At the time of diagnosis, Ph cells from both chronic phase chronic myeloid leukemia and acute lymphoblastic leukemia patients already harbour BCR-ABL kinase domain mutations

Background and Aims. Mutations in the Bcr-Abl kinase domain (KD) are often detected at the time of resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia-positive (Ph+) leukemias. Mutations in rare Ph+ cells have been detected in some imatinib-naïve advanced-phase chronic myeloid leukemia (CML) patients (pts), but it is still unclear whether and with what incidence low level mutations may already be detectable at the time of diagnosis in Ph+ acute lymphoblastic leukemia (ALL) pts and in chronic-phase (CP) chronic myeloid leukemia pts. We therefore analyzed cDNA samples from 24 newly diagnosed pts with Ph+ ALL (n=13) or CP-CML (n=11) who subsequently received TKI therapy (imatinib, dasatinib or nilotinib). Methods. Screening for low level mutations was performed by cloning the Bcr-Abl KD (a.a. 206-524) in a bacterial vector and sequencing 200 independent clones for each pt. Results. All pts had evidence of aberrant KD sequences. Three to twelve different mutations were detected in each pt. Each mutation was present in two to five independent clones. A total of 115 mutations (including 41 silent, 5 nonsense, and 69 missense mutations) were observed. The vast majority (107/115, 93%) have never been reported in association with TKI resistance and are likely not to confer any advantage under TKI selective pressure. Interestingly, 103/115 (90%) mutations were transitions: G>A (n=30), A>G (n=25), C>T (n=25), T>C (n=23). Such a high prevalence of transitions (normally occurring 1.4 times more frequently than transversions) suggests that a specific mechanism generating mutations is active in Ph+ cells (activation- induced cytidine deaminase?). One of the eleven CP-CML pt received hydroxyurea for 6 months before starting imatinib therapy. In this pt, high-sensitivity mutation screening was performed again immediately before imatinib start and showed further accumulation of mutations. Eight Ph+ ALL pts and three CML pts subsequently relapsed with evidence of mutations, but only two with a mutation (T315I) that was already detectable at diagnosis. The remaining thirteen pts are in persistent remission after a follow up ranging from 12 to 52 months, although four of them were harbouring known imatinib-(H396P, D276G, E355G) or dasatinib-(F317L) resistant mutations at low levels. Conclusions. Our observations suggest that: a) Bcr-Abl KD mutations can probably be found at diagnosis in all CP-CML and Ph+ ALL pts; b) mutations seem to arise randomly and most of them are silent or not conferring any growth advantage under the selective pressure of TKIs; c) generation of mutations seems to be linked to Bcr-Abl-driven genetic instability; d) TKI-resistant mutations present at low levels at diagnosis do not always outgrow and lead to relapse, probably because some of them arise in cell clones with limited self-renewal capacity. This warns against high-sensitivity mutation screening of all CML and Ph+ ALL pts before the start of TKI therapy.