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
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