Diagnostic Molecular Pathology of Lymphoid Neoplasms

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1 Diagnostic Molecular Pathology of Lymphoid Neoplasms (Part II) Rational use of molecular testing in lymphomas Beirut, Lebanon Friday December 2, 2011: Hematopathology Session Adam Bagg University of Pennsylvania Philadelphia, USA why Major indications for molecular testing 1

2 why Major indications for molecular testing diagnosis neoplastic vs reactive (and beyond) why Major indications for molecular testing diagnosis neoplastic vs reactive (and beyond) classification based upon the genetic lesion [sine qua non] 2

3 why Major indications for molecular testing diagnosis neoplastic vs reactive (and beyond) classification based upon the genetic lesion [sine qua non] prognosis in otherwise homogeneous diseases why Major indications for molecular testing diagnosis neoplastic vs reactive (and beyond) classification based upon the genetic lesion [sine qua non] prognosis in otherwise homogeneous diseases monitoring/mrd both response and early recurrence 3

4 why Major indications for molecular testing diagnosis neoplastic vs reactive (and beyond) classification based upon the genetic lesion [sine qua non] prognosis in otherwise homogeneous diseases monitoring/mrd both response and early recurrence cryptic abnormalities technical reasons gene rearrangements and PCR V H segments D H segments J H segments C H segments mdgae 1. DJ rearrangement mdgae 2. VDJ rearrangement mdgae 3. High power view V H 2 N D H 3 N J H 5 L FR I CDR I FR II CDR II FR III CDR III FR IV CDRs, FRs and primers 4

5 Specific lymphoma categories Mantle cell lymphoma t(11;14)(q13;q32) upregulates cyclind1 expression s progression through cell cycle testing the defect: DNAPCR FISH FibreFISH IHC RTPCR ~ 10% cyclin D1 negative SOX11?? still = mantle cell lymphoma?? 5

6 Cyclin D1 testing in mantle cell lymphoma DNAPCR FISH IHC RQRTPCR 6

7 Chronic lymphocytic leukemia/sll CLL: Unraveling the heterogeneity Conventional parameters Novel parameters 7

8 CLL: Unraveling the heterogeneity Novel parameters Cytogenetics Cell of origin Cytogenetics 8

9 Cytogenetics del(11q22) ~18% del(13q14) ~55% 12 ~16% del(17p13) ~7% Cytogenetics: molecular del(11q22) ~18% del(13q14)?mirna 12 ~16% del(17p13) ~7% 9

10 Cytogenetics: molecular del(11q22) ATM del(13q14)?mirna 12 ~16% del(17p13) ~7% Cytogenetics: molecular del(11q22) ATM del(13q14)?mirna 12? CDK4 del(17p13) ~7% 10

11 Cytogenetics: molecular del(11q22) ATM del(13q14)?mirna 12? CDK4 del(17p13) p53 Prognostication by cytogenetics Dohner, H et al N Engl J Med 2000, 343:1910. Copyright 2000, Massachusetts Medical Society. All rights reserved. 11

12 Prognostication by cytogenetics Dohner, H et al N Engl J Med 2000, 343:1910. Copyright 2000, Massachusetts Medical Society. All rights reserved. Prognostication by cytogenetics Dohner, H et al N Engl J Med 2000, 343:1910. Copyright 2000, Massachusetts Medical Society. All rights reserved. 12

13 Cell of origin The dogma CLL is a neoplasm of naïve Bcells Cell of origin The dogma CLL is a neoplasm of naïve Bcells but a KARma has run over this DOGma 13

14 Chronic lymphocytic leukemia * * pregc nonmutated IgH naïve poorer prognosis postgc mutated IgH memory better prognosis Impact of SHM on prognosis Binet A ~7yrs ~25yrs Median survival P= Hamblin, TJ Blood 1999, 94: Copyright 1999 reproduced with permission of American Society of Hematology (ASH). 14

15 Chronic lymphocytic leukemia * * pregc nonmutated IgH naïve poorer prognosis (CD38) postgc mutated IgH memory better prognosis (CD38) Chronic lymphocytic leukemia * * pregc nonmutated IgH naïve poorer prognosis ZAP70 postgc mutated IgH memory better prognosis 15

16 Chronic lymphocytic leukemia * * pregc nonmutated IgH naïve poorer prognosis ZAP70 postgc mutated IgH memory better prognosis? mir15/16 ZAP70 by IHC on PB H&E CD79a CD3 16

17 ZAP70 by IHC on PB H&E CD79a CD3 ZAP70 ZAP70 by IHC on PB H&E CD79a CD3 ZAP70 SHM SHM 17

18 Diffuse Large Bcell Lymphoma (DLBCL) Morphologic: centroblastic immunoblastic Tcell/histiocyterich anaplastic plasmablastic lymphomatoid granulomatosis type Clinicopathologic: primary mediastinal (thymic) large Bcell lymphoma primary CNS lymphoma primary effusion lymphoma primary cutaneous large Bcell lymphoma (of the leg!) intravascular large cell lymphoma 18

19 DLBCL: Gene expression profiling Separation of DLBLs into two broad groups: germinal center activated Bcell Reprinted by permission from Macmillan Publishers Ltd: Alizadeh, A et al Nature 2000, 403: GCBDLBCL ABCDLBCL Frequency ~50% ~30% Key genes CD10 BCL6 AMYB LMO2 PKCb1 Cyclin D2 BCL2 IRF/MUM1 Oncogenic events t(bcl2) NFkB IGH SHM Ongoing Fixed Centro:Immunoblastic ~20:1 ~2:1 5year survival ~60% ~35% Median survival ~10 years ~2 years 19

20 DLBCL: Gene expression profiling That s all very nice and impressive, but DLBCL: Gene expression profiling That s all very nice and impressive, but Highly complex (10, 000 s of genes) Expensive Need fresh/frozen tissue 20

21 DLBCL: Gene expression profiling That s all very nice and impressive, but Highly complex (10, 000 s of genes) Expensive Need fresh/frozen tissue So, what s a humble, informationoverloaded pathologist to do? DLBCL: Gene expression profiling That s all very nice and impressive, but Highly complex (10, 000 s of genes) Expensive Need fresh/frozen tissue So, what s a humble, informationoverloaded pathologist to do? Wait for the dust to settle and it might be and use IHC (and only 3 markers at that!) 21

22 CD10 22

23 CD10 CD10 GCB 23

24 CD10? GCB CD10? GCB BCL6 24

25 CD10? GCB BCL6 CD10? GCB BCL6 nongcb 25

26 CD10 GCB? BCL6 nongcb? CD10 GCB? BCL6 nongcb? MUM1 26

27 CD10 GCB? BCL6 nongcb? MUM1 CD10 GCB? BCL6 nongcb? MUM1 27

28 CD10 GCB? BCL6 nongcb? MUM1 CD10 GCB? GCET1 BCL6 BCL2 nongcb? FOXP1 MUM1 28

29 CD10 GCB? GCET1 BCL6 BCL2 nongcb? FOXP1 MUM1 CD10 GCB? GCET1 BCL6 BCL2 nongcb? FOXP1 MUM1 29

30 Diffuse large Bcell lymphoma: what s new in WHO (2008) Incorporation of borderline categories: grey zone lymphomas diffuse large Bcell lymphoma Burkitt lymphoma Bcell lymphoma, unclassifiable, with features intermediate between diffuse large Bcell lymphoma and Burkitt lymphoma (BCLUWFIBDLBCLABL) primary mediastinal Bcell lymphoma classical Hodgkin lymphoma Bcell lymphoma, unclassifiable, with features intermediate between diffuse large Bcell lymphoma and classical Hodgkin lymphoma (BCLUWFIBDLBCLACHL) EBV positive DLBCL of the elderly: polymorphic histology elderly >50 30

31 Diffuse large Bcell lymphoma: BCLUWFIBDLBCLABL Characteristic BL BCLUetc DLBCL Morphology only smallmedium sized cells yes common no only large cells no no common mixture no sometimes rare Proliferation (Ki67) >90% and homogeneous yes common rare <90% or heterogeneous no sometimes common BCL2 expression negative/weak yes sometimes sometimes strong no sometimes sometimes Diffuse large Bcell lymphoma: BCLUWFIBDLBCLABL Characteristic BL BCLUetc DLBCL Genetic features MYC rearrangement yes common rare IG/MYC yes sometimes rare nonig/myc no sometimes rare BCL2 but MYC no rare sometimes BCL6 but MYC no rare sometimes double hit no sometimes rare MYCsimple karyotype yes rare rare MYCcomplex karyotype rare common rare 31

32 Diffuse large Bcell lymphoma: BCLUWFIBDLBCLABL BL BCLUWFIBDLBCLABL attempts to maintain purity of BL and DLBCL DLBCL helpful for pathologists no pressure to force into a diagnostic box avoid classifying the unclassifiable but don t abuse the BCLUWFIBDLBCLABL category!! also allowed some wiggle room in diagnosing BL frustrating for clinicians but our intentions are good 32

33 Pitfalls and caveats: Ig and TCR PCR False positives Pitfalls and caveats: Ig and TCR PCR False positives contamination 33

34 Pitfalls and caveats: Ig and TCR PCR False positives contamination pseudoclonality (small biopsies) Pitfalls and caveats: Ig and TCR PCR False positives contamination pseudoclonality (small biopsies) reactive/inflammatory scenarios H. pylori gastritis (but ) Hepatitis C (but ) Viral infections (HIV, mumps, EBV, CMV) Sjögren syndrome Rheumatoid arthritis 34

35 Pitfalls and caveats: Ig and TCR PCR False positives contamination pseudoclonality (small biopsies) reactive/inflammatory scenarios H. pylori gastritis (but ) Hepatitis C (but ) Viral infections (HIV, mumps, EBV, CMV) Sjögren syndrome Rheumatoid arthritis canonical (TCRg) Pitfalls and caveats: Ig and TCR PCR False positives contamination pseudoclonality (small biopsies) reactive/inflammatory scenarios H. pylori gastritis (but ) Hepatitis C (but ) Viral infections (HIV, mumps, EBV, CMV) Sjögren syndrome Rheumatoid arthritis canonical (TCRg) immune reconstitution post BMT immune response to tumor 35

36 Pitfalls and caveats: Ig and TCR PCR False positives contamination pseudoclonality (small biopsies) reactive/inflammatory scenarios H. pylori gastritis (but ) Hepatitis C (but ) Viral infections (HIV, mumps, EBV, CMV) Sjögren syndrome Rheumatoid arthritis canonical (TCRg) immune reconstitution post BMT immune response to tumor clonal dermatitis Pitfalls and caveats: Ig and TCR PCR False negatives 36

37 Pitfalls and caveats: Ig and TCR PCR False negatives Preanalytic variables [degradation, fixation, representative sampling] Technical consensus primers using CDR3 IgH primers only Biologic pre GC/ intra/post GC precursor Bcells partial DJ oligoclonal (~1/3 precursor BALL) ongoing rearrangements at relapse somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas) Pitfalls and caveats: Ig and TCR PCR False negatives Preanalytic variables [degradation, fixation, representative sampling] Technical consensus primers using CDR3 IgH primers only Biologic pre GC/ intra/post GC precursor Bcells partial DJ oligoclonal (~1/3 precursor BALL) ongoing rearrangements at relapse somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas) 37

38 Pitfalls and caveats: Ig and TCR PCR False negatives Preanalytic variables [degradation, fixation, representative sampling] Technical consensus primers using CDR3 IgH primers only Biologic pre GC/ intra/post GC precursor Bcells partial DJ oligoclonal (~1/3 precursor BALL) ongoing rearrangements at relapse somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas) Pitfalls and caveats: Ig and TCR PCR False negatives Preanalytic variables [degradation, fixation, representative sampling] Technical consensus primers using CDR3 IgH primers only Biologic pre GC/ intra/post GC precursor Bcells partial DJ oligoclonal (~1/3 precursor BALL) ongoing rearrangements at relapse somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas) 38

39 Pitfalls and caveats: Ig and TCR PCR False negatives Preanalytic variables [degradation, fixation, representative sampling] Technical consensus primers using CDR3 IgH primers only Biologic pre GC/ intra/post GC precursor Bcells partial DJ oligoclonal (~1/3 precursor BALL) ongoing rearrangements at relapse somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas) Pitfalls and caveats: Ig and TCR PCR False negatives Preanalytic variables [degradation, fixation, representative sampling] Technical consensus primers using CDR3 IgH primers only Biologic pre GC/ intra/post GC precursor Bcells partial DJ oligoclonal (~1/3 precursor BALL) ongoing rearrangements at relapse somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas) 39

40 Coming full circle? genomics Protein consequence IHC CD10 BCL2 BCL6 BCL10 ZAP70 ALK The last slide Powerful but one piece of the puzzle 40

41 The last slide Powerful but one piece of the puzzle Positive result: not always = neoplastic The last slide Powerful but one piece of the puzzle Positive result: not always = neoplastic Negative result: not always = not neoplastic 41

42 The last slide Powerful but one piece of the puzzle Positive result: not always = neoplastic Negative result: not always = not neoplastic Integrate: with morphologic, immunophenotypic, clinical data The last slide Powerful but one piece of the puzzle Positive result: not always = neoplastic Negative result: not always = not neoplastic Integrate: with morphologic, immunophenotypic, clinical data Decision to perform/ability to interpret: contextual 42

43 The last slide Powerful but one piece of the puzzle Positive result: not always = neoplastic Negative result: not always = not neoplastic Integrate: with morphologic, immunophenotypic, clinical data Decision to perform/ability to interpret: contextual More rational, biologicallybased diagnosis: more appropriate, targeted Rx Any questions 43

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