Long-Term Mutation Follow-up of Philadelphia-Chromosome Positive Leukemia Patients Treated with Second-Generation Tyrosine Kinase Inhibitors after Imatinib Failure Shows That Newly Acquired Bcr-Abl Kinase Domain Mutations Leading to Relapse Are Mainly Detected during the First Year.

Resistance to imatinib in Philadelphia-positive (Ph+) leukemia patients is often associated with selection of point mutations in the Bcr-Abl kinase domain (KD). Dasatinib and nilotinib are second-generation tyrosine kinase inhibitors (TKIs) with different binding modes with respect to imatinib, that have been shown to confer in vitro and in vivo activity against many Bcr-Abl mutated forms. However, both dasatinib and nilotinib have been shown to retain some ‘Achilles heels’, and they include both imatinib-resistant mutations (e.g., T315I) and some novel, inhibitor-specific ones. Selection of either type of KD mutations has frequently been observed in patients (pts) who relapse after an initial response to dasatinib or nilotinib and represents one of the major hurdles on the road to successful treatment of imatinib-resistant pts. We have monitored Abl KD mutation status in a total of 121 pts who received dasatinib (n= 78) or nilotinib (n=43) as 2^nd TKI after imatinib failure since February 2005. Fifty-eight (48%) pts had chronic phase (CP) chronic myelogenous leukemia (CML), 63 pts (52%) had accelerated phase (AP) or blast crisis (BC) CML or Ph+ acute lymphoblastic leukemia (ALL). Median age was 55 years (range, 18–76); median time from diagnosis was 49 months (range, 4–181); median time on imatinib was 32 months (range, 4–66). Median follow-up of all pts who received a 2^nd TKI is 7 months (range, 1–38). Median follow-up of pts who are still on 2^nd TKI treatment is 32 months (range, 28–38). Relapses after an initial response have so far been observed in 46/121 pts. Thirty-eight out of these 46 pts had AP/BC CML or Ph+ ALL at the time 2^nd TKI was started. Forty-one out of 121 (34%) pts have experienced relapse after an initial response during the first 12 months of 2^nd TKI treatment (median time to relapse, 6,5 months; range 4–12 months), while only five of the 45 (11%) pts who were still on 2^nd TKI treatment after >12 months have relapsed (at 13, 15, 18, 20 and 33 months, respectively). Interestingly, none of these 5 pts had never achieved more than a minor cytogenetic response (CgR), and 4/5 pts were receiving a reduced TKI dose because of toxicity. In 36/46 (78%) cases, relapse was associated with newly acquired Abl KD mutations. In particular 26/30 (87%) pts who relapsed on dasatinib and 10/16 (63%) pts who relapsed on nilotinib had evidence of a newly acquired KD mutation presumably responsible for treatment failure. Newly acquired mutations in pts who relapsed on dasatinib as 2^nd TKI were T315I (n= 12 pts) F317L (n= 8 pts) T315A (n=3 pts); V299L (n=3 pts); F317I (n=2 pts); 2 pts had multiple mutations. Newly acquired mutations in pts who relapsed on nilotinib as 2^nd TKI were E255K (n=3); E255V (n=2); Y253H (n=2); T315I (n=1); F359V (n=1); F359C (n=1). Sixteen pts (but none of those harboring the T315I) switched to dasatinib or nilotinib or high-dose imatinib as 3^rd TKI and this rescued hematologic or even cytogenetic responses in a proportion of cases. Our observations suggest that:

• newly acquired mutations leading to relapse in Ph+ leukemia pts receiving dasatinib or nilotinib as 2^nd TKI usually arise rapidly; the likelihood of mutation selection consistently decreases over time, and seems mainly confined to advanced phase pts and to pts with no or minor CgR;

• almost all (87%) cases who developed resistance to dasatinib had newly acquired KD mutations - suggesting that the higher potency with respect to imatinib can overcome Bcr-Abl gene amplification and that Src kinase inhibition may turn off Bcr- Abl-independent resistance mechanisms;

• a lower incidence (63%) of newly acquired KD mutations was observed in pts who developed resistance to nilotinib;

• with the exception of T315I, there is little if no overlap between dasatinib and nilotinib-resistant mutants, which may allow to regain responses by switching TKIs.