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

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. 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 myeloma100%0%0% B, T, and NK lineage of lymphoid malignancies

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. The five major classes of heavy chain are IgM, IgG, IgA, IgD, and IgE. 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. (junctional region) stepwise rearrangement of V, D, and J gene segments Genes encoding antigen receptors are unique: -high diversity -developing lymphocytes through V(D)J rearrangement. The production of variable regions of light and heavy antibody genes by DNA rearrangement.

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

13. T-cell receptor gene rearrangement 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 The variable domain of both the TCR α-chain and β-chain have three hypervariable or complementary determining regions (CDRs)

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 α β γ δ moleculesmolecules 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. PCR D iscrimination 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 B-cell lymphoma Suspected lymphoma of unknown origin Suspected T-cell lymphoma IGH V(D)J FR1, FR2, FR3 IGH DJ(A) IGK-VJ and DE TCRGVJ (A and B) TCRB V(D)J (A and B) TCRB DJ

20. Van Dongen et al Leucemia 2003

21. PCR GeneScan analysis region

22. Example BIOMED-2 multiplex IGH VH-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 12/2004 2/2009 Biopsie FR3: 152 bp 12/2004 Biopsie FR3: 152 bp 2000 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 control 2700 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. ChromosomeGenesEffectOccurrencePrognosis aberrationinvolved t(1;19)(q23;p13)E2A-PBX1fusion 30% pre-B-ALL intermediate t(4;11)(q21;q23)MLL-AF4fusion 40% infant ALLpoor t(9;22)(q34;q11)BCR-ABLfusion 35% adult ALL poor t(12;21)(p13;q22)TEL-AML1fusion 25% childhood ALL good del(1p32)SIL-TAL1TAL1  15% childhood T-ALL intermediate t(8;14)(q24;q32)IGH-MYCMYC  90% Burkitt’s lymphomagood t(11;14)(q13;q32)BCL1-IGHCyclin D1  >90% MCL poor t(14;18)(q32;q21)BCL2-IGHBCL2  >90% FCLintermediate t(2;5)(p23;q35)NPM-ALKfusion 50% ALCLintermediate Prognostic value of chromosomal aberrations

42. FISH Excellent diagnostic tool for detection of well-defined chromosome aberrations Can also be used on tissue sections Requires limited handlings usage 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)