Lymphomes diffus Lymphomes diffus àà grandes cellules Bgrandes cellules BClassification des diffClassification des diff éérentes entitrentes entit ééss

T. J. MolinaT. J. Molina

Diffuse large B Diffuse large B cellcell lymphomaslymphomas

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

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%

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 subtypesT-cell rich large B-cell lymphomaPrimary DLBCL of the CNS Primary cutaneous DLBCL, leg typeEBV positive DLBCL of the elderly

Other lymphomas of large B-cellsPrimary mediastinal (thymic) large B-cell lymphomaIntravascular large B-cell lymphomaDLBCL associated with chronic inflammation (EBV)Lymphomatoid granulomatosis (EBV)ALK positive DLBCL (ALK )Plasmablastic lymphoma (EBV)Large B-cell lymphoma arising in HHV-8 associatedmulticentric Castleman disease (HHV8)Primary Effusion lymphoma (HHV8, EBV)

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

Common morphological variantsCentroblasticImmunoblasticAnaplastic

Rare morphological variantsMolecular subgroups

Germinal centre B-cell like (GC-B)Activated B-cell like (ABC)

Immunohistochemical subgroupsCD5+DLBCLGerminal 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 lymphomaMarginal zone lymphomaNLPHL (Nodular Lymphocyte

Predominance Hodgkin lymphoma )

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

OTHER DIFFICULT DIFFERENTIAL DIAGNOSES

Mantle cell lymphoma , aggressive , pleomorphic subtypeCyclin D1 DLBCL do exist

CD5+ DLBCL is a phenotypical subtype

Is this a transformed small B-cell lymphoma?Follicular Lymphoma

Marginal zone lymphoma

Centroblastic

Centroblastic multilobated

Immunoblastic

Anaplastic

Plasmablastic

DLBCL NOS , Molecular subgroups

• GC like DLBCL

• Activated like DLBCL

• Other (According to Wright/type III).

Alizadeh et al, 2000

Cell of Originsignature

Lenz , NEJM, 2008

Molecular subgroups defined by GEP or QRTPCR impacts on survivalIndepedently from IPI among R-CHOP treated DLBCL patients

Classification OMS des DLBCL NOS

• Immunohistochemical subgroups– Germinal center B-cell (GC-B)

– Non germinal center B-cell (n-GCB)

– CD5 positive DLBCL

Lymphome à grandes cellules B du médiastin à part (entité)

C Hans et al, Blood 2004

PN Meyer, 2011

GC/nGC according to Hans’algorithmis not reproducibly prognostic in

DLBCL treated by R-CHOP

• Nyman 2007,

• Saito 2007,

• Copie-Bergman 2009,

• Ott G 2010,

• Gutierrez Garcia 2011,

• Salles G, 2011

Prognostic forMeyer N, JCO, 2011

Not prognostic forGuttierez Garcia, Blood 2011

Autres algorithmes

• Combiner FISH + IHC : Immunofish, Copie-Bergman, 2009– Mum-1 (30%), foxp1 (0 vs P), FISH bcl6 : 2 parmi trois marqueurs

positifs

Multivariate analysisIPI : p= 0.04nGC score : p=0.04

Copie-Bergman et al, JCO, 2009

ImmunoFISH négatif

ImmunoFISH positif

Ces biomarqueurs peuvent ils être prédictifs?

• Identifier la population cible, la plus susceptible de répondre

• Guide parmi les options thérapeutiques

– C Thieblemont : BioCoral

– O3-2B: RACVBP vs RCHOP

Primary Mediastinal Large B-celllymphoma (PMLBCL)

• Variant of DLBCL derived from a putative thymic B-cell (asteroid variant of thymicmedullary B cell)

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

• No expression of immunoglobulins but expression of pu-1, bob2, oct 1

• Activation of NF-κB with nuclear expression of c-rel

• Functional rearrangement of IgH withoutintraclonal variation.

• No rearrangement of bcl-2 and rare bcl-6 rearrangement

Thymus IF CD79a / MAL

Copie Bergman, 2002

The gene expression signature of PrimaryMediastinal B Cell Lymphoma

A Rosenwald et al. J Exp Med, 2003A Rosenwald et al. J Exp Med, 2003

RosenwaldJEM, 2003

Georg Lenz, M.D., and Louis M. Staudt,.N Engl J Med 2010; 362:1417-1429

Mutations of SOCS1 in classicalHodgkin lymphoma are frequent(Wenger et al, Oncogene, 2006)

8/19 HL patients and 3/5 cell lines

A20 is a tumor suppressor gene in CHL And PMBL.( Schmitz R, J Exp Med, 2009)

A20 which negatively regulates NF-KB ismutated in 40% of PMBL and CHL; in CHL, mostly in EBV negative cases.

MHC Cl II transactivator CIITA is rearranged in 38% PMBL and 15%CHL

STEIDL C , Nature, 2011

• Translocation partner : in PMBL, half of the cases PDL1, PDL2 with overexpression and downregulation of MHC cl II.

• CIITA rearrangement in 4%DLBCL (11% testis) and in 15% NLPHL.

T-cell Histiocyte Rich large B-cell lymphoma

- Histopathology

• difuse infiltration

• large B-cellsLimited numbers (< 10% cells)Scattered cellsNo sheetsCb, Ibl, anaplastic,rare RS-like cells

• Numerous reactive cellsT lymphocytes histiocytes

- Immunophenotype• tumor cells

CD20+, EMA +/-CD15-, CD30-, LMP1-

• Reactive cells

CD3 +Low number of PD1+

• No FDC network

- diagnostic différentiel• CHL • NLPHL•T cell lymphoma

CD20

T-cell/ histiocyte rich large B-cell lymphomas

Immunohistochemical study

• CD20+

• CD30 :2/46

• EMA: 34/38

• CD15 : 0/38

• few CD57+ cells

Specific Clinical Characteristics of Patients With T-Cell/Histiocyte-Rich Large B-Cell Lymphoma

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

Bouabdallah et al, J Clin Oncol 2006; Anthracyclin based therapyWithout rituximab

• HU 133A et U 133 B, 33 000 gènes• 176 patients (série de validation : 221)• Unsupervised clustering (three profiles highly

reproducible)– oxydative Phosphorylation– BCR proliferation cluster– Host immune response

• No prognostic vaue

Monti, Blood, 2004

Microenvironment and DLBCL

Gamma interferonInduced lysosomalthiolreductase

FewCD1a or CD123;

LM B PRIMITIF DES SEREUSES

- Définition• Lymphome se développant

essentiellement dans lescavités des séreuses: pleurale

péricardiqueabdominale

• En l’absence habituelle de massetumorale

• ≠≠≠≠ extension 2 d d’un LM à grandescellules B

- Clinique•Epanchement pleural sans tumeursolide

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

- CliniqueAdulteLésions cutanéesSymptomes neurologiquesHépatosplénomégaliePancytopénie, CIVD

- Histopathologie• Grandes cellules (Cb, Ib, anaplasiques)• Dans les petits vaisseaux

sinus (moelle osseuse, rate)sinusoïdes (foie)capillaires (peau, cerveau, poumon)

• Histiocytes avec Erythrophagocytose

- ImmunophénotypePan 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, infiltr ant

la paroi des vaisseaux, réalisant un aspect angiocentrique

– Autres localisations : cerveau (26%), rein(32%), foie (29%),peau (25-50%)

CD20

LMP1

MIB1 EBER

Other subtypes/entities

• Primary DLBCL of the CNS– Intracerebral and/or intraocular,mum-1+

• Primary cutaneous DLBCL, leg type– N-GC phenotype, female 7th decade

• EBV positive DLBCL of the elderly– More than 50 year-old, noimmunodeficiency, mum1+, polymorphic

subtype• DLBCL associated with chronic inflammation (EBV)

– Pyothorax associated lymphoma, osteomyelite chronic– More than 10 years inflammation– CD20, CD79a often positive, CD138+/-, T- cell markers

Other subtypes/entities

• ALK Positive DLBCL– Lymph nodes, Immunoblast with plasma cell

differentiation, CD138, EMA, IgA, CD20 neg, CD79a neg, CD45 neg,mum1+.

• Plasmablatic lymphoma– Oral cavity ,other extranodal sites, HIVpositive, CD138,

CD30, EMA, EBER, IgG• Large B-cell lymphoma arising in HHV-8 associated

multicentric Castleman disease (HHV8)– IgM plasmablast EBER neg IgM lambda present in mantle zone areas

at the beginning

Borderline cases

B-cell lymphoma, unclassifiable, with featuresintermediate between DLBCL and Burkitt lymphoma

B-cell lymphoma, unclassifiable, with featuresintermediate between DLBCL (PMBL) and classicalHodgkin lymphoma

LYMPHOME DE BURKITTsous-types, cliniques et génétiques

- EndémiqueAfrique et autres régions tropicalesEnfant++, Mâchoire++Association à EBV: > 90%Rôle de la malaria

- SporadiqueLocalisation intraabdominale+Ganglion, plus fréquent chez l’adulteAssociation à EBV: 20%

- Associé à une immunodéficienceLa plupart HIV+Souvent ganglionnaireAssociation à EBV: 40%

EBER

- Histopathologie

• Infiltration diffuse• Aspect de ciel étoilé• Cellules cohésives• Cellules de taille moyenne• Fine couronne cytoplasmique

basophile avec des vacuoleslipidiques

• Noyau rond, +/- irrégulier• Chromatine en mottes• Multiples (2-5) nucléoles

centraux, de taille moyenne• Nombreuses mitoses

LYMPHOME DE BURKITT

LYMPHOME DE BURKITT

- Immunophénotype

Pan B+, sIgM+,

bcl-6+

CD10+, bcl2- , Ki67 > 90%

CD5-, CD23-, TdT-

- Biologie moléculaire

• t(8;14)(q24;32) et variantes

• réarrangement de c-myc

CD10

Mib-1

BURKITT LYMPHOMA

CD10

Mib-1

CD10

MIB1 BCL2

Sporadic Burkitt, Child, EBV -

MIB1BCL2

Diffuse Large B-cell Lymphoma or Intermediate BL/DLBC L?

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 worseprognosis compared to paired DLBCL – May qualify for intermediate features between BL and

DLBCL

Controversy

• Intermediate Molecular Burkitt have been definedby 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 thosecases that might benefit from alternative therapy thanclassicalDLBCL.

Hummel, NEJM, 2006

Borderline cases

B-cell lymphoma, unclassifiable, with featuresintermediate between DLBCL and Burkitt lymphoma

B-cell lymphoma, unclassifiable, with featuresintermediate between DLBCL (PMBL) and classicalHodgkin lymphoma

B-cell lymphoma, unclassifiable, withfeatures intermediate between ClassicalHodgkin Lymphoma (CHL) and Diffuse

large B- cell Lymphoma (DLBCL).• Composite or Sequential

– Both entities within the same patient at the same time (C)

– To differentiate from metachronously occurrence of NSHL and PMBCL (S)

• Intermediate features– between CHL and PMBL

Intermediate Features areExceptional !!

Most often PMBL or CHL

Or other diagnosis…..

In big centers specialised inHematopathology includingLocal cases and consult cases

Max : 1-2 cases per year.

CD20 CD3

CD30

TiA1

CD7EMA

ALK 1

TTake Home Message

• Except PMLBL, entities of DLBCL are rare+++

• A CD5+ agressive B cell lymphoma :exclude MMantle cell lymphoma aggressive variant

before assessing CD5+ DLBCL

• DLBCL are not rarely transformed

• FISH is important in addition to immunohistochemistry, specially Myctranslocation– Burkitt/ Double hit lymphoma

Department of Pathology