Quantification of minimal residual disease in patients with BCR‐ABL‐positive acute lymphoblastic leukaemia using quantitative competitive polymerase chain reaction

Abstract

Summary. We analysed 20 patients with BCR-ABL-positive acute lymphoblastic leukaemia (ALL) by quantitative competitive polymerase chain reaction (QC-PCR) to study the kinetics of the leukaemic clone. Consecutive samples of 16 patients (minor-bcr, n 10; major-bcr, n 6) were analysed after conventional chemotherapy and/or bone marrow transplantation (BMT). DNA competitor templates co-amplifying with either p210 or p190 BCR-ABL cDNA were used for quanti®cation of leukaemia-speci®c BCR-ABL mRNA. In all samples, total ABL transcripts were measured as internal control, and the percentage of BCR-ABL/ABL molecules was calculated. Following induction chemotherapy the number of BCR-ABL transcripts was reduced by a maximum of 2±3 logs. In most patients, additional chemotherapy did not lead to further reduction of BCR-ABL mRNA. In two patients, conventional chemotherapy plus autologous BMT in complete haematological remission resulted in a total reduction of the transcript level of more than 3 logs. In two other patients, allogeneic BMT caused a transient reduction of the BCR-ABL transcripts below the detection level of our method (<1 blast cell in 10 5 normal cells) for a period of 7 and 11 months, respectively. The achievement of PCR negativity did not guarantee sustained remission. Both patients relapsed and BCR-ABL transcript levels rose by more than 1 log prior to frank relapse. Our data demonstrate that quanti®cation of BCR-ABL mRNA allows the evaluation of the dynamics of the leukaemic clone and thus is valuable for the evaluation of minimal residual leukaemia following various therapies and the early detection of increasing BCR-ABL transcripts prior to relapse.

Standardized MRD quantification in European ALL trials: Proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008

“…The second most frequent fusion transcript involves break points in a 5.8-kb region spanning exons 12-16, known as the major break-point cluster region (M-bcr) and is typical of chronic myeloid leukemia (CML) but is also seen in 30% of patients with Ph þ ALL. 34,36,37,56 Detection of BCR-ABL fusion transcripts by reverse transcription PCR is relevant not only for diagnostic purposes but also in particular for quantitative monitoring of MRD. 34 Several studies have shown that levels of MRD in the bone marrow (BM) of patients with Ph þ ALL, assessed by RQ-PCR after induction and after consolidation treatment, is a powerful indicator of prognosis.…”

“…Flow cytometric and molecular methods that allow sensitive detection and specific quantification of residual leukemic cells have been developed. [28][29][30][31][32][33][34][35][36][37][38][39][40] Standardization of methodologies and definitions of common MRD terms become increasingly important, not only to ensure comparability of MRD data between different MRD laboratories within a single MRD-based treatment protocol but also to provide a sound basis for the comparison of MRD results between different treatment protocols, including those that evaluate the effectiveness of new agents.…”

Serial minimal residual disease (MRD) analysis as a predictor of response duration in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) during imatinib treatment

“…24,25 In one small study of five patients employing quantitative PCR, increases of 42 log in PB of two patients was closely followed by relapse while patients without such pronounced increase remained in CR for prolonged periods. 28 However, absolute quantitative parameters reflecting MRD levels and dynamics have not yet been established statistically to predict probability of relapse within a certain time period.…”

“…Although a relatively low interassay variability has been found under specific experimental conditions, 28,31 the interassay variance may be substantially higher in the setting of a multicenter trial, in which sample shipment may impair RNA quality. Furthermore, the thresholds that were identified in our study need to be validated in future prospective trials.…”

“…27 In addition, increasing Bcr-Abl levels after BMT were indicative of imminent relapse. 28 Specific criteria for interpreting MRD results in Ph+ALL patients receiving imatinib therapy remain to be established.…”