Lymphomes diffus à grandes cellules B

Classification, facteurs pronostiques

Thierry Jo Molina

Hôpital Universitaire Necker Enfants Malades

Cours national de DES Janvier 2016

Diffuse large B cell lymphomas

Definition

Lymphomas defined by a neoplasm of large lymphoid

B-cells with nuclear size equal to or exceeding normal

macrophage nuclei or more than twice the size of a

normal lymphocyte

That has a diffuse growth pattern

Morphological, biological and clinical studies have subdivided DLBCL into • Morphological variants • Molecular and immunophenotypical

subgroups • Subtypes • Distinct disease entities

WHO 2008 Classification of DLBCL

Diffuse large B-cell lymphoma subtypes

T-cell rich large B-cell lymphoma

Primary DLBCL of the CNS (EBV)

Primary cutaneous DLBCL, leg type

EBV positive DLBCL of the elderly

Other lymphomas of large B-cells

Primary mediastinal (thymic) large B-cell lymphoma

Intravascular large B-cell lymphoma

DLBCL associated with chronic inflammation (EBV)

Lymphomatoid granulomatosis (EBV)

ALK positive DLBCL (ALK)

Plasmablastic lymphoma (EBV)

Large B-cell lymphoma arising in HHV-8 associated

multicentric Castleman disease (HHV8)

Primary Effusion lymphoma (HHV8, EBV)

Diffuse Large B-cell lymphomas, not otherwise specified (NOS)

Common morphological variants

Centroblastic

Immunoblastic

Anaplastic

Rare morphological variants

Molecular subgroups

Germinal centre B-cell like (GC-B)

Activated B-cell like (ABC)

Immunohistochemical subgroups

CD5+ DLBCL

Germinal Centre B-cell like (GCB)

Non Germinal centre B-cell like (non-GCB)

Importance of assessing if this lymphoma arises de

novo or is developing from

B-CLL (Richter Syndrome )

Follicular lymphoma

Marginal zone lymphoma

NLPHL (Nodular Lymphocyte

Predominance Hodgkin lymphoma)

Crucial role of the size of the sample +++++

Borderline cases

B-cell lymphoma, unclassifiable, with features

intermediate between DLBCL and Burkitt lymphoma

B-cell lymphoma, unclassifiable, with features

intermediate between DLBCL and classical

Hodgkin lymphoma

Cb monomorphe

Cb multilobé

Immunoblastique

Immunophénotype

• Les cellules tumorales expriment :

– CD20, CD79a, mais peuvent perdre certains

marqueurs

– immunoglobuline intracytoplasmique quand il y a une

différenciation plasmocytaire

– CD30 dans les variantes anaplasiques et dans quelques

formes non anaplasiques

– CD10 (25-50%), CD5 (10%)

– Ki67; habituellement plus de 40%

LYMPHOME B RICHE EN LYMPHOCYTES T et / ou EN HISTIOCYTES

- Histopathologie

• Infiltration diffuse

• Grandes cellules B tumorales

petit nombre (< 10% cellules)

dispersées, en petits nids, Cb,

Ibl, anaplasiques,

rares cellules RS-like

• Cellules réactionnelles

nombreuses

lymphocytes T

histiocytes

- Immunophénotype • Cellules tumorales

CD45+

Pan B+

EMA+/-

CD15-, CD30-, LMP1-

• Cellules réactionnelles

Antigènes T associés

Peu de cellules CD57 +

• Pas de réseau de CFD

- diagnostic différentiel • Maladie de Hodgkin à cellularité mixte

• Paragranulome nodulaire / diffus

• LM T CD20

LYMPHOME B RICHE EN LYMPHOCYTES T et / ou EN HISTIOCYTES

Specific Clinical Characteristics of Patients With T-Cell/Histiocyte-

Rich Large B-Cell Lymphoma Bouabdallah, J Clin Oncol, 2003

TCRBCL DLBCL p

(n = 50) (n = 150)

Sex, male/female, n 44/6 82/68 < .0001

Cervical 34% 33% .90

Axillary 52 24 .0002

Mediastinal 30 46 .02

Lumboaortic 54 38 .02

Mesenteric 32 24 .30

Inguinal 30 21 .20

Pelvis 38 16 .002

Spleen 60 17 < .0001

Bone marrow 31 26 .50

Liver 33 11 .001

Gastrointestinal tract 0 10 .005

Bone 4 12 .01

LM A GRANDES CELLULES B PRIMITIF DU MEDIASTIN ( THYMIQUE )

- Clinique

• Adulte jeune, F > H

• Tumeur médiastinale antérieure

- Histopathologie

• LM diffus

• Grandes cellules +/- cytoplasme clair

•Cbl, Ibl, parfois de type RS

• Sclérose

• Lymphocytes réactionnels ,histiocytes,

plasmocytes, éosinophiles

-Cellule d’origine

Cellule B intrathymique

Immunophénotype et génotype

– Pas d ’expression de CD5

– Expression de CD30 (partielle) et CD23

– Expression rare de CD10 et bcl-6 (de Leval, 2001)

– Pas de réarrangement de bcl-2 ou bcl-6

– Surexpression de MAL.

LM B PRIMITIF DES SEREUSES

- Définition

• Lymphome se développant

essentiellement dans les

cavités des séreuses: pleurale

péricardique

abdominale

• En l’absence habituelle de masse

tumorale

• extension 2d d’un LM à grandes

cellules B

- Clinique

•Primitif

HIV+, EBV+, HHV8+

Immunophénotype

• Absence d ’expression de CD19, CD20, CD79a,

cIg

• expression de CD45

• expression aberrante de CD3

• protéine latente HHV8, EBER1+, LMP1-

LM A GRANDES CELLULES B INTRAVASCULAIRE

- Clinique Adulte

Lésions cutanées

Symptomes neurologiques

Hépatosplénomégalie

Pancytopénie, CIVD

- Histopathologie • Grandes cellules (Cb, Ib, anaplasiques)

• Dans les petits vaisseaux

sinus (moelle osseuse, rate)

sinusoïdes (foie)

capillaires (peau, cerveau, poumon)

• Histiocytes avec Erythrophagocytose

- Immunophénotype Pan B+

CD20

Lymphome B de type granulomatose

lymphomatoïde

– Lésion lymphoproliférative angiocentrique et

angiodestructrice, extranodale, comportant des

cellules lymphoïdes B de grande taille

associées à l ’EBV et des lymphocytes T le plus

souvent nombreux

» grade histologique (I, II, III) selon la

proportion de grandes cellulesB.

» Le Grade III est considéré comme une

variante de lymphome diffus à grandes

cellules B

– Evolution en 2 à 5 ans du grade I au grade III.

L ’Alpha-interferon pourrait contrôler les

grades I et II.

– Masses pulmonaires nécrotiques

• Nombreux lymphocytes T réactionnels ,

histiocytes et polynucléaires neutrophiles

• Vasculite lymphocytaire et nécrose fibrinoïde

• grandes cellules lymphoïdes B EBV+ de

morphologie centroblastique ou immunoblastique

– dispersées ou de topographie périvasculaires, infiltrant

la paroi des vaisseaux, réalisant un aspect

angiocentrique

– Autres localisations : cerveau (26%), rein

(32%), foie (29%),peau (25-50%)

CD20

LMP1

MIB1 EBER

Borderline cases

B-cell lymphoma, unclassifiable, with features

intermediate between DLBCL and Burkitt lymphoma

B-cell lymphoma, unclassifiable, with features

intermediate between DLBCL (PMBL) and classical

Hodgkin lymphoma

Intermediate Features are

Exceptional !!

Most often PMBL or CHL

Or other diagnosis…..

In big centers specialised in

Hematopathology including

Local cases and consult cases

Max : 1-2 cases per year.

Renseignements cliniques

• Homme de 85 ans

• Polyadénopathies

• Altération de l’état général

• Biopsie d’un ganglion cervical droit (ganglion de

2.5x1.5x1.5 cm)

Lymphome diffus à grandes cellules B de sous type

immunohistochimique « centre germinatif »?

Algorithme

d’immunohistochimie

Dangereux!

Ne jamais oublier

CD5

CD5

Cycline D1

FISH : DR Isabelle Radford Weiss, Necker

Probable rearrangement dans les regions 3’ de la cycline D1

Diagnostic proposé (OMS 2008)

• Lymphome à cellules du manteau

– Variante agressive

• Sous- type pléomorphe

Proposition d’algorithme pour les

lymphomes diffus à grandes cellules B NOS

• CD20

• CD5

• Mib1

• CD10, BCL6, MUM1, (BCL2, MYC) – Si CD5 +

• Cycline D1 (clone SP4)+++++ – Si positif , manteau variante agressive

– Si négatif ou positivité minoritaire, DLBCL

– Si positif hétérogène mais importante , FISH Cycline D1.

– Il existe des rares cas de lymphome du manteau CD5 négatifs.

Réarrangement de gènes et DLBCL

• t(14;18) BCL2/IgH dans 15-20% des cas

• Réarrangement de bcl-6 dans 25-30% des cas

• Réarrangement de C-MYC 5-10%

DLBCL, Molecular subgroups

• GC like DLBCL

• Activated like DLBCL

Alizadeh et al, 2000

Cell of Origin

signature

Lenz , NEJM, 2008

Molecular subgroups defined by GEP or QRTPCR impacts on survival

Indepedently from IPI among R-CHOP treated DLBCL patients

Lenz, New Engl J Med, 2008, 359, 2313-23

DLBCL NOS

• Immunohistochemical subgroups

– Germinal center B-cell (GC-B)

– Non germinal center B-cell (n-GCB)

C Hans et al, Blood 2004

Program DLBCL IHC

Submitted, 2015

• 03-1B, 2B, 3B, 39B, 6B, 7B

• CD10, BCL6 MUM1, MYC (40%), BCL2 (50%,

70%) , MYC/BCL2

• TMA : 670 patients

– 237 RACVBP, 433 R-CHOP/RMiniCHOP

Double Expressor DLBCL:

MYC/ BCL2 IHC in DLBCL is a

major prognostic factor in DLBCL

• Oct 2012, JCO

– MYC antibody potential surrogate to FISH

– Observed in around 21% of DLBCL

– Negative impact on prognosis after adjusting for

high risk features in multivariable model including

elevated IPI

• Editorial M Preundschuch,

• NA Johnson et al,

• TM Green et al

Johnson, 2012

TM Green ,

2012

TM Green , 2012

Program DLBCL IHC

ASH 2014

• 03-1B, 2B, 3B, 39B, 6B, 7B

• CD10, BCL6 MUM1, MYC (40%), BCL2 (50%,

70%) , MYC/BCL2

• TMA : 670 patients

– 237 RACVBP, 433 R-CHOP/RMiniCHOP

MYC

• 577 cases

– 40% cutoff : positive in 29.5%

– 70% cutoff : positive in 14.2%

– No preferential expression among GC or nGC

– Worse PFS if MYC positive (40% threshold) (p<0.01) and OS (p<0.01)

MYC+ BCL2+

• Threshold BCL2 50% : 557 patients (125

MYC+BCL2+). (60% NGC, p= 0.1)

• Theshold BCL2 70% : 557 patients (116

MYC+BCL2+); (61.2% NGC, p=0.06)

MYC+ BCL2+ 70

Multivariate Analysis

OS

PFS

Parameter Modality tested Hazard Ratio 95% Lower Confidence Limit for Hazard Ratio

95% Upper Confidence Limit for Hazard Ratio

Pr > ChiSq

Double positive Myc (>=40%) & Bcl2(>=70%)

Yes 1.228 0.843 1.789 0.2843

IPI in class 2 2.148 1.105 4.172 0.0241

3 4.295 2.385 7.734 <.0001

4-5 7.454 4.185 13.279 <.0001

Hans score n-GC 1.552 1.081 2.228 0.0172

Parameter Modality tested Hazard Ratio 95% Lower Confidence Limit for Hazard Ratio

95% Upper Confidence Limit for Hazard Ratio

Pr > ChiSq

Double positive Myc (>=40%) & Bcl2(>=70%)

Yes 1.261 0.901 1.767 0.1768

IPI in class 2 1.955 1.179 3.244 0.0094 3 3.035 1.904 4.837 <.0001 4-5 4.825 3.051 7.630 <.0001

Hans score n-GC 1.591 1.160 2.181 0.0040

• No preferential GC/nGC expression

BCL2≥70%

Multivariate Analysis

OS

PFS

Parameter Modality tested Hazard Ratio 95% Lower Confidence Limit for Hazard Ratio

95% Upper Confidence Limit for Hazard Ratio

Pr > ChiSq

Bcl2 positivity (>=70%)

Yes 1.477 1.053 2.072 0.0240

IPI in class 2 2.230 1.214 4.096 0.0097

3 3.744 2.141 6.545 <.0001

4-5 6.744 3.928 11.579 <.0001

Hans score n-GC 1.638 1.163 2.307 0.0047

Parameter Modality tested Hazard Ratio 95% Lower Confidence Limit for Hazard Ratio

95% Upper Confidence Limit for Hazard Ratio

Pr > ChiSq

Bcl2 positivity (>=70%)

Yes 1.477 1.053 2.072 0.0240

IPI in class 2 2.230 1.214 4.096 0.0097

3 3.744 2.141 6.545 <.0001

4-5 6.744 3.928 11.579 <.0001

Hans score n-GC 1.638 1.163 2.307 0.0047

Multivariate Analysis R-CHOP

OS

PFS

Parameter Modality tested Hazard Ratio 95% Lower Confidence Limit for Hazard Ratio

95% Upper Confidence Limit for Hazard Ratio

Pr > ChiSq

Bcl2 positivity (>=70%)

Yes 1.477 1.053 2.072 0.0240

IPI in class 2 2.230 1.214 4.096 0.0097

3 3.744 2.141 6.545 <.0001

4-5 6.744 3.928 11.579 <.0001

Hans score n-GC 1.638 1.163 2.307 0.0047

Parameter Modality tested Hazard Ratio 95% Lower Confidence Limit for Hazard Ratio

95% Upper Confidence Limit for Hazard Ratio

Pr > ChiSq

Bcl2 positivity (>=70%)

Yes 1.477 1.053 2.072 0.0240

IPI in class 2 2.230 1.214 4.096 0.0097

3 3.744 2.141 6.545 <.0001

4-5 6.744 3.928 11.579 <.0001

Hans score n-GC 1.638 1.163 2.307 0.0047

Explanations ?

• Patients included in trials/from pathological files

– Not the same type of patients, DLBCL, DHL?

– Follow up and clinical data are more relevant in

clinical trials.

• TM Green Study

Explanations ?

-N Johnson, Oct 2012

– Multivariate analysis A2

• MYC/BCL2 T/V : 0, 036 /0, 048 (OS); 0.13/0.067 (PFS)

• IPI : 0, 001/0.013 (0S); 0.001/0.003 (PFS)

• ABC subtype : 0.017/0.67 (OS); 0.001/0.62 (PFS)

Borderline cases

B-cell lymphoma, unclassifiable, with features

intermediate between DLBCL and Burkitt lymphoma

B-cell lymphoma, unclassifiable, with features

intermediate between DLBCL (PMBL) and classical

Hodgkin lymphoma

Borderline cases, Provisional entity WHO 2008

B-cell lymphoma, unclassifiable, with features

intermediate between DLBCL and Burkitt lymphoma

highly aggressive clinical behavior

complex caryotypes

features overlapping between DLBCL and BL

BURKITT LYMPHOMA

CD10

Mib-1

MIB1 BCL2

Diffuse Large B-cell Lymphoma or Intermediate BL/DLBCL?

Recommandation WHO

• Look for myc rearrangement

• Look for double hit lymphoma ((bcl2/myc,

bcl6/myc, bcl2/bcl6/myc).

• Double hit particularly myc/bcl2 have a worse

prognosis compared to paired DLBCL

– May qualify for intermediate features between BL and

DLBCL

Controversy

• Intermediate Molecular Burkitt have been defined

by GEP and correlated with genome complexity.

(Hummel, NEJM, 2006)

• Not by atypical morphology and /or phenotype as

in WHO

– might increase according to the WHO numbers of

intermediate features

– Therefore, lack of consensus criteria to define those

cases that might benefit from alternative therapy than

classical DLBCL.

Proposal for Adult Intermediate Cases Salaverria I, JCO, 2011

• MYC-negative mature aggressive B-cell lymphomas with typical Burkitt

morphology and/or phenotype

• MYC-positive mature aggressive B-cell lymphomas lacking typical Burkitt

morphology and/or phenotype

– (a) IG-MYC–positive mature B-cell aggressive lymphomas with simple

karyotype

– (b) IG-MYC–positive mature aggressive B-cell lymphomas with complex

karyotype (limited set of FISH probes?)

– (c) Non-IG-MYC–positive mature aggressive B-cell lymphomas

– (d) Double-hit–positive mature aggressive B-cell lymphomas (subgroup of

b and c)

• MYC-negative aggressive B-cell lymphomas with features intermediate between

BL and DLBCL

• Then evaluation for clinical presentation and evolution in large intergroup

series.

FISH GHEDI Study : PI C. Copie Bergman,2015 Blood

• Patients 776 DLBCL patients enrolled onto LNH-03 GELA trials age 18 to 95 years, de novo, previously untreated excluded: previous history of indolent lymphoma R- chemotherapy (R-CHOP =483/R-ACVBP=293) 574 MYC evaluable • Centrally reviewed DLBCL cases • FISH analysis TMA blocks prepared from FFPE DLBCL cases BCL2, BCL6, MYC, IGK, IGL split-signal FISH DNA probes (DAKO),

IGH/MYC/CEP8 tri-color DF FISH probe (Vysis),IgK/MYC, IgL/MYC

• Immunohistochemistry - CMYC (clone Y69) - CD10, BCL6, MUM1

HE CD20

BCL2 IGH/MYC/CEP8

RESULTS (2) Prognostic Impact of BCL2-R , BCL6-R and BCL2/BCL6-R

BCL2-R n= 82/515 (15,9%)

BCL6-R n=129/541 (23,8%)

No significant impact of BCL2-R, BCL6-R or BCL2/BCL6-R on EFS, PFS and OS.

BCL2/BCL6 n= 15/580 (3%)

RESULTS Prognostic Impact of MYC-R n=51

.0058

74%GCB

26%non GCB

RESULTS Prognostic Impact of MYC-R SH n=19

.0339

RESULTS Prognostic Impact of MYC-R DH n=32

.0457

IMPORTANCE OF MYC PARTNER GENE

P <0.001

.0002

IMPORTANCE OF MYC PARTNER GENE

P <0.001

.0175

IMPORTANCE OF MYC PARTNER GENE

P <0.001

.0023

• MYC-IG DLBCL, but not MYC-non-IG DLBCL, had a significant shorter OS as compared to MYC translocation negative DLBCL.

• This adverse prognostic effect of MYC-IG on OS was maintained in MYC-SH and MYC-DH

• Multivariate analysis showed that, in addition to IPI, MYC-IG predicted a poor OS and PFS in DLBCL, independently from COO.

• 92% of the MYC-R are DLBCL-NOS and only 8% may qualify for BCLu

IMPORTANCE OF MYC PARTNER GENE

Prognostic factors varied

according to therapy

Interest of ABC Signature (Dunleavy K, Blood, 2009)

Bortezomib + Chemotherapy induces a better OS among ABC DLBCL

Suggesting the importance of inhibiting NF-kB pathway in such DLBCL

LNH 03-2B

Recher C et al, Lancet, 2011.

• 18-65 ans

• IPI 1 factor

• RACVBP induces a significant better OS than

RCHOP

– Aim : Comparing GCB/non GCB algorithm between

RCHOP and RACVBP.

Clinical study

PFS OS

GC DLBCL

PFS OS

Non-GCB DLBCL

PFS OS

Parameter Modality

analysed

Hazard

Ratio

Lower limit Upper limit P

Progression-Free Survival

Mass >10 cm Yes 1.83 0.94 3.57 0.08

Interaction between treatment arm and Hans

algorithm 1 4.26 1.21 15.05 0.02

LDH >Normal 1.54 0.53 4.52 0.43

Stage III-IV 1.03 0.34 3.11 0.96

Nb of extra-nodal sites >1 1.68 0.78 3.61 0.19

R-CHOP vs. R-ACVBP for GCB patients 0.75 0.31 1.81 0.52

R-CHOP vs. R-ACVBP for non-GCB patients 3.21 1.29 8.00 0.01

Overall Survival

Mass >10 cm Yes 3.35 1.43 7.89 0.01

Interaction between treatment arm and Hans

algorithm 1 13.38 1.87 95.74 0.01

LDH >Normal 0.88 0.20 3.85 0.86

Ann Arbor stage III-IV 0.59 0.13 2.68 0.50

Nb of extra-nodal sites >1 1.71 0.56 5.20 0.35

R-CHOP vs. R-ACVBP for GCB patients 0.46 0.13 1.65 0.23

R-CHOP vs. R-ACVBP for non-GCB patients 6.09 1.37 27.03 0.02

Patients With non- GCB DLBCL Benefit

From R-ACVBP regimen over R-CHOP.

Exact mechanism unknown, role of MTX as

inhibitor of NF-kB activation?

TJ Molina et al, J Clin Oncol, 2014

LYSA & LYSARC

GHEDI study

PI F Jardin

K Leroy, C Copie (FISH), TJM (IHC), JPJais, Hervé

Tilly, C Haioun, GA Salles.

PI of the clinical trials, Clinicians,Pathologists,

Statisticians, Biologists.

LYSARC staff and LYSA-Pathology

DLBCL : Conclusions • DHL lymphomas (Genetic break)5% of DLBCL and Double

Expressor(Protein)21% are linked to different subtypes of DLBCL – GCB for DHL FISH and Non GCB for DHScore lymphoma

• Importance of the MYC partner (IgH, K, L) for DHL prognosis whether SH or DH

• BCLu are rare among DHL DLBCL

• Prognosis of MYC+BCL2+lymphoma on multivariate analysis among patients included in clinical trial not clear.

• In practical – MYC FISH should combine presence of IG partner to be important as a

prognostic tool; More important than DHL overall.

– BCL2 protein is a strong prognostic factor in addition to GC/nGC among patients treated with R-CHOP.

– MYC protein in our hands is not a strong prognostic factor nor it is robust enough to act as a pre-test for MYC or MYCIg translocation