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

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The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1-positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (⌬4-7) and by removal of exons 2 through 7 (⌬2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the ⌬4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the ⌬2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronicphase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAGmediated recombination. (Blood. 2009;