defined lymphoma entities in the current who classification
TRANSCRIPT
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