cDNA synthesis for BCR-ABL1 detection at the MMR level: the importance of using the appropriate kit

Biological Procedures Online

Jianxiang Chi²  Chryso Pierides²  Andrie Mitsidou²  Andri Miltiadou¹  Petroula Gerasimou¹  Paul Costeas¹ 

[1] Karaiskakio Foundation, Nicosia, Cyprus

[2] The Center for the Study of Haematological Malignancies, Nicosia, Cyprus

关键词: MRD;    MMR;    CML;    cDNA synthesis;    BCR-ABL;   

Others  :  1128601
DOI  :  10.1186/s12575-015-0014-x

 received in 2014-11-07, accepted in 2015-01-03,  发布年份 2015

【 摘 要 】

Background

The synthesis of complementary DNA (cDNA) for use in the detection of BCR-ABL1 at the Major Molecular Response (MMR) level is a well-established method used by clinical laboratories world-wide. However, the quality of cDNA provides sensitivity challenges and consequently affects the detection of Minimal Residual Disease (MRD).

Results

Herein, we evaluated six commercially available kits for the synthesis of cDNA according to amplification success rate, linearity and ABL1 copy number. Based on our results, the Invitrogen SuperScript® III Reverse Transcriptase kit performed better, among the ones used in this study, for the cDNA synthesis, followed by the First Strand cDNA Synthesis Kit for RT-PCR (AMV), available from Roche Applied Sciences.

Conclusions

Accurate and sensitive testing for the detection of abnormal transcripts, allows the correct stratification and treatment of patients. Hence, the use of a suitable kit for the cDNA synthesis is of great importance. This study provides a comprehensive point of reference for clinical laboratories in an attempt to optimize BCR-ABL1 detection. We propose that the Invitrogen SuperScript® III Reverse Transcriptase kit is the most suitable, among the ones used in this study, for the cDNA synthesis to be used for the detection of BCR-ABL1 at the MMR level in a CML MRD assay.

【 授权许可】

   
2015 Chi et al.; licensee BioMed Central.

【 图 表 】

Figure 3.

【 参考文献 】

• [1]Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C, et al.: Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002, 346:645-652.
• [2]Faderl S, Talpaz M, Estrov Z, Kantarjian HM: Chronic myelogenous leukemia: biology and therapy. Ann Intern Med 1999, 131:207-219.
• [3]Baccarani M, Dreyling M: Chronic myeloid leukaemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2010, 21(Suppl 5):v165-v167.
• [4]McGahon A, Bissonnette R, Schmitt M, Cotter KM, Green DR, Cotter TG: BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death. Blood 1994, 83:1179-1187.
• [5]Jamieson CH, Ailles LE, Dylla SJ, Muijtjens M, Jones C, Zehnder JL, et al.: Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N Engl J Med 2004, 351:657-667.
• [6]Daley GQ, Van Etten RA, Baltimore D: Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science 1990, 247:824-830.
• [7]Fainstein E, Marcelle C, Rosner A, Canaani E, Gale RP, Dreazen O, et al.: A new fused transcript in Philadelphia chromosome positive acute lymphocytic leukaemia. Nature 1987, 330:386-388.
• [8]Armitage JO: Bone marrow transplantation. N Engl J Med 1994, 330:827-838.
• [9]Biernaux C, Loos M, Sels A, Huez G, Stryckmans P: Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals. Blood 1995, 86:3118-3122.
• [10]Olavarria E, Kanfer E, Szydlo R, Kaeda J, Rezvani K, Cwynarski K, et al.: Early detection of BCR-ABL transcripts by quantitative reverse transcriptase-polymerase chain reaction predicts outcome after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood 2001, 97:1560-1565.
• [11]Singh Z, Medlin S, Usmani SZ: Molecular Monitoring and Treatment of Chronic Myeloid Leukemia (CML). J Clin Exp Pathol 2012, 2:1-9.
• [12]Cilloni D, Renneville A, Hermitte F, Hills RK, Daly S, Jovanovic JV, et al.: Real-time quantitative polymerase chain reaction detection of minimal residual disease by standardized WT1 assay to enhance risk stratification in acute myeloid leukemia: a European LeukemiaNet study. J Clin Oncol 2009, 27:5195-5201.
• [13]Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML, et al.: Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003, 349:1423-1432.
• [14]Radich JP, Gooley T, Bryant E, Chauncey T, Clift R, Beppu L, et al.: The significance of bcr-abl molecular detection in chronic myeloid leukemia patients “late,” 18 months or more after transplantation. Blood 2001, 98:1701-1707.
• [15]Branford S, Fletcher L, Cross NC, Muller MC, Hochhaus A, Kim DW, et al.: Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials. Blood 2008, 112:3330-3338.
• [16]Cortes J, Talpaz M, O’Brien S, Jones D, Luthra R, Shan J, et al.: Molecular responses in patients with chronic myelogenous leukemia in chronic phase treated with imatinib mesylate. Clin Cancer Res 2005, 11:3425-3432.
• [17]Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio G, Apperley J, et al.: Chronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNet. J Clin Oncol 2009, 27:6041-6051.
• [18]Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF, et al.: European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood 2013, 122:872-884.
• [19]Baccarani M, Castagnetti F, Gugliotta G, Palandri F, Soverini S: Response definitions and European Leukemianet Management recommendations. Best Pract Res Clin Haematol 2009, 22:331-341.
• [20]Cross NC, Feng L, Chase A, Bungey J, Hughes TP, Goldman JM: Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation. Blood 1993, 82:1929-1936.
• [21]Hochhaus A, Lin F, Reiter A, Skladny H, Mason PJ, van Rhee F, et al.: Quantification of residual disease in chronic myelogenous leukemia patients on interferon-alpha therapy by competitive polymerase chain reaction. Blood 1996, 87:1549-1555.
• [22]Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J, et al.: Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006, 108:28-37.
• [23]Marin D: Initial choice of therapy among plenty for newly diagnosed chronic myeloid leukemia. Hematol Am Soc Hematol Educ Progr 2012, 2012:115-121.
• [24]Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N, et al.: Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program. Leukemia 2003, 17:2318-2357.