In Chronic Myeloid Leukemia (CML) Patients with Complete Cytogenetic Response to Imatinib, BCR-ABL Kinase Domain Mutations Are Relatively Rare and Not Consistently Associated with Subsequent Relapse.

Background: Imatinib mesylate (IM) induces complete cytogenetic remission (CCR) in 82% of newly diagnosed patients with CML in chronic phase. Most of these patients display minimal residual disease by nested RT-PCR for BCR-ABL. A previous study suggested that kinase domain (KD) mutations in BCR-ABL may be responsible for disease persistence in a significant percentage (9/13) of CCR patients (Chu et al. Blood, 2005). As the relapse rate in this small cohort was high, we decided to investigate the incidence of KD mutants in a more representative group of patients with stable CCR.

Methods: We performed nested RT-PCR for BCR-ABL in 72 CCR patients, using 2 sets of primers spanning the breakpoint and entire kinase domain, with ABL as a control gene. Mutation analysis was performed with direct sequencing and denaturing-HPLC of BCR-ABL amplicons. Median time on IM in these patients was 32 months (range, 8–65). In case of wild-type sequence, PCR products were subcloned and a median of 16 individual clones (range, 11–20) sequenced.

Results: Amplification was successful in 42/72 patients (58%). We detected 11 different point mutations in 9/42 (21%), of which 3 were novel (C305S, T212R, N231D). The remaining 8 mutants are known to be IM-resistant. C305S was found only by analysis of subclones, while all other mutations were detected upon initial analysis. 5/9 patients with a mutation had a corresponding rise in BCR-ABL transcripts by qRT-PCR. In the other 4, low or undetectable levels of BCR-ABL were maintained. For 2 of these, follow-up mutation analysis detected wild-type BCR-ABL, indicating a decrease of the mutant clone below the detection threshold. For patient 5, with G250E, an initial rise in BCR-ABL was followed by a continual decrease and stable CCR. Although most of the mutants detected were expected to lead to hematologic relapse, at a median follow-up of 12 months, this occurred in only one case.

Conclusions: KD mutations were detected in a modest proportion of CCR patients (12% of all patients and 21% of BCR-ABL-positive patients). The relatively high rate of PCR negativity may be due to the long amplicons (1.4kb). Our data imply that, in the majority of patients, disease persistence must be mediated by mechanisms other than KD mutations. In addition, some mutants failed to persist upon follow-up analysis, suggesting that the mutations occurred in transiently amplifying cells that are not capable of prolonged survival.