Abstract
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).
AFTER IMATINIB ON DASATINIB No. Evaluable for Response to dasatinib No. clones sequenced No. unmutated Clones (%) No. Evaluable Pts No. clones sequenced No. unmutated Clones (%) DASATINIB RESPONSE 68 680 311 (46) 32 305 94 (30) No CG Response 30 309 149 (48) 13 120 17 (14) CG Response Minor 11 91 32 (35) 5 47 4 (8) Partial 7 69 38 (55) 3 28 13 (46) Complete 20 211 92 (43) 11 110 60 (55)
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.
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