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Minimal Residual Disease in B Cell Malignancies

Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia

Abstract

Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that ‘real-time’ quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline JH TaqMan probes to be used in combination with one of six corresponding germline JH primers and one allele specific oligonucleotide (ASO) primer complementary to the junctional region. In nine cases in which both approaches were compared, at least similar (n = 4) or slightly higher (n = 5) maximal sensitivities were obtained using an ASO primer. The ASO primer approach reached maximal sensitivities of at least 10−4 in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions had to be increased to remove or diminish background signals; this only concerned the frequently occurring JH4, JH5 and JH6 gene rearrangements. After optimization of the conditions (mainly by increasing the annealing temperature), only occasional aspecific amplification signals were observed at high threshold cycle (CT) values above 42 cycles and at least six cycles above the CT value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline JH TaqMan probes and six corresponding germline JH primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies.

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References

  1. Coustan-Smith E, Behm FG, Sanchez J, Boyett JM, Hancock ML, Raimondi SC, Rubnitz JE, Rivera GK, Sandlund JT, Pui CH, Campana D . Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia Lancet 1998 351: 550–554

    CAS  PubMed  Google Scholar 

  2. Cave H, van der Werff ten Bosch, Suciu S, Guidal C, Waterkeyn C, Otten J, Bakkus M, Thielemans K, Grandchamp B, Vilmer E . Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer – Childhood Leukemia Cooperative Group New Engl J Med 1998 339: 591–598

    CAS  PubMed  Google Scholar 

  3. van Dongen JJM, Seriu T, Panzer-Grumayer ER, Biondi A, Pongers-Willemse MJ, Corral L, Stolz F, Schrappe M, Masera G, Kamps WA, Gadner H, van Wering ER, Ludwig WD, Basso G, De Bruijn MA, Cazzaniga G, Hettinger K, van der Does-van den Berg A, Hop WC, Riehm H, Bartram CR . Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood Lancet 1998 352: 1731–1738

    CAS  Google Scholar 

  4. Gruhn B, Hongeng S, Yi H, Hancock ML, Rubnitz JE, Neale GA, Kitchingman GR . Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome Leukemia 1998 12: 675–681

    CAS  PubMed  Google Scholar 

  5. van Dongen JJ, Szczepanski T, De Bruijn MA, van den Beemd MW, de Bruin-Versteeg S, Wijkhuijs JM, Tibbe GJ, van Gastel-Mol EJ, Groeneveld K, Hooijkaas H . Detection of minimal residual disease in acute leukemia patients Cytokines Mol Ther 1996 2: 121–133

    CAS  PubMed  Google Scholar 

  6. Seriu T, Hansen-Hagge TE, Erz DH, Bartram CR . Improved detection of minimal residual leukemia through modifications of polymerase chain reaction analyses based on clonospecific T cell receptor junctions Leukemia 1995 9: 316–320

    CAS  PubMed  Google Scholar 

  7. Steenbergen EJ, Verhagen OJ, van Leeuwen EF, van den Berg H, Behrendt H, von dem Borne AE, van der Schoot CE . IgH/TCR delta PCR oligonucleotide liquid hybridization, a fast and sensitive assay for monitoring minimal residual disease in childhood B-precursor ALL Leukemia 1995 9: 216–222

    CAS  PubMed  Google Scholar 

  8. Pongers-Willemse MJ, Seriu T, Stolz F, d'Aniello E, Gameiro P, Pisa P, Gonzalez M, Bartram CR, Panzer-Grumayer ER, Biondi A, San Miguel JF, van Dongen JJM . Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia Leukemia 1999 13: 110–118

    CAS  PubMed  Google Scholar 

  9. Szczepanski T, Langerak AW, Wolvers-Tettero ILM, Ossenkoppele GJ, Verhoef G, Stul M, Petersen EJ, de Bruijn MAC, van't Veer MB, van Dongen JJM . Immunoglobulin and T cell receptor gene rearrangement patterns in acute lymphoblastic leukemia are less mature in adults than in children: implications for selection of PCR targets for detection of minimal residual disease Leukemia 1998 12: 1081–1088

    CAS  Google Scholar 

  10. Szczepanski T, Beishuizen A, Pongers-Willemse MJ, Hählen K, van Wering ER, Wijkhuijs JM, Tibbe GJM, De Bruijn MAC, van Dongen JJM . Cross-lineage T-cell receptor gene rearrangements occur in more than ninety percent of childhood precursor-B-acute lymphoblastic leukemias: alternative PCR targets for detection of minimal residual disease Leukemia 1999 13: 196–205

    CAS  PubMed  Google Scholar 

  11. Cross NC . Quantitative PCR techniques and applications Br J Haematol 1995 89: 693–697

    CAS  PubMed  Google Scholar 

  12. Cave H, Guidal C, Rohrlich P, Delfau MH, Broyart A, Lescoeur B, Rahimy C, Fenneteau O, Monplaisir N, d'Auriol L . Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of delta and gamma T-cell receptor genes Blood 1994 83: 1892–1902

    CAS  PubMed  Google Scholar 

  13. Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA . Quantitation of targets for PCR by use of limiting dilution Biotechniques 1992 13: 444–449

    CAS  PubMed  Google Scholar 

  14. Ouspenskaia MV, Johnston DA, Roberts WM, Estrov Z, Zipf TF . Accurate quantitation of residual B-precursor acute lymphoblastic leukemia by limiting dilution and a PCR-based detection system: a description of the method and the principles involved Leukemia 1995 9: 321–328

    CAS  PubMed  Google Scholar 

  15. Neale GAM, Coustan Smith E, Pan Q, Chen X, Gruhn B, Stow P, Behm FG, Pui CH, Campana D . Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia Leukemia 1999 13: 1221–1226

    CAS  Google Scholar 

  16. Heid CA, Stevens J, Livak KJ, Williams PM . Real time quantitative PCR Genome Res 1996 6: 986–994

    CAS  PubMed  Google Scholar 

  17. Holland PM, Abramson RD, Watson R, Gelfand DH . Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase Proc Natl Acad Sci USA 1991 88: 7276–7280

    CAS  PubMed  Google Scholar 

  18. Pongers-Willemse MJ, Verhagen OJ, Tibbe GJ, Wijkhuijs AJ, de Haas V, Roovers E, van der Schoot CE, van Dongen JJM . Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes Leukemia 1998 12: 2006–2014

    CAS  PubMed  Google Scholar 

  19. Mensink E, van de Locht A, Schattenberg A, Linders E, Schaap N, Geurts vK, De Witte T . Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR Br J Haematol 1998 102: 768–774

    CAS  PubMed  Google Scholar 

  20. Luthra R, McBride JA, Cabanillas F, Sarris A . Novel 5′ exonuclease-based real-time PCR assay for the detection of t(14;18)(q32;q21) in patients with follicular lymphoma Am J Pathol 1998 153: 63–68

    CAS  PubMed  Google Scholar 

  21. Marcucci G, Livak KJ, Bi W, Strout MP, Bloomfield CD, Caligiuri MA . Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay Leukemia 1998 12: 1482–1489

    CAS  PubMed  Google Scholar 

  22. Pallisgaard N, Clausen N, Schroder H, Hokland P . Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript Genes Chromosomes Cancer 1999 26: 355–365

    CAS  PubMed  Google Scholar 

  23. Emig M, Saussele S, Wittor H, Weisser A, Reiter A, Willer A, Berger U, Hehlmann R, Cross NC, Hochhaus A . Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR Leukemia 1999 13: 1825–1832

    CAS  Google Scholar 

  24. Verhagen OJHM, Wijkhuijs AJM, van der Sluijs-Gelling AJ, Szczepanski T, van der Linden-Schrever BEM, Pongers-Willemse MJ, van Wering ER, van Dongen JJM, van der Schoot CE . Suitable DNA isolation method for the detection of minimal residual disease by PCR techniques Leukemia 1999 13: 1298–1299

    CAS  PubMed  Google Scholar 

  25. van Dongen JJM, Wolvers-Tettero IL . Analysis of immunoglobulin and T cell receptor genes. Part I: Basic and technical aspects Clin Chim Acta 1991 198: 1–91

    CAS  PubMed  Google Scholar 

  26. Beishuizen A, Verhoeven M-AJ, Mol EJ, Breit TM, Wolvers-Tettero ILM, Van Dongen JJM . Detection of immunoglobulin heavy-chain gene rearrangements by Southern blot analysis. Recommendations for optimal results Leukemia 1993 7: 2045–2053

    CAS  Google Scholar 

  27. Steenbergen EJ, Verhagen OJ, van den Berg H, van Leeuwen EF, Behrendt H, Slater RR, von dem Borne AE, van der Schoot CE . Rearrangement status of the malignant cell determines type of secondary IgH rearrangement (V-replacement or V to DJ joining) in childhood B precursor acute lymphoblastic leukemia Leukemia 1997 11: 1258–1265

    CAS  PubMed  Google Scholar 

  28. Szczepanski T, Pongers-Willemse MJ, Langerak AW, Harts WA, Wijkhuijs AJM, van Wering ER, van Dongen JJM . Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6–19 and DH7–27 gene usage, can result in complete V–D–J rearrangements. and are rare in T-cell receptor αβ lineage Blood 1999 93: 4079–4085

    CAS  PubMed  Google Scholar 

  29. Willems PMW, Verhagen OJHM, Segeren C, Veenhuizen P, Guikema J, Wiemer E, Groothuis L, de Jong T, Kok H, Bloem A, Bos N, Vellenga E, Mensink EJBM, Sonneveld P, Lokhorst H, van der Schoot CE, Raymakers RAP . A consensus strategy to quantitate malignant cells in Myeloma patients validated in a multicentre study Blood (in press)

  30. Cook GP, Tomlinson IM . The human immunoglobulin VH repertoire Immunol Today 1995 16: 237–242

    CAS  Google Scholar 

  31. Tomlinson IM, Cook GP, Walter G, Carter NP, Riethman H, Buluwela L, Rabbitts TH, Winter G . A complete map of the human immunoglobulin VH locus Ann NY Acad Sci 1995 764: 43–46

    CAS  PubMed  Google Scholar 

  32. Corbett SJ, Tomlinson IM, Sonnhammer ELL, Buck D, Winter G . Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, ‘minor’ D segments or D–D recombination J Mol Biol 1997 270: 587–597

    CAS  PubMed  Google Scholar 

  33. Steenbergen EJ, Verhagen OJ, van Leeuwen EF, von dem Borne AE, van der Schoot CE . Distinct ongoing Ig heavy chain rearrangement processes in childhood B-precursor acute lymphoblastic leukemia Blood 1993 82: 581–589

    CAS  Google Scholar 

  34. Beishuizen A, Verhoeven MA, van Wering ER, Hahlen K, Hooijkaas H, van Dongen JJM . Analysis of Ig and T-cell receptor genes in 40 childhood acute lymphoblastic leukemias at diagnosis and subsequent relapse: implications for the detection of minimal residual disease by polymerase chain reaction analysis Blood 1994 83: 2238–2247

    CAS  Google Scholar 

  35. Steward CG, Goulden NJ, Katz F, Baines D, Martin PG, Langlands K, Potter MN, Chessells JM, Oakhill A . A polymerase chain reaction study of the stability of Ig heavy-chain and T-cell receptor delta gene rearrangements between presentation and relapse of childhood B-lineage acute lymphoblastic leukemia Blood 1994 83: 1355–1362

    CAS  Google Scholar 

  36. Sanz I . Multiple mechanisms participate in the generation of diversity of human H chain CDR3 regions J Immunol 1991 147: 1720–1729

    CAS  PubMed  Google Scholar 

  37. Wasserman R, Galili N, Ito Y, Reichard BA, Shane S, Rovera G . Predominance of fetal type DJH joining in young children with B precursor lymphoblastic leukemia as evidence for an in utero transforming event J Exp Med 1992 176: 1577–1581

    CAS  PubMed  Google Scholar 

  38. Yamada M, Wasserman R, Reichard BA, Shane S, Caton AJ, Rovera G . Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes J Exp Med 1991 173: 395–407

    CAS  PubMed  Google Scholar 

  39. Steenbergen EJ, Verhagen OJ, van Leeuwen EF, Behrendt H, Merle PA, Wester MR, von dem Borne AE, van der Schoot CE . B precursor acute lymphoblastic leukemia third complementarity-determining regions predominantly represent an unbiased recombination repertoire: leukemic transformation frequently occurs in fetal life Eur J Immunol 1994 24: 900–908

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Dutch Cancer Foundation/ Koningin Wilhelmina Fonds (grant SNWLK 97–1567) and PE Biosystems (Nieuwerkerk a/d IJssel, The Netherlands). We thank Dr T Szczepanski for support in the design of the TaqMan probes and for critical reading of the manuscript. We thank the Dutch Childhood Leukemia Study Group (DCLSG) for kindly providing ALL cell samples. Board members of the DCLSG are IM Appel, H van den Berg, JPM Bökkerink, MCA Bruin, JJ Groot-Loonen, SSN de Graaf, K Hählen, PM Hoogerbrugge, WA Kamps, FAE Nabben, JA Rammeloo, T Revesz, AYN Schouten-van Meeteren, AJP Veerman, M van Weel- Sipman and RS Weening.

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Verhagen, O., Willemse, M., Breunis, W. et al. Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia. Leukemia 14, 1426–1435 (2000). https://doi.org/10.1038/sj.leu.2401801

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