Defined lymphoma entities in the current WHO classification

Luca Mazzucchelli Istituto cantonale di patologia, Locarno

Bellinzona, January 29-31, 2016

Evolution of lymphoma classification

  Rappaport

  Lukes and Collins (immunophenotype)

  Kiel Classification (Europe)

  Working Formulation (USA)

  REAL Classification (1992)

  WHO classification (2001)

  Update of WHO classification (2008)

  Next lymphoma classification (2016)

Requisites of a classification

  Easy to apply   Minimal intra- and inter-observer variability   Must give relevant clinical information relating to pathogenesis

and prognosis   Must be validated in prospective studies   Must be a dynamic process that can integrate clinical

(prognosis, therapy) and pathological advances (immunology, genetics)

WHO classification: selection of problematic issues

  DLBCL represent a group of lymphomas with similar histological features. They may have a very distinctive clinical behaviour and, consequentely, they need a different treatment approach. Role of MYC alterations and next generation sequencing (NGS) in the routine diagnostic

  EBV in DLBCL (DLBCL of the elderly)

  High-grade transformation of low-grade B-cell lymphoma

  Lymphoplasmacytic lymphoma defined by morphology, clinical features, or molecular tests

  Anaplastic large cell lymphomas: ALK positive, ALK negative, and primary cutaneous

DLBCL categories

  DLBCL specifed by site Primary CNS, testicular, skin leg-type, primary mediastinal lymphoma

  DLBCL with characteristic histologic, immunophenotypic or genotypic features

T-cell rich B cell lymphoma, CD5+ DLBCL, ALK +

  DLBCL associated with EBV o HHV8 infection Post-transplant lymphoproliferative disease, EBV + DLBCL of the elderly

lymphomas with chronic infection,…

  DLBCL, NOS

  Lymphoma non classifiable

  Molecular subgroups   Germinal centre B-cell-like (GCB)   Activated B-cell-like (ABC)   Unclassified

  Common morphologic variants   centroblastic   immunoblastic   anaplastic

  Cytogenetic alterations   Bcl2   Bcl6   C-myc

  EBV-Status   Mutation-patterns

Diffuse large B-cell lymphoma, NOS

Diffuse large B-cell lymphoma

A Alizadeh AA, et al.: Distinct type of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000, 403:503

Decision tree for classification of DLBCL based on immunohistochemistry

CD10 MUM1

bcl6

GCB

GCB Non-GCB

Non-GCB +

+

+ -

-

-

Hans et al. Blood 2004

Immunohistochemical algorithms (Hans, Blood 2004) in the CHOP-treatment era

  Blood 2004; 103:275   Mod Pathol 2005; 18:1113   Arch Pathol Lab Med 2006;

130:1819   J Pathol 2006; 208:714   Blood 2007; 109:4930   Eur J Haematol 2007;

79:501   J Clin Oncol 2006; 24:4135

  Histopathology 2007; 51:70   Blood 2003; 101:78   J Clin Oncol 2005; 23:7060   Haematologica 2007;

92:778   J Clin Oncol 2008; 26:447   Ann Oncol 2007; 18:931

It works... It doesn‘t work...

Immunohistochemistry is …..laboratory specific

 Pre-analytic  Time to fixation  Time of fixation  Type of fixation

 Analytic  Test validation  Type of antigen retrieval  Test reagents  Standardized procedures  Automated methods

 Post-analytic  Interpretation criteria  Reporting elements  Quality assurance procedures

Markers used in various researchers’ algorithms to predict the cell of origin in DLBCL

Testoni et al, Annals of Oncology advance access, January 2015

Studies GCB DLBCL ABC DLBCL Hans et al 2004 CD10>30%

BCL6>30% MUM1>30%

Murris et al 2006 CD10>30% MUM1>30% BCL2>50%

Nyman et al 2007 MUM1>30% FOXP1 >80%

Natkunam et 2008 LMO2>30%

Choi et al 2009 GCET1>80% CD10>10% BCL6>30%

MUM1>30% FOXP1 >80%

Meyer et al 2011 GCET1>80% CD10>10% LMO2>30%

MUM1>30% FOXP1 >80%

Visco et al 2012 CD10>30% BCL6>30%

FOXP1>60%

  Gene expression profiling may divide DLBCLs in prognostically important subgroups

  The application of the Hans‘ immunohistochemical algorithm for OS prediction gives contradictory results, but it is easy to apply, and remains the most used algorithm in daily practice

  The application of new immunohistochemical algorithms must be validated in prospective studies, but unlikely it will affect the daily practice

G Ott et al. Blood 2010; 116(23):4916 Extension of the study published in Heamatologica 2009; 15:5494

Immunoblastic lymphoma Immunoblastic lymphoma with plasmablastic features

G Ott et al. Blood 2010; 116(23):4916

G Ott et al. Blood 2010; 116(23):4916

Limits of clinical studies on aggressive B-cell lymphoma

  Definition of the histopathological diagnosis (heterogeneity of the populations studied, «heterogeneity» of pathologists)

  Retrospective – prospective studies   Definition of the methods and cutoff levels   Quality of the data

Target genes of MYC

Chromosomal translocations involving MYC

 MYC-IG rearrangements   IGH   IGKappa   IGLambda

 MYC non-IG rearrangements  BCL6  PAX5  BCL11A   IKAROS   others

  MYC-R occurs in 3-16% of DLBCL   The presence of a MYC-R is a strong adverse prognostic

factor in CHOP and R-CHOP treated patients   MYC-R in combination with IPI and patient‘s age accurately

predict clinical outcome   The presence of MYC-R can not be predicted by

lymphoma‘s morphology   MYC is frequently found with other genetic abnormalities

(Double-Hit lymphoma)

J Clin Oncol 2010; 28:3360 Blood 2009; 114:3533 J Clin Pathol 2009; 62:754

Lymphoma MYC Rearrangement

BL Present in >90% of cases; Primary genetic event. Translocation involves IG partner genes: 85% t(8;14), 15% t(2;8) or t(8;22).

DLBCL Present in 7-14% of de novo cases. Usually a secondary genetic event. Translocation involves IG (70%) and non-IG (30%) partner genes. Associated with “double hit” lymphoma.

BCLU Present in 35-50% of cases. Translocation rarely involves IGH as a partner gene. Translocation involves IGk, IGλ or non-IG partner genes. Associated with “double hit” lymphoma.

PBL Present in approximately 50% of cases. Translocation involves IG partner genes in the majority of cases, usually IGH.

Adv Anat Pathol 2011, 18:219-228

Diffuse large B-cell lymphomas of immunoblastic type are a major reservoir for MYC-IGH translocations.

Horn H, et al. Am J Surg Pathol 2015

  MYC-R in 13/39 (33%) IB-DLBCL and in 5/68 (7%) non-IB-DLBCL

  IGH was the translocation partner in all IB-DLBCL (reported in the literature in 50-70% of all DLBCL)

  Sole abnormality in the majority of IB-DLBCL (77%) whereas DH are reported in 58-85% of MYC-R DLBCL

Double hit B-cell lymphomas

  B-cell lymphomas characterized by a recurrent chromosomal translocation in combination with a MYC/8q24 breakpoint

  DH lymphomas are rare (0-12%)

  Most DH lymphomas have a BCL2+/MYC+ combination

  There are no unifying morphological features of DH lymphomas

  Most DH lymphomas have a GC phenotype (CD10+, bcl6+, bcl2+, high ki67)

Double hit B-cell lymphomas

  Complex karyotypes   Gene expression profile

intermediate between BL and DLBCL

  Frequent non IGH partner of MYC

  Median age 51-65 years   Highly aggressive clinical

behaviour   Elevated LDH, bone marrow

and CNS involvement, high IPI score

  Resistent to chemotherapy (median survival of only 0,2-1,5 years)

How can the aggressive course of DH lymphomas be explained?

  DH lymphomas often have a very complex karyotype   DH lymphomas are not a distinct entity but probably represent

an heterogeneous group of lymphomas (i.e. de novo or transformation of follicular lymphoma)

BCL2 is anti-apoptotic without mediating proliferative signals

MYC drives cells in active and proliferative state Mature B cells

PROLIFERATION APOPTOSIS

Prognostic role of MYC-R DLBCL

  JCO 2010; 28:3360   Heamatologica 2013;

98:1554   Mod Pathol 2014; 27: 958   Am J Surg Pathol

2015;39:61

  Am J Surg Pathol 2012; 36-612

  J Clin Oncol 2012; 30:3452   Eur J Haematol 2014;92:48

It works... It doesn‘t work...

MYC-IG rearrangements are negative predictors of survival in DLBCL patients treated with immunochemotherapy: a GELA/LYSA study. C Copie-Bergman et al Blood, prepublished online, September 2015

MYC-IG but not MYC non-IG is predictor of poor PFS and OS

Absence of MYC-DH prognosis significance by multivariate analysis

  193 patients with DLBCL treated with R-CHOP   FISH identified DH in 6% of patients   High MYC and BCL2 expression observed in 29% of the

patients   The immunohistochemical D-H Score defined a large subset of

DLBCL with poor outcome

Double-Hit Score

MYC +>40% = score 1 BCL2 + <70% = score 0

MYC +>40% = score 1 BCL2 +>70% = score 1

= DHS 1

= DHS 2

Green TM et al 2012, JCO 30:3460

MYC-FISH BCL2-FISH

DH-FISH DH-FISH

Green TM et al 2012, JCO 30:3460

Impact on survival - FISH

Green TM et al 2012, JCO 30:3460

Impact on survival - IHC

MYC and BCL2 immunohistochemistry

Studies Patients Cut off Results

Green TM et al, JCO 2012, 30:3460

193 116 (validation)

BCL2 70% MYC 40%

FISH 6% IHC 29%

Johnson NA et al, JCO 2012, 30:3452

167 140 (validation)

BCL2 50% MYC 40%

FISH 5% IHC 21%

Horn H et al, Blood Jan 2013

442 BCL2 0% MYC 40%

FISH 4,9% IHC 15%

Evaluation of MYC protein expression in DLBCL

 Monoclonal antibody (clone Y69, N-terminal domain) detecting MYC expression in FFPE   Different cut-off to consider a case as positive (most used is ≥ 40%)   Staining heterogeneity in DLBCL   Correlation between MYC-R and protein expression in more than 70% of cases.   Worse prognosis: MYC-R and high protein expression   High MYC protein expression in around 20% of cases without MYC-R   Other mechanisms than rearrangement implicated in MYC expression

classical MYC breakpoint

1G1R1F

alternative MYC breakpoint

1G2F

DLBCL

MYC subclonality

11/23(48%) DLBCL

1G2R1F 2G1R1F 2G2R1F 1G0R1F

1G1R2F 1G1R3-6F

2G1R2F 1G2R2F 1G2R3-6F

classical MYC breakpoint

1G1R1F

alternative MYC breakpoint

1G2F

BL

0/13 BL

NO MYC subclonality

Subclonal populations involving MYC

IHC +

  MYC rearrangements can be detected in 10% of DLBCL

  MYC-R is more frequent in a subset of DLBCL (IB-DLBCL 33%)

  MYC-R occurs with different partner

  MYC-R partners or the presence of additional translocations may have a prognostic impact (MYC-IG worse prognosis)

  Low MYC protein expression may be related to subclonality/heterogeneity of DLBCL or non-IG translocation partners

  MYC-R and MYC protein overexpression (>50%) are associated with bulky disease, fail to achieve complete remission with R-CHOP therapy and have poorer DFS

  Interpretation of study results must consider the heterogeneity of DLBCL in general, but also heterogeneity/subclonality within a single lymphoma

EBV positive diffuse large B-cell lymphoma of the elderly

  EBV+ clonal B-cell lymphoid proliferation that occurs in patients > 50 years, without any known immunodeficiency or prior lymphomas.

  Lymphomatoid granulomatosis, plasmablastic lymphoma, primary effusion lymphoma, DLBCL associated with chronic inflammation, post-transplant lymphoproliferative disorders must be excluded

Methods of EBV detections

IHC EBER Viral load

EBV and DLBCL

  EBV positive DLBCL, both in the elderly and in young groups show significantly worse OS and PFS than negative cases.

  No significant difference of outcome between different age groups with EBV-positive DLBCL.

  Clinicopathologic, immunophenotyping, genetic profiling, microRNA profiling between EBV pos. DLBCL in patients younger or older than 50 years are not distinctive.

  PFS and OS are significantly worse for patients with EBV DNA load above 700 copies/µg, and for EBER-positive patients

  Even among EBER-negative patients, higher EBV DNA load conferr worse PFS and OS

Lu TX et al. 2015, Sci Rep 5:12168; Ok CY et al. 2015, Oncotarget 613933; Okamoto et al 2015, Hematol Oncol

  There is sufficient evidence that the arbitrary age cutoff for EBV-positive DLBCL should be eliminated in the WHO classification

  EBV-DNA load and EBER are potential biomarker associated with prognosis of DLBCL