Biomedical papers - Ahead of Print

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. X:X | 10.5507/bp.2017.006

Clonality testing of lymphoproliferative disorders in a large cohort of primary and consultant biopsies

Michaela Svachovaa, Martin Tichya, Patrik Flodra, Jana Steigerovaa,b, Zdenek Kolara,b, Jan Bouchala,b
a Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Czech Republic
b Institute of Molecular and Translation Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic

Background: Lymphoproliferative disease often presents the clinician and pathologist with a diagnostic dilemma, particularly in the early course of the disease.

Methods: We used modified BIOMED-2 protocols to detect monoclonal expansions of immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) genes in 957 formalin-fixed paraffin-embedded samples from 717 patients. To eliminate false-positive results, heteroduplex analysis was used after PCR reactions. The impact of different fixatives on DNA quality and performance of PCR was assessed.

Results: In the class of B lymphomas we detected clonal IgH rearrangement in nearly 80% of cases and in the class of T lymphomas in 64% of cases. Performance of the assays was 94.7% and 92.5% for IgH and TCR clonality, respectively. Clonality rates in various B and T lymphomas were in concordance with previous studies. We also present 10 difficult cases where PCR analysis of IgH and TCR gene rearrangements significantly contributed to a decision on the correct diagnosis.

Conclusion: These results confirm that the PCR-based analysis is suitable as a routine method and is helpful in establishing a diagnosis in morphologically unclear cases.

Keywords: clonality; IgH and TCR gene rearrangement; lymphoid proliferation; polymerase chain reaction; BIOMED-2

Received: July 14, 2016; Accepted: February 24, 2017; Prepublished online: March 14, 2017


References

  1. Medeiros LJ, Carr J. Overview of the role of molecular methods in the diagnosis of malignant lymphomas. Arch Pathol Lab Med 1999;123:1189-207. Go to PubMed...
  2. Langerak AW, Groenen PJ, Jm van Krieken JH, van Dongen JJ. Immunoglobulin/T-cell receptor clonality diagnostics. Expert Opin Med Diagn 2007;1:451-61. Go to original source... Go to PubMed...
  3. Evans PA, Pott Ch, Groenen PJ, Salles G, Davi F, Berger F, Garcia JF, van Krieken JH, Pals S, Kluin P, Schuuring E, Spaargaren M, Boone E, Gonzalez D, Martinez B, Villuendas R, Gameiro P, Diss TC, Mills K, Morgan GJ, Carter GI, Milner BJ, Pearson D, Hummel M, Jung W, Ott M, Canioni D, Beldjord K, Bastard C, Delfau-Larue MH, van Dongen JJ, Molina TJ, Cabecadas J. Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia 2007;21:207-14. Go to original source... Go to PubMed...
  4. Ramasamy I, Brisco M, Morley A. Improved PCR method for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell neoplasms. J Clin Pathol 1992;45:770-5. Go to original source... Go to PubMed...
  5. Elenitoba-Johnson KS, Bohling SD, Mitchell RS, Brown MS, Robetorye RS. PCR analysis of the immunoglobulin heavy chain gene in polyclonal processes can yield pseudoclonal bands as an artifact of low B cell number. J Mol Diagn 2000;2:92-6. Go to original source... Go to PubMed...
  6. Tai YC, Peh SC. Feasibility of T-cell receptor gamma (TCRgamma) gene angement on formalin-fixed, paraffin-embedded tissues by PCR assays. Singapore Med J 2003;44:250-5. Go to PubMed...
  7. Bielawski K, Zaczek A, Lisowska U, Dybikowska A, Kowalska A, Falkiewicz B. The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis. Int J Mol Med 2001;8:573-8. Go to original source... Go to PubMed...
  8. Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 2002;161:1961-71. Go to original source... Go to PubMed...
  9. Theriault C, Galoin S, Valmary S, Selves J, Lamant L, Roda D, Rigal-Huguet F, Brousset P, Delsol G, Al Saati T. PCR analysis of immunoglobulin heavy chain (IgH) and TcR-gamma chain gene rearrangements in the diagnosis of lymphoproliferative disorders: results of a study of 525 cases. Mod Pathol 2000;13:1269-79. Go to original source... Go to PubMed...
  10. Gurbity TP, Bagdi E, Groen NA, Budel LM, Abbou M, Krenacs L, Dinjens WN. Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5'-->3' DNA polymerase and 3'-->5' exonuclease activity. Virchows Arch 2003;443:643-8. Go to original source... Go to PubMed...
  11. Tonegawa S. Somatic generation of antibody diversity. Nature 1983;302:575-81. Go to original source... Go to PubMed...
  12. Trainor KJ, Brisco MJ, Story CJ, Morley AA. Monoclonality in B-lymphoproliferative disorders detected at the DNA level. Blood 1990;75:2220-2. Go to PubMed...
  13. Corneo B, Moshous D, Gungor T, Wulffraat N, Philippet P, Le Deist FL, Fischer A, de Villartay JP. Identical mutations in RAG1 or RAG2 genes leading to defective V(D)J recombinase activity can cause either T-B-severe combined immune deficiency or Omenn syndrome. Blood 2001;97:2772-6. Go to original source... Go to PubMed...
  14. Bassing CH, Swat W, Alt FW. The mechanism and regulation of chromosomal V(D)J recombination. Cell 2002;109 Suppl:S45-55. Go to original source... Go to PubMed...
  15. van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, Garcia-Sanz R, van Krieken JH, Droese J, Gonzalez D, Bastard C, White HE, Spaargaren M, Gonzalez M, Parreira A, Smith JL, Morgan GJ, Kneba M, Macintyre EA. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003;17:2257-317. Go to original source... Go to PubMed...
  16. Vanasse GJ, Concannon P, Willerford DM. Regulated genomic instability and neoplasia in the lymphoid lineage. Blood 1999;94:3997-4010. Go to PubMed...
  17. Messier TL, O'Neill JP, Hou SM, Nicklas JA, Finette BA. In vivo transposition mediated by V(D)J recombinase in human T lymphocytes. EMBO J 2003;22:1381-8. Go to original source... Go to PubMed...
  18. Inlay M, Xu Y. Epigenetic regulation of antigen receptor rearrangement. Clin Immunol 2003;109:29-36. Go to original source... Go to PubMed...
  19. Boule JB, Rougeon F, Papanicolaou C. Terminal deoxynucleotidyl transferase indiscriminately incorporates ribonucleotides and deoxyribonucleotides. J Biol Chem 2001;276:31388-93. Go to original source... Go to PubMed...
  20. Bagg A. Immunoglobulin and T-cell receptor gene rearrangements: minding your B's and T's in assessing lineage and clonality in neoplastic lymphoproliferative disorders. J Mol Diagn 2006;8:426-9-quiz526-7. Go to original source... Go to PubMed...
  21. Lorenzen J, Jux G, Zhao-Hohn M, Klockner A, Fischer R, Hansmann ML. Detection of T-cell clonality in paraffin-embedded tissues. Diagn Mol Pathol 1994;3:93-9. Go to original source... Go to PubMed...
  22. Benhattar J, Delacretaz F, Martin P, Chaubert P, Costa J. Improved polymerase chain reaction detection of clonal T-cell lymphoid neoplasms. Diagn Mol Pathol 1995;4:108-12. Go to original source... Go to PubMed...
  23. Kusic B, Dominis M, Dzebro S, Antica M. Molecular insight into the diagnosis of lymphoma. Int J Mol Med 2003;12:667-71. Go to original source... Go to PubMed...
  24. Bagg A, Braziel RM, Arber DA, Bijwaard KE, Chu AY. Immunoglobulin heavy chain gene analysis in lymphomas: a multi-center study demonstrating the heterogeneity of performance of polymerase chain reaction assays. J Mol Diagn 2002;4:81-9. Go to original source... Go to PubMed...
  25. Svachova M, Tichy M. PCR analysis of immunoglobulin heavy chain and TCR gene rearrangements in diagnosis of lymphoproliferative disorders on formalin-fixed, paraffin-embedded tissues. Neoplasma 2008;55:36-41. Go to PubMed...
  26. Mitha N, McGlennen RC. Methods to detect clonal gene rearrangements in lymphomas and leukemias. Methods Mol Med 2001;49:189-209. Go to PubMed...
  27. McCarthy KP, Sloane JP, Kabarowski JH, Matutes E, Wiedemann LM. A simplified method of detection of clonal rearrangements of the T-cell receptor-gamma chain gene. Diagn Mol Pathol 1992;1:173-9. Go to PubMed...
  28. McCarthy KP, Sloane JP, Kabarowski JH, Matutes E, Wiedemann LM. The rapid detection of clonal T-cell proliferations in patients with lymphoid disorders. Am J Pathol 1991;138:821-8. Go to PubMed...
  29. Gleissner B, Maurer J, Thiel E. Detection of immunoglobulin heavy chain genes rearrangements in B-cell leukemias, lymphomas, multiple myelomas, monoclonal and polyclonal gammopathies. Leuk Lymphoma 2000;39:151-5. Go to original source... Go to PubMed...
  30. Nihal M, Mikkola D, Wood GS. Detection of clonally restricted immunoglobulin heavy chain gene rearrangements in normal and lesional skin: analysis of the B cell component of the skin-associated lymphoid tissue and implications for the molecular diagnosis of cutaneous B cell lymphomas J Mol Diagn 2000;2:5-10. Go to original source... Go to PubMed...
  31. Groenen PJ, Langerak AW, van Dongen JJ, van Krieken JH. Pitfalls in TCR gene clonality testing: teaching cases. J Hematop 2008;1:97-109. Go to original source... Go to PubMed...
  32. Gonzalez M, Gonzalez D, Lopez-Perez R, Garcia-Sanz R, Chillon MC, Balanzategui A, Mateos MV, Alaejos I, Langerak AW, Orfao A, Van Dongen JJ, San Miguel JF. Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies. Haematologica 1999;84:779-84. Go to PubMed...
  33. Diss TC, Pan L, Peng H, Wotherspoon AC, Isaacson PG. Sources of DNA for detecting B cell monoclonality using PCR. J Clin Pathol 1994;47:493-6. Go to original source... Go to PubMed...
  34. Nikiforova MN, Hsi ED, Braziel RM, Gulley ML, Leonard DG, Nowak JA, Tubbs RR, Vance GH, Van Deerlin VM. Detection of clonal IGH gene rearrangements: summary of molecular oncology surveys of the College of American Pathologists. Arch Pathol Lab Med 2007;131:185-9. Go to PubMed...
  35. Campo E, Swerdlow SH, Harris NL, Pileri S, Stein H, Jaffe ES. The 2008 WHO classification of lymphoid neoplasms and beyond: evolving concepts and practical applications. Blood 2011;117:5019-32. Go to original source... Go to PubMed...
  36. Miettinen M, Lasota J. Polymerase chain reaction based gene rearrangement studies in the diagnosis of follicular lymphoma--performance in formaldehyde-fixed tissue and application in clinical problem cases. Pathol Res Pract 1997;193:9-19. Go to original source... Go to PubMed...
  37. Langerak AW, Groenen PJ, Bruggemann M, Beldjord K, Bellan C, Bonello L, Boone E, Carter GI, Catherwood M, Davi F, Delfau-Larue MH, Diss T, Evans PA, Gameiro P, Garcia Sanz R, Gonzalez D, Grand D, Hakansson A, Hummel M, Liu H, Lombardia L, Macintyre EA, Milner BJ, Montes-Moreno S, Schuuring E, Spaargaren M, Hodges E, van Dongen JJ. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia 2012;26:2159-71. Go to original source... Go to PubMed...
  38. Segal GH, Jorgensen T, Scott M, Braylan RC. Optimal primer selection for clonality assessment by polymerase chain reaction analysis: II. Follicular lymphomas. Hum Pathol 1994;25:1276-82. Go to original source... Go to PubMed...
  39. Hartmann S, Helling A, Döring C, Renné Ch. Clonality testing of malignant lymphomas with the BIOMED-2 primers in a large cohort of 1969 primary and consultant biopsies. Pathology - Research and Practice 2013;209:495-502.
  40. Kuppers R, Klein U, Hansmann ML, Rajewsky K. Cellular origin of human B-cell lymphomas. N Engl J Med 1999;341:1520-9. Go to original source... Go to PubMed...
  41. Brauninger A, Kuppers R, Spieker T, Siebert R, Strickler JG, Schlegelberger B, Rajewsky K, Hansmann ML. Molecular analysis of single B cells from T-cell-rich B-cell lymphoma shows the derivation of the tumor cells from mutating germinal center B cells and exemplifies means by which immunoglobulin genes are modified in germinal center B cells. Blood 1999;93:2679-87. Go to PubMed...
  42. Child FJ, Woolford AJ, Calonje E, Russell-Jones R, Whittaker SJ. Molecular analysis of the immunoglobulin heavy chain gene in the diagnosis of primary cutaneous B cell lymphoma. J Invest Dermatol 2001;117:984-9. Go to original source... Go to PubMed...
  43. Venkatraman L, Catherwood MA, Patterson A, Lioe TF, McCluggage WG, Anderson NH. Role of polymerase chain reaction and immunocytochemistry in the cytological assessment of lymphoid proliferations. J Clin Pathol 2006;59:1160-5. Go to original source... Go to PubMed...
  44. Berget E, Helgeland L, Molven A, Vintermir OK. Detection of clonality in follicular lymphoma using formalin-fixed, paraffin embedded tissue samples and BIOMED-2 immunoglobulin primers. J Clin Pathol 2011;64:37-41. Go to original source... Go to PubMed...
  45. McClure RF, Kaur P, Pagel E, Ouillette PD, Holtegaard CE, Treptow CL, Kurtin PJ. Validation of immunoglobulin gene rearrangement detection by PCR using commercially available BIOMED-2 primers. Leukemia 2006;20:176-9. Go to original source... Go to PubMed...
  46. Kosari F, Shishehbor F, Saffar H, Sadeghipour A. PCR-based clonality analysis in diffuse large B-cell lymphoma using BIOMED-2 primers of IgH (FR3) on formalin-fixed paraffin embedded tissue. Arch Iran Med 2013;16:526-9. Go to PubMed...
  47. Kanzler H, Kuppers R, Hansmann ML, Rajewsky K. Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells. J Exp Med 1996;184:1495-505. Go to original source... Go to PubMed...
  48. Seitz V, Hummel M, Marafioti T, Anagnostopoulos I, Assaf C, Stein H. Detection of clonal T-cell receptor gamma-chain gene rearrangements in Reed-Sternberg cells of classic Hodgkin disease. Blood 2000;95:3020-4. Go to PubMed...
  49. Hebeda KM, Van Altena MC, Rombout P, Van Krieken JH, Groenen PJ. PCR clonality detection in Hodgkin lymphoma. J Hematop 2009;2:34-41. Go to original source... Go to PubMed...
  50. Burack WR, Laughlin TS, Friedberg JW, Spence JM, Rothberg PG. PCR assays detect B-lymphocyte clonality in formalin-fixed, paraffin-embedded specimens of classical hodgkin lymphoma without microdissection. Am J Clin Pathol 2010;134:104-11. Go to original source... Go to PubMed...
  51. Zaki MA, Wada N, Kohara M, Ikeda J, Hori Y, Fujita S, Ogawa H, Sugiyama H, Hino M, Kanakura Y, Morii E, Aozasa K. Presence of B-cell clones in T-cell lymphoma. Eur J Haematol 2011;86:412-9. Go to original source... Go to PubMed...
  52. Whittaker SJ, Smith NP, Jones RR, Luzzatto L. Analysis of beta, gamma, and delta T-cell receptor genes in mycosis fungoides and Sezary syndrome. Cancer 1991;68:1572-82. Go to original source... Go to PubMed...
  53. Ponti R, Quaglino P, Novelli M, Fierro MT, Comessatti A, Peroni A, Bonello L, Bernengo MG. T-cell receptor gamma gene rearrangement by multiplex polymerase chain reaction/heteroduplex analysis in patients with cutaneous T-cell lymphoma (mycosis fungoides/Sezary syndrome) and benign inflammatory disease: correlation with clinical, histological a Br J Dermatol 2005;153:565-73. Go to original source... Go to PubMed...
  54. Gallardo F, Costa C, Bellosillo B, Solé F, Estrach T, Servitje O, García-Muret MP, Barranco C, Serrano S, Pujol RM. Lymphomatoid Papulosis Associated with Mycosis Fungoides: Clinicopathological and Molecular Studies of 12 Cases. Acta Derm Venereol 2004;84:463-8. Go to original source... Go to PubMed...
  55. Steinhoff M, Hummel M, Anagnostopoulos I, Kaudewitz P, Seitz V, Assaf C, Sander C, Stein H. Single-cell analysis of CD30+ cells in lymphomatoid papulosis demonstrates a common clonal T-cell origin. Blood 2002;100:578-84. Go to original source... Go to PubMed...
  56. Langerak AW, Szczepanski T, van der Burg M, Wolvers-Tettero IL, van Dongen JJ. Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations. Leukemia 1997;11:2192-9. Go to original source... Go to PubMed...
  57. Zhou XG, Sandvej K, Gregersen N, Hamilton-Dutoit SJ. Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement. Mol Pathol 1999;52:104-10. Go to original source... Go to PubMed...