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

ABL1 kinase domain (AKD) mutations are the most important mechanism of resistance to tyrosine kinase inhibitors (TKIs) in CML. Direct sequencing (DS) techniques detect AKD mutations in 20%–40% of imatinib-resistant pts. Therefore, most pts fail TKI therapy for unknown reasons. We evaluated the incidence and clinical consequences of AKD mutations among 70 pts in CML-CP after imatinib failure (13 intolerant) enrolled in a phase I study of dasatinib. Mutations were studied by DS of nested PCR-amplified BCR-ABL1 products and by DNA expansion of specific clones (DESC) followed by DNA sequencing of ≥10 clones. Patients had received imatinib at 400 mg/d (n=60), 600 mg/d (n=8), or 800 mg/d (n=2). Prior to dasatinib, AKD mutations were detected in 61/70 (87%) pts by DESC, including 38 (54%) with mutations in ≥20% of sequenced clones. Mutations were only detected in 18/38 (47%) by DS. Overall, 125 mutations at 113 amino acid positions were detected by DESC (78 previously unreported). Mutations conferring resistance to >1μM imatinib (M244V, G250E, Q252H, Y253H, E255K/V, F359V, H396R, and T315I) were detected in 30 (43%) pts by DESC, but only in 5 (7%) by DS. Two or more mutations within the same clone (polymutants) were detected in 29/70 (41%) pts by DESC, with clones expressing 2 (n=38), 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. Pts received dasatinib for a median of 19 months (range, 2–52), of whom 68 (97%) are evaluable for response. DESC available in 32 pts during dasatinib therapy revealed 20 additional mutations not present at dasatinib start (19 amino acid positions), including 5 previously not reported (all in polymutant clones). Dasatinib-resistant mutations (L248V/R, Q252H, E255K, V299L, T315I/A, and F317L/C/I/S/V) were detected in 10/32 (31%) cases (5 with T315I) by DESC (but only in 3/16 [19%] by DS). Of these 16 pts 13 died (all in BP) and 3 are alive in CP carrying E255K, T315I, and F317L respectively.

The percentage of clones with unmutated BCR-ABL1 before dasatinib decreased significantly compared to those present after a median of 16 wks (range, 4–84) during dasatinib (p=0.001), particularly in pts carrying highly dasatinib-resistant mutants. No differences were seen in the proportion of unmutated BCR-ABL1–expressing clones between pts with no cytogenetic (CG) response and those who achieved a partial (PCyR) or a complete CG (CCyR) response prior to dasatinib therapy. Conversely, DESC during dasatinib therapy showed the proportion of unmutated clones was lower among pts who failed to achieve a CG response compared to those who had a PCyR or CCyR (p=0.0001).

In conclusion, DESC demonstrates a high prevalence of AKD mutations among pts who fail imatinib, revealing heightened BCR-ABL1 genomic instability in this setting. This high incidence of mutations might partly explain TKI resistance in pts found to carry unmutated BCR-ABL1 by DS. The latter might be mediated by generation of resistant polymutant clones that perpetuate a “mutator phenotype” and by exhaustion of clones carrying unmutated BCR-ABL1 alleles.