ NHL (N° 46)CD5+ NHL (N° 46) 25 (54%)25 (54%) 19 (41.3%)19 (41.3%)

NHL (CD5-) (N° 51)NHL (CD5-) (N° 51) 8 (15.6%) 8 (15.6%) 6 (11.7%) 6 (11.7%)

Treatment-Free Survival CD38+ pts: cut-off=20%

p<.0001

CD38 neg

CD38 pos

Gentile et al, BJH 2005

p<.0001

Treatment-Free Survival in Rai Stage 0 Patients According to CD38 Positivity (cut-off=20%)

CD38 neg

CD38 pos

Gentile et al, BJH 2005

Change over Time of Remission Criteria

– Moving away from palliative treatmentclearance of blood major marker

– Moving towards a curative approach

clearance of bone marrow– No longer on a morphologic definition of

remission, but the use of immunophenotypic and genetic monitoring of minimal residual disease

High risk17p13 deletions or p53 mutations, 11q22-q23

deletions and unmutated IgVH

unmutated IgVH ± 12q trisomy ± ZAP-70 ± CD38+

Standard risk

BIOLOGICAL WORK-UP FOR ‘YOUNGER’ CLL

Correct diagnostic definition & dd(ndr, should always take place!)

Phase II pilot study to evaluate a therapeutic strategy diversified accoring

to the biologic profile of patients with CLL with advanced stage and/or

progressive disease aged <60 years 

LLC0405 GIMEMA Protocol

“YOUNG” CLL PATIENTS WITH PROGRESSIVE DISEASE

Fludarabine + Campath-1H

NR

agedonor

Auto, AlloTransplantation orFurther Campath

MCR

PBSC

W & W

CR PR

HIGH RISK

OffStudy

agedonor

“YOUNG” CLL PATIENTS WITH PROGRESSIVE DISEASE

Fludarabine + Cyclophosphamide x 4

Standard risk

Campath

CR-CIR

W & W

PBSC

W & W

NR MCR

NR CR CIR PR

W & W

PR

Off study

FC x 2

PROBLEMATICHE

• Ottimale inquadramento diagnostico e diagnosi differenziale

• Stratificazione prognostica alla diagnosi su base biologica almeno fino a 65 anni

problema di numerosità, di tecnologie e competenze, di riproducibilità, di costi

• Possibilità di monitorizzare la MRM (immunologica e molecolare)

clinicalclinicalimmunedeficiencyimmunedeficiency

cellular and humoralcellular and humoralimmune impairmentimmune impairment

Inhibition of Inhibition of CD40L expressionCD40L expression

Stimulation ofStimulation ofIg productionIg production

T B

CLL

CLL

ImpairedImpairedinteractionsinteractions

with B and APCwith B and APC

Immune defects

Tumor cell activation,Tumor cell activation,proliferation and inhibitionproliferation and inhibition

of apoptosisof apoptosis

B-CLL cell accumulation

InappropriateInappropriateproduction ofproduction of

autoreactive antibodiesautoreactive antibodies

Autoimmune phenomena

IL-4IL-4

IL-4IL-4

IL-4IL-4

IL-4IL-4

IgIg

CD40CD40

CD40LCD40L

membrane and soluble membrane and soluble mediator interactionsmediator interactions

genomicgenomiccharacterization, characterization,

new drugsnew drugs

Changes in approach to CLL – From patients to genes

Last decades’ evolution in the understanding of CLL

BNKT

1970s 1970s →→ 80s 80s 1980s 1980s →→ 90s 90s

2000 2000 →→

1995 00s1995 00s NEW THERAPIES

BIOLOGICALLY-BASED PROGNOSTIC STRATIFICATION

AcknowledgmentsAcknowledgments::

A. Guarini, S. De Propris, S. Chiaretti, I. Dalla Starza, A. Guarini, S. De Propris, S. Chiaretti, I. Dalla Starza,

E. Ghia, F. Mancini, R. Maggio, M. Mancini, I. Del E. Ghia, F. Mancini, R. Maggio, M. Mancini, I. Del

Giudice, F.R. Mauro – Giudice, F.R. Mauro – RomeRome

G. Castoldi, A. Cuneo – G. Castoldi, A. Cuneo – FerraraFerrara

G. Gaidano, D. Capello – G. Gaidano, D. Capello – NovaraNovara

J. Ritz – J. Ritz – BostonBoston

GIMEMA networkGIMEMA network