Mutational Analysis of Chronic Myeloid Leukemia (CML) Clones Reveals Heightened BCR-ABL1 Genetic Instability and Wild-Type BCR-ABL1 Exhaustion in Patients Failing Sequential Imatinib and Dasatinib Therapy.

Abstract ABL kinase domain (AKD) mutations are the main mechanism of resistance in patients (pts) with CML who fail tyrosine kinase inhibitors (TKIs) therapy, being found in 20%–40% of cases by direct sequencing (DS). Therefore, many pts fail TKI therapy for unknown reasons. We evaluated the development of AKD mutations among 61 CML pts after imatinib-intolerance (n=10) or -resistance (n=51) enrolled in a phase I study of dasatinib by DS of nested PCR-amplified BCR-ABL1 products as well as by DNA expansion of specific clones (DESC) followed by DNA sequencing of at least 10 clones. Prior to imatinib (400 mg daily in 47, 600 in 13, and 800 in 1), 54 pts were in chronic (CP), 2 in accelerated (AP), and 5 in blastic (BP) phase. At the end of imatinib therapy, 26 pts were in CP, 14 in AP, and 21 in BP. AKD mutations (in ≥1 out of 10 sequenced clones) were detected in 58/61 (95%) pts by DESC (4 pts with wild type [WT] BCR-ABL1) but only in 23/55 (42%) by DS. Overall, 118 AKD mutations at 112 amino acid positions were detected by DESC, of which 77 had never been previously reported. Mutations conferring resistance to >1μM imatinib (M244V, G250E, Q252H, Y253H, E255K/V, F359V, H396R, and T315I) were detected in 20 (34%) pts by DESC, but only in 5 (8%) by DS. Combinations of mutations within the same clone (polymutants) were detected in 33/58 (57%) pts by DESC, with clones expressing 2 (n=41), 3 (n=11), 4 (n=1), or even 5 (n=2) distinct mutations. By contrast, only 1 pt was found to carry 2 different mutations (M244V and M351T) by DS. Dasatinib was subsequently given to 56/61 (92%) pts (53 evaluable for response) for a median of 17 months (range, 1 to 48). DESC available in 15 pts during dasatinib therapy revealed 16 additional mutations (15 amino acid positions), including 5 previously not reported (all in polymutants). Dasatinib-resistant mutations (L248V/R, Q252H, E255K, V299L, T315I/A, and F317L/C/I/S/V) were detected in 7/15 (47%) cases (2 with T315I) by DESC but only in 2/15 (13%) by DS. Of these 15 pts, only 3 (1 CP, 1 AP, and 1 BP) are alive. The proportion of clones harboring WT BCR-ABL1 prior to and during dasatinib therapy decreased significantly (p=0.003), particularly in pts harboring highly dasatinib-resistant mutants. Notably, pts without cytogenetic (CG) response on dasatinib had a lower proportion of WT clones compared with those who achieved at least a partial cytogenetic response (p=0.02). AFTER IMATINIB ON DASATINIB Evaluable Patients No. Clones No. WT Clones (%) Evaluable Patients No. Clones No. WT Clones (%) DASATINIB RESPONSE 53 598 268 (48) 15 130 26 (20) No CG Response 25 242 128 (49) 7 58 5 (9) CG Response Minor 10 79 33 (42) 4 35 9 (26) Partial 6 57 35 (61) 2 18 8 (44) Complete 12 123 46 (37) 2 19 4 (21) In summary, DESC greatly increases the sensitivity of detection of AKD mutants compared to DS and reveals heightened BCR-ABL1 genetic instability among pts failing TKIs, which could explain TKI resistance in pts not carrying resistant mutations by DS. This might be mediated by generation of resistant polymutant clones that perpetuate a “mutator phenotype” leading to WT BCR-ABL1 exhaustion.