Clonal Evolution of BCR-ABL1 Kinase Domain Mutation In Tyrosine Kinase Inhibitor Treatment Patient

Abstract 597

Resistance against tyrosine kinase inhibitors (TKI) has been an important issue in treatment for chronic myeloid leukemia (CML) and Ph-positive acute lymphoblastic leukemia (ALL), and it mainly result from BCR-ABL1 kinase domain mutation. Many studies have focused on the issue, however, characteristics of origin and evolution of BCR-ABL1 mutant clones remains unclear. In this study, we analyzed BCR-ABL1 mutations within the kinase domain for 189 bone marrow or peripheral blood samples, which were collected from 151 cases of CML or Ph-positive ALL. All samples were taken prior to and in the process of TKI treatment, followed by nested PCR and sequencing. Partial of the samples were cloned and sequenced to determine the composition and proportion of mutant clones. Quantification of BCR-ABL1 fusion gene expression was performed according to Europe Against Cancer Program protocol. We reported here that (i) Totally 43 patients harbored 27 BCR-ABL1 mutations, consisting of 24 point mutations, two frameshift mutations (1086_1087delAG/D363fsX380 and 1330delG/D444fsX452) and one in-frame insertion mutation (1073-1074insAAA/357-358insK); (ii) Both deletion mutations only occurred transiently with very low level of BCR-ABL1 expression (0.020% and 0.013% respectively), whereas the insertion mutation was detected in a nearly 100% of mutant clone with completely resistant against the Imatinib; (iii) One G250E and one L370Q mutant clones were detected in 4.1% (2/49) of the patients with BCR-ABL1 expression at low level (0.55% and 0.16% respectively) prior to TKI therapy. Following TKI treatment, resistant mutations were detected in 75.4% (43/57) of patients who presented drug resistance clinically; (iv) Coexistence of multiple mutations in a single clone or temporal evolution of different mutation clones were found in 6 patients, 4 of whom underwent reduction of drug dosage. One clone carrying E355G and T315I mutations simultaneously was detected in patient No.1, and proportion of the double mutant clone increased following incidence of TKI resistance. Two different clones carrying G250E and M351T respectively were detected in Patient No.2. The proportion of M351T clone with less resistance decreased, and the proportion of G250E clone with moderate resistance went up during Imatinib therapy. A similar phenomenon was seen in Patient No.6 carrying the mutation of Q252H and T315I. Two different clones carrying E275D and D444fsX452 respectively were detected at low proportion (11.3% and 15.1% respectively) and low BCR-ABL1 expression 0.013% in Patient No.3, and surprisingly, the two clones disappeared and a T315I clone emerged and became dominant a month after with CML progressed toward the blast crisis; (v) Three different TKI-resistant clones, Y253H, F359V and F486S, were simultaneously detected with a high BCR-ABL1 expression (142%) in a Ph positive ALL (Patient No.4), who is in follow-up presently; (vi)Two different clones carrying G250E and T315I respectively were detected in Patient No.5. The proportion of G250E clone with moderate resistance decreased, whereas frequency of the T315I clone with highly resistance increased following Imatinib and Dasatinib administration. This patient received allo-geneic bone marrow transplantation (BMT) due to TKI resistance, and thereafter the D363fsX380 mutation without kinase activity was transiently detected with very low level of BCR-ABL1 expression, and Dasatinib sensitivity was restored; (vii) Clone and sequencing also find that mutant clones were randomly occurred with low percentage even in pre TKI treatment samples and K562 cell line. These mutations could be common or uncommon resistant mutations, and also can be synonymous mutations or even loss of kinase activity mutations. Taken together, our data indicate that (i) Origin of mutant clones may be random in samples with and without TKI treatment; (ii) The mutation clones always have lower kinase activity than wild type clone, thus it can not evolution into the major clone in the absence of TKI; (iii) The TKI-resistant clone may acquire proliferating advantage, and evolve into the dominant clone under the pressure of TKI; and (iv) We further propose that temporary interruption of TKI and switching to non-selective therapy can be a valid therapeutic option for anti-resistant strategy.