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.
Authors: Sherbenou Daniel W. , Wong Matthew J. , Humayun Adil , McGreevey Laura S. , Yang Rui , Heinrich Michael C. , Press Richard D. , Druker Brian J. , Deininger Michael W. 
Published: April 2020

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.

Characteristics of CCR patients with a KD mutation. Patient No. Age at Study (years) Time on IM (months) CD34+ Mutation Detected CD34-/MNC Mutation Detected Time to Last Follow-up (months) Current Cytogenetic Status Follow-up Sample Mutation Status No Amp: No PCR amplification. NA: No sample available. Mutations detected by nested RT-PCR and sequencing (1), or D-HPLC (2), or both in parallel (3). Subcloning results are shown in parentases. 3 84 40 NA T212R3(15,15) 11 5% Ph+ In Progress 5 27 28 No Amp G250E3(17/17) 14 CCR G250E 19 52 18 N231D2 WT3(C305S:3/20) 5 CCR NA 20 43 43 Y253F, M244V2 M244V3(20/20) NA NA NA 21 24 63 WT3 (16/16) G321E, E355G2 NA NA NA 28 80 31 NA T315I1 (15/15) 12 CCR In Progress 30 51 39 NA Y253H1 (16/16) 10 CCR WT 31 39 43 NA T315I2 17 CCR No Amp 33 69 32 NA F359V2 5 100% Ph+ NA