Antigen receptor (immunoglobulin and T-cell receptor) gene rearrangements: Utility in Routine Diagnostic Hematopathology

Transcription

1 Antigen receptor (immunoglobulin and T-cell receptor) gene rearrangements: Utility in Routine Diagnostic Hematopathology DIAGNÓSTICO PRÁTICO DOS LINFOMAS São Paulo, Brasil 02 DE SETEMBRO DE 2011 Adam Bagg University of Pennsylvania Philadelphia, USA

2 Irene sends her apologies

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4 Molecular targets

5 Rearrangements - physiologic - pathologic Molecular targets

6 Rearrangements - physiologic - pathologic Molecular targets

7 Molecular targets Rearrangements - physiologic - pathologic creation of a novel chimeric gene upregulated/overexpression of a protooncogene A B A B C A B t(9;22) bcr-abl qualitative t(8;14) IgH + c-myc quantitative

8 Molecular targets Rearrangements - physiologic - pathologic homogeneity vs heterogeneity creation of a novel chimeric gene present vs absent upregulated/overexpression of a protooncogene A B A B C A B t(9;22) bcr-abl qualitative t(8;14) IgH + c-myc quantitative

9 Molecular targets Rearrangements - physiologic - pathologic homogeneity vs heterogeneity Mutations creation of a novel chimeric gene present vs absent upregulated/overexpression of a protooncogene A B A B C A B t(9;22) bcr-abl qualitative t(8;14) IgH + c-myc quantitative

10 Molecular targets Rearrangements - physiologic - pathologic homogeneity vs heterogeneity Mutations Additions creation of a novel chimeric gene present vs absent upregulated/overexpression of a protooncogene Losses - deletions - silencing A C B A A B B t(9;22) bcr-abl qualitative t(8;14) IgH + c-myc quantitative

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12 Antigen receptor (Ig and TCR) gene rearrangements

13 Antigen receptors Antigen Immunoglobulin T-cell receptor B-cell T-cell

14 IgH gene rearrangement and PCR V H D H J H C H µδγαε

15 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement

16 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement

17 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement µδγαε

18 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement µδγαε 2. V-DJ rearrangement

19 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement µδγαε 2. V-DJ rearrangement

20 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement µδγαε 2. V-DJ rearrangement µδγαε

21 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement µδγαε 2. V-DJ rearrangement µδγαε 3. High power view V H 2 N D H 3 N J H 5

22 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement µδγαε 2. V-DJ rearrangement µδγαε 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

23 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement µδγαε 2. V-DJ rearrangement µδγαε 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

24 IgH gene rearrangement and PCR V H D H J H C H µδγαε 1. DJ rearrangement µδγαε 2. V-DJ rearrangement µδγαε 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

25 IgH gene rearrangement and PCR gel-based PCR product detection Size Poly Mono Mono Mono Poly Neg

26 TCRγ gene rearrangement and PCR V γ J γ C γ J γ C γ P1 P 1 1 P2 2 2 VJ rearrangement P2 2 2 High power view V γ 6 N J γ P2 Multiplex PCR Vγ1-8 Vγ9 Vγ10 Vγ11 JγP1 JγP Jγ1 JγP2 Jγ2

27 TCRγ gene rearrangement and PCR Atypical T-cell lymphoproliferations

28 TCRγ gene rearrangement and PCR Atypical T-cell lymphoproliferations capillary electrophoresis-based PCR product detection

29 TCRγ gene rearrangement and PCR Atypical T-cell lymphoproliferations capillary electrophoresis-based PCR product detection reactive

30 TCRγ gene rearrangement and PCR Atypical T-cell lymphoproliferations capillary electrophoresis-based PCR product detection reactive neoplastic

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32 Other methods for AR GR Detection heteroduplex analysis SSCP DGGE microchip melting curve multichannel electrophoresis ligase chain reaction IGH PCR: usually straight-forward TRG PCR: more of an issue Interpretation relative peak height relative peak ratio height ratio peak height ratio best-fit normal distribution algorithm

33 Better Basis to Behold B- (and T-) cell clones: BIOMED 2 5 IGH 3 VJ 2 DJ hierarchy of IG GR 100% IG-lambda+ 97 primers 14 tubes 418 reactions 8 IG 6 TCR 2 IGK 1 IGL 3 TRB 2 TRG 1 TRD 1 VJ 1 Kde 1 VJ 2 VJ 1 DJ TRB analysis - 38 primers reactions will be IGK+ but ~5-10% IG-kappa+ will be IGL+ IGK and IGL also subjected to SHM, but? less so Kde reaction not affected by SHM

34 Better Basis to Behold B- (and T-) cell clones: BIOMED 2 97 primers 14 tubes 418 reactions 8 IG 6 TCR 5 IGH 2 IGK 1 IGL 3 TRB 2 TRG 3 VJ 2 DJ 1 VJ 1 Kde 1 VJ 2 VJ 1 DJ Incomplete DJ rearrangements ~30% IGH DJ+ in B-cell neoplasms up to 60% IGH DJ+ in myeloma ~60% TRB DJ+ in T-cell neoplasms excellent targets: - VJ primers will miss - since not transcribed, no SHM! 1 TRD

35 Better Basis to Behold B- (and T-) cell clones: BIOMED 2 Biomed Cambridge Tissue Frozen Fixed* T-cell lymphomas 94%** 98% B-cell lymphomas 99% 96% Pre-GC B (MCL) GC B (FL) 100% 84% 100% 30%*** * >300bp DNA product, excluded 21% cases ** 99% if exclude ALCL *** 13% with FR3 only [addition of IGK 100%] FR1 and FR2 better than FR3 in frozen

36 BIOMED 2 IG proposed algorithms Biomed 2 Frozen Cambridge Fixed 3 IGH VJ 1 st 98% 2 IGK 1 IGH VJ (FR2) 1 st 91% 2 IGK 2 IGH DJ 2 nd 99% 1 IGL 2 IGH VJ (FR1 & FR3) 2 nd 96% 1 IGL

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38 Antigen receptor gene rearrangements Useful in the following situations:

39 Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations

40 Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue

41 Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype

42 Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype T-cell lymphoproliferations

43 Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype T-cell lymphoproliferations baseline for MRD

44 Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype T-cell lymphoproliferations baseline for MRD BM: precursor B-cells

45 Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype T-cell lymphoproliferations baseline for MRD BM: precursor B-cells But not that helpful in:

46 Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype T-cell lymphoproliferations baseline for MRD BM: precursor B-cells But not that helpful in: diagnosing specific entities

47 Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype T-cell lymphoproliferations baseline for MRD BM: precursor B-cells But not that helpful in: diagnosing specific entities unraveling the heterogeneity

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49 Pitfalls and caveats: Ig and TCR PCR False positives

50 Pitfalls and caveats: Ig and TCR PCR False positives - contamination

51 Pitfalls and caveats: Ig and TCR PCR False positives - contamination - pseudoclonality (small biopsies)

52 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

53 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 (TCRγ)

54 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 (TCRγ) - immune reconstitution post BMT - immune response to tumor

55 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 (TCRγ) - immune reconstitution post BMT - immune response to tumor - clonal dermatitis

56 Pitfalls and caveats: Ig and TCR PCR False negatives

57 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/ precursor B-cells partial DJ oligoclonal (~1/3 precursor B-ALL) ongoing rearrangements at relapse intra/post GC somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas)

58 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/ precursor B-cells partial DJ oligoclonal (~1/3 precursor B-ALL) ongoing rearrangements at relapse intra/post GC somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas)

59 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/ precursor B-cells partial DJ oligoclonal (~1/3 precursor B-ALL) ongoing rearrangements at relapse intra/post GC somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas)

60 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/ precursor B-cells partial DJ oligoclonal (~1/3 precursor B-ALL) ongoing rearrangements at relapse intra/post GC somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas)

61 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/ precursor B-cells partial DJ oligoclonal (~1/3 precursor B-ALL) ongoing rearrangements at relapse intra/post GC somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas)

62 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/ precursor B-cells partial DJ oligoclonal (~1/3 precursor B-ALL) ongoing rearrangements at relapse intra/post GC somatic hypermutation (primary/ongoing) (follicular lymphoma, myeloma) IgH deletions (~1/10 lymphomas)

63 Any questions

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