Serial Analysis of BCR-ABL Kinase Domain Mutations Prior to and During Imatinib Treatment for Newly Diagnosed and Recurrent BCR-ABL Positive Acute Lymphoblastic Leukemia.

Patients with newly diagnosed and recurrent Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) differ profoundly in their responsiveness to imatinib: whereas nearly all imatinib-naïve patients with de novo Ph+ALL initially achieve a complete remission (CR), eventually followed by secondary resistance, imatinib rarely induces a CR in patients with disease recurrence after chemotherapy. Mutations in the BCR-ABL tyrosine kinase domain (KD) are commonly associated with acquired imatinib resistance, but their role in primary resistance is not known. Recently, we determined that the same mutation found at relapse could already be detected prior to first imatinib exposure in 30% to 40% of patients with de novo Ph+ALL but surprisingly was not associated with an inferior response to imatinib monotherapy, which resulted in a CR rate exceeding 90%. Possible reasons for the inferior response of patients with recurrent Ph+ALL include a higher frequency of pre-existing mutations, a preponderance of mutations with greater transforming activity, a larger mutant clone or the contribution of non-mutational resistance mechanisms. To elucidate the potential role of these resistance mechanisms, we compared patients with de novo Ph+ALL and patients who had failed prior chemotherapy with respect to the frequency and pattern of pre-existing mutations, the level of mutant clones, the outgrowth kinetics of mutations during the first 4 weeks of imatinib monotherapy and the concordance between mutations detected prior to imatinib and at the time of relapse.

By denaturing high-performance liquid chromatography (D-HPLC; sensitivity 0.1-1%), ligation PCR (sensitivity 0.1-0.005%) and cDNA sequencing, we examined bone marrow and/or peripheral blood samples collected pre-treatment, during therapy and at relapse from 91 patients with Ph+ lymphoid leukemias who were enrolled in the initial phase II studies of imatinib for treatment of BCR-ABL positive leukemias after chemotherapy failure (n=65), or in a prospective, randomized clinical trial assessing front-line imatinib therapy in elderly patients with newly diagnosed Ph+ALL (n=26).

Prior to imatinib treatment, the frequency of TKD mutations did not differ significantly between patients with de novo and recurrent Ph+ ALL, respectively (30 % vs. 46%; p=n.s.) as detected by highly sensitive DHPLC and ligation PCR. P-loop mutations predominated in both patient groups (92% and 85% of detected mutations). The T315I gatekeeper mutation was identified in 4 patients, mutations in the activation loop were uncommon. Following only four weeks imatinib, the prevalence of detectable mutations increased substantially in patients with recurrent (to 75%) but not with de novo Ph+ALL (38%). Moreover, the median mutated bcr-abl transcript levels after 4 weeks of imatinib treatment were significantly higher in patients with recurrent than with de novo Ph+ALL (median 10%, [range 0-100] versus median 0%;range [0-7]). Conspicuously, we observed good concordance between the type of bcr-abl mutation detected prior to imatinib and at relapse.

The incidence, pattern and levels of TKD mutations detected prior to imatinib and at relapse are similar in advanced and newly diagnosed Ph+ALL. In contrast, the outgrowth kinetics of cells with TKD mutations were far more rapid in patients with recurrent disease. These data provide clinical-translational evidence for a cooperative effect between mutational and non-mutational resistance mechanisms in Ph+ALL, and highlight the importance of early mutation monitoring using highly sensitive methodology.

Hochhaus:Novartis : Research Funding. Ottmann:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria.