High Frequency of Point Mutations of BCR/ABL Gene in CML Patients Resistant to Imatinib Therapy - Report of a Novel Mutation.

Imatinib is a drug that competitively targets the adenosine 5′-thriphosphate (ATP)-binding site of the kinase domain of ABL with high specificity and has been stablished as the new standard care in CML. However, resistance to imatinib develops in all phases of the disease, mainly in accelerated phase (AP) and blast crisis (BC). Mutations in the kinase domain of BCR/ABL are the most frequent mechanisms associated to resistance in vivo.

Aims: detect mutations in the kinase domain of BCR/ABL in CML patients with primary or secondary resistance to Imatinib.

Patients and Methods: we evaluated five patients in blast crisis followed in our institution with primary (1) or secondary resistance (4) 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. For the first PCR, primers PCR1 were used and for the second PCR, PCR2 primers; PCR 1 - BCRF (sense - 5′ TGA CCA ACT CGT GTG TGA AAC TC) and ABLKinaseR (anti-sense - 5′ TCC ACT TCG TCT GAG ATA CTG GAT T); PCR 2 ABLKinaseF (sense - 5′ CGC AAC AAG CCC ACT GTC T) and ABLKinaseR (anti-sense - 5′ TCC ACT TCG TCT GAG ATA CTG GAT T). PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank).

Results: We found mutations in four of five CML patients in blast crisis with secondary resistance to Imatinib (E255K, E279K, F359V). E279K mutation was found in a patient in myeloid blast crisis which become resistant to imatinib after 18 months of therapy. He died of progressive disease(PD). This mutation was not previously described and additional studies are necessary to categorize it. Recently H-bonds were reported between Arg 386, located in A-loop and Asp276/Glu279 located in β3-αC-loop which stabilizes the inactive conformation of the A-loop, necessary for imatinib binding. E255K mutation was found in two patients with lymphoid BC. This mutation is located in P-loop and is related to a poor prognosis. These two patients died with PD. F359V mutation is found outside P-loop and related to a longer survival. In fact, the patient with this mutation was in second CP when mutation was detected and had a good response to dose increase. Later, she was submitted to non-myeloablative BMT and is still alive. Mutations were not identified in one patient, a case with primary resistance, corroborating literature data. In conclusion, BCR/ABL kinase domain mutations are frequent in advanced phases of the disease and have prognostic implications. We described a novel mutation which may have clinical relevance. Early detection of BCR/ABL mutations may help in clinical decisions.