on behalf of the GIMEMA Working Party on Chronic Myeloid Leukemia Abstract Purpose: ABL kinase domain mutations have been implicated in the resistance to the BCR-ABL inhibitor imatinib mesylate of Philadelphia-positive (Ph+) leukemia patients. Experimental Design: Using denaturing high-performance liquid chromatography and sequencing, we screened for ABL kinase domain mutations in 370 Ph+ patients with evidence of hematologic or cytogenetic resistance to imatinib. Results: Mutations were found in 127 of 297 (43%) evaluable patients. Mutations were found in 27% of chronic-phase patients (14% treated withimatinib frontline; 31% treated with imatinib post-IFN failure), 52% of accelerated-phase patients, 75% of myeloid blast crisis patients, and 83% of lymphoid blast crisis/Ph+ acute lymphoblastic leukemia (ALL) patients. Mutations were associated in 30% of patients with primary resistance (44% hematologic and 28% cytogenetic) and in 57% of patients with acquired resistance (23% patients who lost cytogenetic response; 55% patients who lost hematologic response; and 87% patients who progressed to accelerated phase/blast crisis). P-loop and T315I mutations were particularly frequent in advanced-phase chronic myeloid leukemia and Ph+ ALL patients, and often accompanied progression from chronic phase to accelerated phase/blast crisis. Conclusions: We conclude that (a) amino acid substitutions at seven residues (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) account for 85% of all resistance-associated mutations; (b) the search for mutations is important both in case of imatinib failure and in case of loss of response at the hematologic or cytogenetic level; (c) advanced-phase chronic myeloid leukemia and Ph+ ALL patients have a higher likelihood of developing imatinib-resistant mutations; and (d) the presence of either P-loop orT315I mutations in imatinib-treated patients should warn the clinician to reconsider the therapeutic strategy.Imatinib mesylate (1 -5) is a potent and selective inhibitor of the oncogenic BCR-ABL tyrosine kinase, which is deregulated in as many as 95% of chronic myeloid leukemia (CML) patients and in f20% of adult Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. Despite its striking efficacy, however, resistance is observed in a proportion of patients, especially those with Ph+ ALL or advanced-stage CML.Through the contribution of several research groups, the past 4 years have brought us considerable knowledge on the molecular mechanisms underlying resistance to imatinib (reviewed in ref. 6). Reactivation of BCR-ABL tyrosine kinase activity within the leukemic clone is most commonly associated with the emergence of point mutations in the ABL kinase domain that impair imatinib binding without affecting ATP

Our results show that, irrespective of the hematologic response, monitoring for emerging mutations in the first months of therapy may play a role in detecting patients with worse prognosis, for whom a revision of the therapeutic strategy should be considered.

Nilotinib has a higher binding affinity and selectivity for BCR-ABL with respect to imatinib and is an effective treatment of chronic myeloid leukemia (CML) after imatinib failure. In a phase 2 study, 73 early chronic-phase, untreated, Ph ؉ CML patients, received nilotinib at a dose of 400 mg twice daily. The primary endpoint was the complete cytogenetic response (CCgR) rate at 1 year. With a median follow-up of 15 months, the CCgR rate at 1 year was 96%, and the major molecular response rate 85%. Responses were rapid, with 78% CCgR and 52% major molecular response at 3 months. During the first year, the treatment was interrupted at least once in 38 patients (52%). The mean daily dose ranged between 600 and 800 mg in 74% of patients, 400 and 599 mg in 18% of patients, and was less than 400 mg in 8% of patients. Dose interruptions were mainly due to nonhematologic and biochemical side effects. Myelosuppression was irrelevant. One patient progressed to blastic crisis after 6 months; one went off-treatment for lipase increase grade 4 (no pancreatitis). Nilotinib is safe and very active in early chronic-phase CML. These data support a role for nilotinib for the frontline treatment of CML. This study was registered at ClinicalTrials.

Dasatinib and nilotinib are tyrosine kinase inhibitors (TKIs) developed to overcome imatinib resistance in Philadelphiapositive leukemias. To assess how BcrAbl kinase domain mutation status evolves during sequential therapy with these TKIs and which mutations may further develop and impair their efficacy, we monitored the mutation status of 95 imatinib-resistant patients before and during treatment with dasatinib and/or nilotinib as second or third TKI. We found that 83% of cases of relapse after an initial response are associated with emergence of newly acquired mutations. However, the spectra of mutants conferring resistance to dasatinib or nilotinib are small and nonoverlapping, except for IntroductionResistance to the tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) in chronic myeloid leukemia (CML) and Philadelphiapositive (Ph ϩ ) acute lymphoblastic leukemia (ALL) is often caused by selection of mutations in the Bcr-Abl kinase domain (KD), [1][2][3][4][5][6][7][8][9] altering residues that are directly or indirectly critical for IM binding. To overcome this problem, novel TKIs have been developed. Dasatinib and nilotinib [10][11][12][13] have been the first ones to enter clinical evaluation and to receive marketing approval in IMresistant patients. Preclinical experience with these agents showed that they are active against several IM-resistant Bcr-Abl mutants, with the exception of T315I. 10,11,14 However, both TKIs have been hypothesized to retain their own "Achilles heels." Recent studies have tried to profile mutations that will probably emerge under dasatinib and nilotinib, inducing random mutagenesis and then selecting for cell clones retaining viability when cultured in the presence of the inhibitors. [15][16][17][18] Results suggested that, besides T315I, other mutants might be critical. To assess how Bcr-Abl KD mutation status evolves under the selective pressure of sequential therapy with novel TKIs and which mutations among those predicted by in vitro studies may indeed develop in patients who relapse on dasatinib or nilotinib, we have monitored the mutation status of 95 IM-resistant patients before and during sequential treatment with one or both of these agents. Methods Patients and definitionsThis report focuses on 95 patients (Table 1) who were referred to our laboratory for mutation analysis at the time of IM failure and who received up to 2 subsequent TKIs (dasatinib and/or nilotinib). Thirty-eight patients had chronic-phase (CP) CML, 46 patients had advanced-phase CML (accelerated-phase [AP], n ϭ 11; myeloid blast crisis [BC], n ϭ 18; lymphoid BC, n ϭ 17), and 11 patients had Ph ϩ ALL. For CML patients, IM failure was defined according to European LeukemiaNet recommendations. 19 Similar criteria were applied to define IM failure in Ph ϩ ALL. All 95 patients received a second TKI (dasatinib, n ϭ 55; nilotinib, n ϭ 40). Twenty-six of 95 patients received a third TKI after relapse on second TKI (dasatinib, n ϭ 16; nilotinib, n ϭ 10). For the purpose of this analysis, response to dasat...