Inclusion criteria for the study were patients with CP-CML who were aged ≥20 years, treated with imatinib for at least 24 months and achieved MMR with detectable levels of BCR-ABL1 transcripts by RQ-PCR, and a sensitivity of at least MR4 below the standardized line. Exclusion criteria were patients with a performance status (PS) of grade ≥3 by ECOG definition, with active second primary cancer, with clinically defined pleural effusion, with a history of critical cardiovascular events, including acute myocardial infarction within 6 months, angina pectoris within 3 months, and congestive heart failure within 3 months, with prolonged QTc of ≥450 ms or the possibility of congenital prolonged QT syndrome, with a history of treatment by dasatinib, or with ABL1 mutations (T315I, F317L and V299L) for which dasatinib is ineffective, at the time of study inclusion. Pregnant or breastfeeding women were also ineligible.
RQ-PCR analysis for BCR-ABL1 transcripts
The BCR-ABL1 transcript level in peripheral blood was monitored before and at 1, 3, 6, 9, 12, 15 and 18 months after switching to dasatinib. To measure the BCR-ABL1 transcript level, the RQ-PCR method with at least MR4 sensitivity (i.e., 0.0069% on IS) was carried out by Bio Medical Laboratories (BML, Inc., Tokyo, Japan) as described previously [14, 16, 17]. In this analysis, we defined DMR as a peripheral major BCR-ABL1/ABL1 transcript ratio below the detection limit of RQ-PCR analysis.
Screening for BCR-ABL1 mutations
Before switching to dasatinib, we screened for 25 clinically important BCR-ABL1 mutations at 18 nucleotide positions by RT-PCR using PCR-Invador assay (BML, Inc., Tokyo, Japan) in all enrolled patients [18, 19].
Study design and treatment
After confirmation of MMR with detectable levels of BCR-ABL1 transcripts by RQ-PCR, dasatinib therapy was initiated for eligible patients at a dose of 100 mg once a day. The study treatment was continued until disease progression or development of unacceptable adverse events. During the study, any treatment for CML other than dasatinib was not allowed. Therapy for comorbidities and/or adverse events was permitted. Interruption, dose reduction, or dose re-escalation of dasatinib due to adverse events were allowed.
Evaluation of efficacy
The primary endpoint in this study was the achievement of DMR at 12 months after switching to dasatinib. Secondary endpoints included the dose intensity of dasatinib at 12 months, progression-free survival, safety profiles, and immunophenotypic alterations of peripheral lymphocytes after switching to dasatinib. Progressive disease was defined as loss of complete hematological response, loss of MMR, progression to accelerated/blastic phases, or death from any cause during the treatment period.
Evaluation of safety
Adverse events were monitored and assessed according to the Common Terminology Criteria for Adverse Events version 4.0 in all participating patients throughout the study. Chest X-ray was carried out to check pleural effusion at baseline, at 2 weeks and at 1, 2, 3, 6, 9, 12, 15 and 18 months after switching to dasatinib or more frequently if necessary. An ECG was carried out to screen for arrhythmia and to monitor the QTc interval at baseline, at 2 weeks and at 1, 2, 3, 6, 9, 12, 15 and 18 months after switching to dasatinib or more frequently if necessary.
Monitoring immunophenotypes in peripheral blood lymphocytes during dasatinib treatment
Immunophenotypes of peripheral blood lymphocytes were monitored before, at 2 weeks and at 1, 3, 6, 9 and 12 months after switching to dasatinib by two- or three-color flow cytometry performed by BML Inc. using monoclonal antibodies against the following antigens—CD3, CD4, CD8, CD16, CD56, CD57, CD25 and CD127.
Differences in immunophenotypes in peripheral lymphocytes between paired samples before and at 6 months after switching to dasatinib were evaluated statistically by paired t test using JMP Pro 11.2 (SAS Institute Inc.). A statistically significant result was considered to have a P value of <0.05.
Ethics and study management
The study was conducted by the Kanto CML study group, which consists of 13 institutions in Japan, according to the Declaration of Helsinki, and the protocol was reviewed by institutional review boards or ethics committees for each participating center. Before entry into the study, all candidates were informed about the aim of the present study, and also merits and demerits of switching to dasatinib. Merits include induction of deeper response and leading to future TKI discontinuation. Demerits include newly developed adverse events caused by dasatinib, although certain adverse events due to imatinib treatment may be solved. Written informed consent was obtained from all participating patients before registration. This trial was registered at www.clinicaltrials.gov as # NCT01342679.