Comprehensive comparison of FISH, RT‐PCR, and RQ‐PCR for monitoring the BCR‐ABL gene after hematopoietic stem cell transplantation in CML

Abstract

The reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with fluorescence in situ hybridization (FISH) and real-time quantitative RT-PCR (RQ-PCR) for minimal residual disease (MRD) monitoring in 266 post-transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR-ABL positive samples determined by first-round (1st) RT-PCR, second-round (2nd) RT-PCR, and RQ-PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR-ABL/ABL ratio by RQ-PCR had a mean of 0.000 13 in the 1st RT-PCR-negative samples and 1.42 in the 1st RT-PCR-positive samples (P<0.001), and means of 0.000 39 and 0.51 in the 2nd RT-PCR-negative and -positive samples (P< 0.001). The mean ratios of BCR-ABL/ABL by RQ-PCR were significantly different in N/N (1st/2nd RT-PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ-PCR is confined to the 1st RT-PCR level. In this respect, monitoring of the 1st RT-PCR might be useful for estimating normalized BCR-ABL levels after transplantation. Nested RT-PCR was of limited use, as RQ-PCR quantified the BCR-ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT-PCR. FISH was significantly correlated with RQ-PCR in FISH-positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ-PCR in a few cases with molecular relapse. By analyzing a large number of samples post-transplant, we found that RQ-PCR might be the most useful assay for MRD monitoring; however, FISH and RT-PCR were found to be useful complementary tools.

Comparison of genetic changes between interphase and metaphase nuclei in monitoring CML and APL treatment using DC-FISH technique

“…[8][9][10][11] For patients with CML and APL, techniques for monitoring response to treatment are clinically important to identify, as early as possible, the patients who are at the a high risk of relapse, since some therapeutic approaches such as allogenic bone marrow transplantation may result in long-term disease-free survival. 4,[12][13][14][15] The determination and quantification of residual leukemic cells in patients with CML and APL in remission exposes those who might be at risk of relapse and could require close clinical monitoring. Prognostically valuable structural and numerical chromosomal aberrations may sometimes go undetected by using conventional GTG-banding.…”

Preemptive treatment of minimal residual disease post transplant in CML using real-time quantitative RT-PCR: a prospective, randomized trial

“…Our previous study showed that Q-RT-PCR agreed well with first-round RT-PCR, but not with second-round RT-PCR. 10 Our purpose for defining MRel in the present study was to facilitate decision-making with respect to therapeutic intervention and immunotherapy soon after transplantation, taking into account the risk of severe toxicity, and we used a Q-RT-PCR cut-off level from firstround RT-PCR rather than from second-round RT-PCR. As the cut-off level of Q-RT-PCR for first-round RT-PCR positivity was 0.0189, we arbitrarily defined MRel as a BCR-ABL/ABL ratio exceeding 10 À2 .…”

“…RT-PCR is the most sensitive of the molecular technology-based assays, but it is limited by its qualitative nature, and thus may not detect early relapse in individual patients. On the other hand, Q-RT-PCR correlates strongly with cytogenetics, FISH, RT-PCR, competitive RT-PCR, and Southern blot, [6][7][8][9][10] and its usefulness for MRD monitoring has been reported by many authors. 6,11,12 An important issue when deciding therapeutic intervention using results from Q-RT-PCR is: how should molecular relapse (MRel) be defined and at what level immunotherapy should be given?…”

Cytogenetic clonal evolution alone in CML relapse post-transplantation does not adversely affect response to imatinib mesylate treatment

“…21,22 To be brief, total RNA was extracted using an RNAqueous Kit (Ambion, Austin, TX, USA) and reverse transcription was performed using 1 mg RNA. The two-step nested procedure was used for the nested RT-PCR amplification (sensitivity, 10…”