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Nilotinib has significant efficacy in patients with newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP) and in patients with CML-CP or CML in accelerated phase (CML-AP) after imatinib failure. We investigated the occurrence of cross-intolerance to nilotinib in imatinib-intolerant patients with CML. Only 1/75 (1%) patients with nonhematologic imatinib intolerance experienced a similar grade 3/4 adverse event (AE), and 3/75 (4%) experienced a similar persistent grade 2 nonhematologic AE on nilotinib. Only 7/40 (18%) patients with hematologic imatinib intolerance discontinued nilotinib, all because of grade 3/4 thrombocytopenia. Ninety percent of imatinib-intolerant patients with CML-CP who did not have complete hematologic response (CHR) at baseline (n = 52) achieved CHR on nilotinib. Nilotinib induced a major cytogenetic response in 66% and 41% of patients with imatinib-intolerant CML-CP and CML-AP (complete cytogenetic response in 51% and 30%), respectively. Minimal cross-intolerance was confirmed in patients with imatinib-intolerant CML. The favorable tolerability of nilotinib in patients with imatinib intolerance leads to alleviation of AE-related symptoms and significant and durable responses. In addition to its established clinical benefit in patients with newly diagnosed CML and those resistant to imatinib, nilotinib is effective and well-tolerated for long-term use in patients with imatinib intolerance. This study is registered at http://www.clinicaltrials.gov as NCT00471497

CML patients should be screened for Abl kinase domain (KD) mutations before changing tyrosine kinase inhibitor (TKI) therapy to ensure that the second-line TKI will be effective against mutations that arose on the first drug. Sanger sequencing (SS) can confidently detect mutations present in >20% of BCR-ABL molecules but will fail to detect minor resistant sub-clones. If missed, these minor sub-clones may be further selected by the second-line TKI and cause treatment failure. The T315I mutation can be particularly problematic as it is resistant to most TKIs except ponatinib. It seems reasonable to detect all mutations if possible, and avoid second line drugs that are known to be ineffective in their presence. We have developed a next generation sequencing strategy (Illumina MiSeq, 2 x 300 bp) that enables confident detection of all Abl KD mutations present at a level of at least 1% in the BCR-ABL cDNA, this level being 3-5x above the background calling-error rate. Two 500 bp PCR products are sufficient to cover the entire kinase domain (aa M237 to E505). With 300 bp paired-end sequencing of the products, a 65 bp region containing the 315 codon (codons 306-326) is sequenced on both strands. By excluding base changes that are not corroborated on both strands, mutations in this region can be detected with a 10-fold higher accuracy (in 0.1% of BCR-ABL molecules). Samples with low disease burden (1% BCR-ABL/ABL positivity) can also be amplified sufficiently with 50 cycles of PCR, minimising artefactual DNA polymerase-induced mutations. Indexing allows the simultaneous analysis of 80 PCR's in a single MiSeq run (Abl KD of 40 patients). Important aspects of the method are: 50% PhiX DNA is added to the library to increase complexity, and the flow cell is seeded at low density (300,000 clusters per mm2) to reduce sequencing errors.Overlapping paired reads are combined to produce a single FASTQ sequence (modified FLASH source code). Any bases in the overlapping region that do not agree with their counterpart on the other strand are labelled "N" and given a quality score (Q score) of 20.Combined sequences are quality parsed (FASTX Tool Kit) to exclude sequences that do not have a Q score of at least 20 at all basesParsed high quality sequences are compared to the reference sequence. We have sequenced the BCR-ABL KD of patients who were sub-optimal responders (BCR-ABL/ABL ratio of >1% at >= 11 months on therapy) in the NCRI SPIRIT 2 trial of first-line imatinib vs dasatinib. Of 60 sub-optimally responding imatinib patients, 6 (10%) had high level mutations (in >20% of BCR-ABL molecules): T315I, L387F, G250E, N331D, M244V x 2. The patients with L387F and G250E were switched to dasatinib and proceeded to respond well. The patient with T315I was also initially switched to dasatinib but failed to respond (BCR-ABL/ABL 29% after 1 year). This patient eventually received ponatinib with good response (BCR-ABL/ABL One patient with M244V was switched to nilotinib. Initially this caused relative selection of a pre-existing M387F mutated clone, this mutation increasing from 11 months). Our study demonstrates the value of using 2 x 300 bp paired-end sequencing to detect high and low level mutations, even in patients with low-level disease burden, to guide the choice of an appropriate second-line TKI. Disclosures Dickson: Ariad: Research Funding. Kennedy:Ariad: Research Funding. Cork:Roche: Research Funding; Ariad: Research Funding; BMS: Research Funding; Novartis: Research Funding. Hedgley:Roche: Research Funding; BMS: Research Funding; Ariad: Research Funding; Novartis: Research Funding. Copland:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Holyoake:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. O'Brien:BMS: Consultancy, Honoraria, Research Funding; Pzifer: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Ramashoye:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Pfeifer and colleagues establish for the first time that BCR-ABL kinase domain mutations that are detectable at very low levels in some imatinib-naive patients with Ph+ ALL emerge as the predominant clones at relapse. The ABL tyrosine kinase inhibitor imatinib has improved the complete remission