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Acquired alterations of IGH and TCR loci in lymphoproliferative disorders

Acquired alterations of IGH and TCR loci in lymphoproliferative disorders. Sabine Franke, PhD CHU Liège, Centre for Human Genetics. Interuniversity course - HUMAN GENETCIS. 20/04/12 ULG. Overview. what are lymphoproliferative disorders? what are IGH and TCR?

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Acquired alterations of IGH and TCR loci in lymphoproliferative disorders

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  1. Acquired alterations of IGH and TCR loci in lymphoproliferative disorders Sabine Franke, PhD CHU Liège, Centre for Human Genetics Interuniversity course - HUMAN GENETCIS 20/04/12 ULG

  2. Overview • what are lymphoproliferative disorders? • what are IGH and TCR? • what are alterations and how to detect them?

  3. Blood cell development 2 major types of lymphocytes: B and T cells

  4. Lymphoproliferative disorders (LPDs)LPDs refer to several conditions in which lymphocytes are produced in excessive quantities. • Chronic lymphocytic leukemia • Acute lymphoblastic leukemia • Hairy cell leukemia • lymphomas • Multiple myeloma • Waldenstrom’s macroglobulinemia • Wiskott-Aldrich syndrome • Post-transplant lymphoproliferative disorder • Autoimmune lymphoproliferative syndrome (ALPS) • ‘Lymphoid interstitial pneumonia’

  5. B, T, and NK lineage of lymphoid malignancies Lymphoid malignancy B lineage T lineage NK lineage Acute lymphoblastic leukemia– children 82 – 86% 14 – 18% < 1%– adults 75 – 80% 20 – 25% < 1% Chronic lymphocytic leukemias 95 – 97% 3 – 5% 1 – 2% Non-Hodgkin lymphomas – nodal NHL 95 – 97% 3 – 5% < 2%– extranodal NHL 90 – 95% 5 – 10% < 2%– cutaneous NHL 30 – 40% 60 – 70% < 2% Multiple myeloma 100% 0% 0%

  6. Blood cell development

  7. Gene rearrangements of the antigen receptor genes occur during the lymphoid proliferation

  8. The ability to produce billions of different antibodies in humans results from the production of variable regions of light and heavy antibody genes by DNA rearrangement. http://www.biology.arizona.edu

  9. Schematic representation of an immunoglobulin

  10. Identify lymphocyte populations derived from one single cell using the unique V-J gene rearrangements present within these antigen receptor loci. • These gene rearrangements generate products that are unique in length and sequence in each cell.

  11. The production of variable regions of light and heavy antibody genes by DNA rearrangement. stepwise rearrangement of V, D, and J gene segments • Genes encoding antigen receptors are unique: • high diversity • developing • lymphocytes through • V(D)J rearrangement. (junctional region)

  12. T-cells T-cells have receptor gene rearrangement.

  13. T-cell receptor gene rearrangement The variable domain of both the TCR α-chain and β-chain have three hypervariable or complementary determining regions (CDRs) • Receptor for antigen on the majority of mature T-cells consists of two polypeptides alpha and beta that are linked by disulphide bonds and are associated with CD3 • A small population of mature T-cells express a different TCR heterodimer in association with CD3. This is composed of two polypeptides designated gamma and delta

  14. So how much variation is possible through recombining gene fragments? Over 15,000,000 combinations of variable, diversity and joining gene segments are possible. Imprecise recombination and mutation increase the variability into billions of possible combinations.

  15. Estimated diversity of human Ig and TCR molecules IgH Igα Igג TCR αβγδ molecules molecules Number gene segments V gene segments ~44 ~43 ~38 ~46 ~47 ~6 ~6 D gene segments 27 2 3 J gene segments 6 5 4 53 13 5 4 Combination diversity >2x10 6 2x10 6 <5000 Junctional diversity ++ ++ + ++ ++ ++++ Total diversity >10 12>10 12 >10 12

  16. Clinically relevant testing • Reactive versus malignant • B-cell versus T-cell malignancy • New lymphoma versus recurrence • Assessment of remission and relapse • Clinically relevant: bone marrow involvement • (relation with prognosis) • Evaluation of treatment effectiveness • detection of minimal residual disease - treatment

  17. PCRDiscrimination between monoclonal and polyclonal Ig/TCR gene PCR products • GeneScanning analysis Fast, accurate, sensitive non-quantitative monitoring of clonal proliferations Need sequence equipment • Heteroduplex analysis Sensitivity ~5-10% available to most laboratories

  18. Design of novel primer sets for detection of Ig/TCR rearrangements BIOMED-2 study (multiplex PCR) Ig genes: IGH: VH-JH and DH-JH IGK: V-J and Kde rearrangements IGL: V-J TCR genes: TCRB: V-J and D-J TCRG: V-J TCRD: V-J, D-D, D-J, and V-D

  19. BIOMED-2 clonality strategy Suspected lymphoma of unknown origin Suspected T-cell lymphoma Suspected B-cell lymphoma TCRGVJ (A and B) TCRB V(D)J (A and B) TCRB DJ IGH V(D)J FR1, FR2, FR3 IGH DJ(A) IGK-VJ and DE

  20. Van Dongen et al Leucemia 2003

  21. PCR GeneScan analysis region region

  22. Example BIOMED-2 multiplex IGHVH-FR2–JH Use of controls

  23. From the patient to the analysis • Selection of material • protocol • Check DNA quality • Clonality analysis

  24. Examples of cases • Lymphoma? • Relapse?

  25. Case 1 • Female • 63 years • Lymphoma in dec. 2004 • FR1 polyclonal, FR3 monoclonal • Feb. 2009 biopsy • Relapse?

  26. GeneScan analysis of controls H2O

  27. Case 1: Relapse? FR3 2/2009 Biopsie FR3: 152 bp 12/2004 Biopsie FR3: 152 bp 2000 12/2004 600

  28. Results Controls ok B-cell targets: IGH(VDJ) FR3 clonal Molecular conclusion: Clonal rearrangement of the IGH gene was detected in this specimen. This gene rearrangement profile is identical to the one detected in the biopsie of 12/2004 and confirms the relapse of the disease.

  29. Case 2 • Female • 81 years • Biopsie • Lymphoma?

  30. Case 2 3/2009 biopsie FR1 327,11 bp 2700 control 100

  31. Results Controls ok B-cell targets: IGH(VDJ) FR1 monoclonal FR2 monoclonal Molecular conclusion: Clonal rearrangements of the IGH gene were detected in this specimen. This gene rearrangement profile fits to the presense of a monoclonal B-cell population/B-NHL.

  32. Analysis of TCRB gene rearrangements

  33. BIOMED-2 multiplex TCRB tube B

  34. Use of clonality analysis 1. Making the diagnosis Normal  reactive  malignant 2. Involvement (staging) 3. Assessment of remission and relapse Normal  reactive  malignant 4. Evaluation of treatment effectiveness - detection of minimal residual disease (MRD) MRD-based risk-group stratification (treatment reduction or treatment intensification)

  35. Detection of abnormality at molecular level • PCR • Southern blot • FISH

  36. Southern blot • Large region • Large quantity of high molecular weight DNA • Less sensitive • Labor intensive

  37. FISH – Fluorescence in situ hybridization • Relative large region • Translocation partner has not te be known • On metaphases and nuclei

  38. Split-signal FISH

  39. Split-signal FISH for human Ig genes

  40. Split-signal FISH for human TCR genes

  41. Prognostic value of chromosomal aberrations Chromosome Genes Effect Occurrence Prognosis aberration involved t(1;19)(q23;p13) E2A-PBX1 fusion 30% pre-B-ALL intermediate t(4;11)(q21;q23) MLL-AF4 fusion 40% infant ALL poor t(9;22)(q34;q11) BCR-ABL fusion 35% adult ALL poor t(12;21)(p13;q22) TEL-AML1 fusion 25% childhood ALL good del(1p32) SIL-TAL1 TAL1  15% childhood T-ALL intermediate t(8;14)(q24;q32) IGH-MYC MYC  90% Burkitt’s lymphoma good t(11;14)(q13;q32) BCL1-IGH Cyclin D1  >90% MCL poor t(14;18)(q32;q21) BCL2-IGH BCL2  >90% FCL intermediate t(2;5)(p23;q35) NPM-ALK fusion 50% ALCL intermediate

  42. FISH • Excellent diagnostic tool for detection of well-defined chromosome aberrations • Can also be used on tissue sections • Requires limited handlingsusage of directly labeled probes • Split-signal FISH has several major advantages– detection of aberrations, independent of partner gene– minimization of false-positive results – identification of partner gene or affected chromosome region, if metaphases are available

  43. Summary • Gene rearrangements of the antigen receptor genes occur during the lymphoid proliferation • These gene rearrangements generate products that are unique in length and sequence in each cell. • Unique length allows by PCR discrimination of monoclonality and polyclonality • Standardized protocol (BIOMED-2)

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