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
Irene sends her apologies
Molecular targets
Rearrangements - physiologic - pathologic Molecular targets
Rearrangements - physiologic - pathologic Molecular targets
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
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
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
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
Antigen receptor (Ig and TCR) gene rearrangements
Antigen receptors Antigen Immunoglobulin T-cell receptor B-cell T-cell
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 4 5 23 1 2 3 4 5 6 µδγαε
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 4 5 23 1 2 3 4 5 6 µδγαε 1. DJ rearrangement
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 5 6 µδγαε 1. DJ rearrangement
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 5 6 µδγαε 1. DJ rearrangement 1 2 3 4 5 45 1 2 3 5 6 µδγαε
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 5 6 µδγαε 1. DJ rearrangement 1 2 3 4 5 45 1 2 3 5 6 µδγαε 2. V-DJ rearrangement
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 5 6 µδγαε 1. DJ rearrangement 1 2 3 5 6 µδγαε 2. V-DJ rearrangement
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 5 6 µδγαε 1. DJ rearrangement 1 2 3 5 6 µδγαε 2. V-DJ rearrangement 1 2 3 5 6 µδγαε
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 5 6 µδγαε 1. DJ rearrangement 1 2 3 5 6 µδγαε 2. V-DJ rearrangement 1 2 3 5 6 µδγαε 3. High power view V H 2 N D H 3 N J H 5
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 5 6 µδγαε 1. DJ rearrangement 1 2 3 5 6 µδγαε 2. V-DJ rearrangement 1 2 3 5 6 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
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 5 6 µδγαε 1. DJ rearrangement 1 2 3 5 6 µδγαε 2. V-DJ rearrangement 1 2 3 5 6 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
IgH gene rearrangement and PCR V H D H J H C H 1 2 3 4 5 45 1 2 3 5 6 µδγαε 1. DJ rearrangement 1 2 3 5 6 µδγαε 2. V-DJ rearrangement 1 2 3 5 6 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
IgH gene rearrangement and PCR gel-based PCR product detection Size Poly Mono Mono Mono Poly Neg
TCRγ gene rearrangement and PCR V γ J γ C γ J γ C γ 1 2 3 4 5 6 7 8 9 10 11 P1 P 1 1 P2 2 2 VJ rearrangement 1 2 3 4 5 6 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
TCRγ gene rearrangement and PCR Atypical T-cell lymphoproliferations
TCRγ gene rearrangement and PCR Atypical T-cell lymphoproliferations capillary electrophoresis-based PCR product detection
TCRγ gene rearrangement and PCR Atypical T-cell lymphoproliferations capillary electrophoresis-based PCR product detection reactive
TCRγ gene rearrangement and PCR Atypical T-cell lymphoproliferations capillary electrophoresis-based PCR product detection reactive neoplastic
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
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 - 325 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
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
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
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
Antigen receptor gene rearrangements Useful in the following situations:
Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations
Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue
Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype
Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype T-cell lymphoproliferations
Antigen receptor gene rearrangements Useful in the following situations: atypical lymphoproliferations limited tissue equivocal immunophenotype T-cell lymphoproliferations baseline for MRD
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
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:
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
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
Pitfalls and caveats: Ig and TCR PCR False positives
Pitfalls and caveats: Ig and TCR PCR False positives - contamination
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
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)
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
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
Pitfalls and caveats: Ig and TCR PCR False negatives
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)
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)
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)
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)
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)
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)
Any questions
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