Low level mutations in the Bcr-Abl kinase domain may already be detected at diagnosis both in patients with Philadelphia-positive acute lymphoblastic leukemia and in patients with chronic phase chronic myeloid leukemia

Abstract

Mutations in the Bcr-Abl kinase domain (KD) are often detected at the time of resistance to tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts). It is still unclear whether and in how many pts low level mutations may already be detectable at the time of diagnosis. We therefore analyzed cDNA samples from 22 newly diagnosed pts with Ph+ ALL (n=13) or chronic phase (CP)-CML (n=9) who subsequently received TKI therapy (imatinib, dasatinib or nilotinib). 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. 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 105 mutations (including 35 silent, 5 nonsense, and 65 missense mutations) were observed. The vast majority (98/105, 93%) of them have never been reported in association with TKI resistance and are likely not to confer any advantage under TKI selective pressure. Interestingly, 93/105 (89%) mutations were transitions: G>A (n=28), A>G (n=23), C>T (n=22), T>C (n=20). 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. One of the nine 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 one with a mutation (T315I) that was already detectable at diagnosis. The remaining eleven pts are in persistent remission after a follow up ranging from 12 to 36 months, although four of them were harbouring known imatinib-(H396P, D276G, E355G) or dasatinib-(F317L) resistant mutations at low levels. 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