Subjects and DNA Extraction
The study was approved by the Research and Ethics Committee of Universiti Sains Malaysia and the Ministry of Health, Malaysia (ethics no. USMKK/PPP/JEPeM [244.3.(4)] and KKM/NHSEC/08/0804/P12-687), which complies with the Declaration of Helsinki of 1964, as revised in 2013. Informed consent was obtained from all patients for being included in the study. Subjects were recruited from various hospitals in Malaysia including Hospital Universiti Sains Malaysia (HUSM), Hospital Raja Perempuan Zainab II (HRPZ), Hospital Pulau Pinang, Hospital Raja Permaisuri Bainun, Universiti Kebangsaan Malaysia Medical Center (PPUKM), Sime Darby Medical Centre and Hospital Umum Sarawak (HUS). In this study, 270 CML patients (139 IM resistant and 131 IM good responders) were involved who were all BCR-ABL non-mutated. The patients selected were Philadelphia chromosome-positive CML patients in chronic, accelerated or blast phase, treated for at least 12 months, with IM (400 and 600 mg, respectively) on frontline treatment. The patients' characteristics are shown in Table 1.
Evaluation of imatinib response: By referring to European LeukemiaNet recommendations for the management of chronic myeloid leukemia 2013 , hematologic, cytogenetic and molecular criteria were accessed. Based on this, patients were grouped into IM good responders and IM-resistant CML patients. Hematologic response was considered as complete when the platelet count was <450 × 109/l; white blood cell count <10 × 109/L; differential without immature granulocytes and with <5% basophils and nonpalpable spleen. The cytogenetic response was defined as complete (0% Ph+ metaphases), partial (1–35% Ph+ metaphases), minor (36–65% Ph+ metaphases), minimal (66–95% Ph+ metaphases) and none (>95–100% Ph+ metaphases) . Molecular response was best assessed according to the International Scale (IS) as the ratio of BCR-ABL1% on a log scale, where 10%, 1%, 0.1%, 0.01%, 0.0032% and 0.001% correspond to a decrease of 1, 2, 3, 4, 4.5 and 5 logs, respectively, below the standard baseline that was used in the IRIS study. A BCR-ABL1 expression of ≤0.1% corresponds to major molecular response (MMR) . CML patients who achieved the above-mentioned response criteria were categorized as IM good responders and those who did not achieve the above response criteria within the specified time frame were categorized under the IM non-responders/resistant group.
Peripheral blood (3 ml) was collected after obtaining written informed consents from the subjects. Genomic DNA was extracted using a DNA extraction kit, QIAGEN QIAamp® DNA Blood Mini kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Genotyping was conducted at Human Genome Centre, Universiti Sains Malaysia.
Genotyping of CYP3A4*18 Polymorphisms
Genotyping of CYP3A4*18 was performed by using the polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP) technique. Amplification of CYP3A4*18 was performed by using forward (CACATCAGAATGAAACCACC) and reverse (AGAGCCTTCCTACATAGAGTCA) primers. PCR reactions were conducted in a 25-μl volume 1× PCR buffer, 2.0 µM magnesium chloride (MgCl2), 0.5 µM dNTPs, 0.4 µM of each primer and 1.0 U AmpliTaq Gold Polymerase. Denaturation was at 95 °C for 2 min, followed by 35 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s and a final extension step at 72 °C for 5 min. The 450-bp PCR products were electrophoresed on a 2% agarose gel at 100 V for 30 min.
Following PCR amplification, 4 µl of 450-bp PCR products were digested with 1.0 unit of a restriction enzyme (Msp1) for 1 h at 37 °C. The digested PCR products were analyzed by electrophoresis on a 2% agarose gel at 90 V for 50 min. The homozygous wild-type allele (TT) was identified by the presence of an undigested band (450 bp), while the heterozygous allele (TC) was confirmed by the presence of three fragments at 450, 282 and 168 bp. The homozygous variant allele (CC) was identified by the presence of two fragments at 282 and 168 bp.
Genotyping of CYP3A5*3 Polymorphisms
Genotyping of CYP3A5*3 was performed by using a PCR–RFLP. The 293-bp DNA fragment that contains the CYP3A5*3 allele was amplified with the primer pair 5′-GGTCCAAACAGGGAAGAAATA-3′ (forward) and 5′-CATGACTTAGTAGACAGATGAC-3′ (reverse). The PCR reactions were conducted in a 25-µl volume of 1× PCR buffer, 2.0 µM MgCl2, 0.5 µM dNTPs, 0.4 µM of each primer and 1.0 U AmpliTaq Gold Polymerase with a denaturation step of 95 °C for 2 min, followed by 35 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s and a final extension step at 72 °C for 5 min. The 293-bp PCR products were run on a 2% agarose gel electrophoresis at 100 V for 30 min.
Following PCR amplification, 4 µl of PCR products was digested by restriction enzyme Ssp1 for 15 min at 37 °C. The digested PCR products were analyzed by electrophoresis on a 3% agarose gel. The homozygous wild-type allele (AA) was identified by the presence of 148, 125 and 20 bp, whereas the homozygous variant allele (GG) was confirmed by the presence of fragments of 168- and 125-bp size. The heterozygous variant allele (AG) was identified by the presence of 168-, 148-, 125- and 20-bp fragments.
Following genotyping, a few samples from each different genotype were randomly selected for sequencing to confirm the expected sequences of each genotype. The PCR product was purified by using a QIAquick PCR purification kit (QIAGEN) before sending it to First BASE Laboratories (Kuala Lumpur, Malaysia) for sequencing.
The frequencies of polymorphic genotypes among IM-resistant and good-response CML patients were compared by using the chi-square test (χ
2). The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a binary logistic regression to investigate the risk association of genotypes with IM response. All statistical tests were two sided, and statistical significance was determined as p < 0.05. SPSS v.20.0 (SPSS Inc., Chicago, IL, USA) was utilized. Pair-wise linkage disequilibrium (LD) indices (r
2) were determined using Haploview v.4.2 .