Detection of BCR-ABL Point Mutations in Patients with Chronic Myeloid Leukemia (CML) Resistant to Imatinib.

Background: Imatinib is now the standard therapy for newly diagnosed CML patients. However, some patients develop resistance, mainly those treated in advanced stages. Mutations in the kinase domain of BCR/ABL are the most frequent mechanisms associated to resistance in vivo and some of them do not respond even to other kinase inhibitors (nilotinib and dasatinib).

Aims: detect mutations in the kinase domain of BCR/ABL in CML patients with primary or secondary resistance to Imatinib and describe their clinical outcome after mutation detection.

Patients and Methods: we evaluated 17 CML patients with primary or secondary resistance to Imatinib. After RNA extraction from peripheral blood samples, amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR, to cover amino acids 244–486. PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank).

Results: We found seven mutations in ten patients resistant to imatinib: 5 in blast crisis (BC), 3 in accelerated phase (AP) and 2 in chronic phase (CP).

Mutations identified: T315I (3), L248V(1), G250E(1), F359V(1), M244V(1), E255K (2) and E279K (1). Five patients were treated with dasatinib (trials CA-180–005, 006, 034 and 035). Patients with T315I mutation (3) did not respond to treatment. One is dead due disease progression and two are currently been treated with hidroxiurea in CP and AP. Patient with mutation M244V is still using dasatinib, presenting major cytogenetic response. Patient with F359V mutation was submitted to a non-myeloablative stem cell transplantation from an identical sibling donor, in AP. The transplantation was uneventful, and no acute or chronic GVHD was observed. On day +57 the chimerism analysis showed a mixed chimerism followed by a hematological relapse. Two lymphocytes infusions were done, on days +77 and +114, with no response. The F359V mutation was still present after the SCT and the patient was included in a phase II trial of dasatinib (CA180-005), which is on going and is in partial hematological response. Patients with other mutations died in blast crisis. In seven patients mutations were not found, but two patients had clonal evolution.

Summary/Conclusions: BCR-ABL mutations are frequent in patients resistant to imatinib. Patients with P-loop mutations presented a worse prognosis with rapid evolution to blast crisis and patients with T315I mutation had no response to dasatinib, as recently described. We identified two patients with non-P-loop mutations with good response to dasatinib. P-loop mutations have been associated with poor prognosis, but its prognostic impact depends on patient selection and treatment received after detection of mutation. Mutations detection is helpful in deciding strategies to overcome resistance to imatinib.