Bone marrow specimens and associated clinicopathological information documented prior to and following chemotherapy were collected from 30 AML patients treated at the Department of Hematology and Oncology at Yuyao People's Hospital (Ningbo, China). There were 13 male and 17 female patients, with a mean age of 47.8±15.4 years (range, 19–76 years). AML was diagnosed in accordance with the revised French-American-British classification, which included classification into subtypes M0-7 (10). In total, 13 different chemotherapy regimens were chosen according to the status of the patients. Among them only 6 patients were treated with one kind of drug, including one male of subtype M5 and four females (two of subtype M3, and one each of subtypes M4 and M6) who were treated with cytarabine (Ara-c), and one female M4 patient who was treated with idarubicin (IDA). The remaining 24 patients were treated with multi-drug chemotherapy regimens: HAA [homo-harringtonine (HHT) + cytarabine (Ara-C) + aclacinomycin (ACLA)]; IA (IDA + Ara-c); AAG [Ara-C + ACLA + granulocyte colony-stimulating factor (G-CSF)]; ATRA combined with arsenic trioxide (AS₂O₃); all trans–retinoic acid (ATRA) combined with AS₂O₃ and daunorubicin (DNR); HA (HHT + Ara-c); IA + HAG; AA (Ara-c + ACLA); HAG (HHT + Ara-c + G-CSF); HAG + IDA; and ATRA + As₂O₃ + HA. The clinical parameters of the patients with AML are summarized in Table I. The Ethics Committee at Yuyao People's Hospital approved the study. Written informed consent was obtained from all patients after the possible consequences of participating in the study had been explained.

Genomic DNA was isolated from bone marrow nucleated cells using a nucleic acid extraction analyzer (Lab-Aid 820; Zeesan Biotech, Xiamen, China). The DNA concentration of each specimen was measured via a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, NC, USA). All DNA samples were modified using the reagents provided in the ZYMO EZ DNA Methylation-Gold kit (Zymo Research Corp., Irvine, CA, USA). Following bisulfite treatment, the converted DNA samples were stored at −20°C.

Modified DNA samples were subjected to MSP using APC and CYP1B1 MSP primers (11). Two pairs of primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) according to the sequences listed in Table II. Methylated primers were used to amplify methylated regions and unmethylated primers to amplify unmethylated regions. Each PCR reaction contained 1.5 µl sodium bisulfite modified DNA, 0.5 µl each primer, 10 µl Zymo TaqTM Premix (Zymo Research, Orange, CA, USA) and 7.5 µl DNAase/RNAase-free water in a final reaction volume of 20 µl. DNA amplification was performed using a Veriti^® PCR machine (Applied Biosystems, Thermo Fisher Scientific, Inc.). Thermocycling conditions were as follows: Initial denaturation step at 95°C for 10 min followed by 35 cycles of amplification, each cycle included a denaturation step at 94°C for 30 sec, an annealing step with a primer-specific temperature for 45 sec and an elongation step at 72°C for 1 min. The final extension step was performed at 72°C for 7 min. The methylation status of each sample was determined using one or two independent experiments. Water blank was used as a negative control. PCR products were analyzed using a Qsep100 DNA Analyzer (Bioptic Inc., Taiwan, China). Samples were considered as methylated or unmethylated according to the presence of clearly visible peaks by the Q-analyzer software (Fig. 1). The sequences and details of the methylated and unmethylated primers are provided in Table II. DNA samples were randomly sequenced using the Applied Biosystems 3730 DNA Analyzer (Thermo Fisher Scientific, Inc.) to confirm complete bisulfite conversion (Fig. 2).

Comparisons between APC2 and CYP1B1 promoter methylation were performed using the correction formula of a χ ² test. Statistical analyses were performed using the SPSS 18.0 Evaluation version software for Windows (SPSS Inc., Chicago, IL, USA). MSP results were compared in samples pre- and post-chemotherapy.

As shown in Table I, the chemotherapy agents used included Ara-C, ATRA, As₂O₃, HHT, G-CSF, IDA, ACLA and DNR. A total of 22 patients in remission and eight patients with a poorer prognosis were treated with these 13 agents, either in single or combined regimens.

MSP was performed on pre- and post-chemotherapy bone marrow samples from 30 AML patients in order to determine whether the chemotherapy treatment was able to alter APC2 and CYP1B1 promoter methylation levels. APC2 promoter methylation status remained unchanged by chemotherapy. In contrast, seven patients demonstrated CYP1B1 promoter changes during chemotherapy.

Seven patients treated with six regimens exhibited CYP1B1 promoter methylation changes, including five samples with hypermethylation and two samples demonstrating hypomethylation. Of the five patients with induced hypermethylation, three patients in remission were treated with Ara-C, HHT + Ara-C and IDA + Ara-C + ACLA + G-CSF + HHT regimens, and two patients treated with As₂O₃ + DNR + ATRA. Ara-C + ACLA + G-CSF regimens resulted in a poor patient prognosis. Two patients with induced hypomethylation were treated with Ara-C + ACLA + G-CSF, and IDA + Ara-C regimens and had a poorer prognosis.

The results of the present study were obtained from three patients diagnosed with the M1 AML subtype, eight with M2, seven with M3, six with M4, three with M5 and three with M6. Outcomes associated with chemotherapy-induced CYP1B1 promoter methylation changes varied according to the subtypes. Chemotherapy-induced methylation changes were more often observed in M3 patients (57.1%, 4/7) compared with patients diagnosed with other subtypes (M1: 33.3%, 1/3; M2: 12.5%, 1/8; M4: 16.7%, 1/6; M5: 0%, 0/3; and M6: 0%, 0/3).

As shown in Table I, of the 24 patients aged ≤60 years, 20 patients exhibited induced hypermethylation and four patients showed induced hypomethylation. Among the six patients aged >60 years old, five exhibited induced hypermethylation and one demonstrated induced hypomethylation. By further categorizing patients according to age, chemotherapy-induced methylation changes were observed to be more often present among AML patients >60 years of age (33.3%, 2/6) compared with patients ≤60 years of age (20.8%, 5/24). Among the patients aged ≤60 years, one patient of the M1 subtype (Ara-C + ACLA + G-CSF; aged 59 years) exhibited an induced hypermethylation and worse consequence, one patient of M3 subtype (As₂O₃ + DNR + ATRA; aged 40 years) showed an induced hypermethylation along with a poor prognosis, two M3 patients (HHT + Ara-C; aged 59 years and Ara-C; aged 30 years) demonstrated induced hypermethylation along with remission and one patient of the M3 subtype (IDA + Ara-C; aged 42 years) exhibited induced hypomethylation along with a poor prognosis. As for patients >60 years of age, one M2 subtype patient (Ara-C + ACLA + G-CSF, aged 76 years) exhibited an induced hypomethylation along with a poor prognosis and one M4 subtype patient (IDA + Ara-C + ACLA + G-CSF + HHT, aged 67 years) demonstrated hypermethylation along with an improved prognosis.

As shown in Table I, 4/13 male and 3/17 female patients demonstrated CYP1B1 promoter methylation status changes. Among them, one M3 subtype male patient (HHT + Ara-C) and one M4 subtype male patient (IDA + Ara-C + ACLA + G-CSF + HHT) displayed induced hypermethylation along with remission. One M3 subtype male patient (As₂O₃ + DNR + ATRA) exhibited induced hypermethylation along with a poor prognosis, and one M2 subtype male patient (Ara-C + ACLA + G-CSF) demonstrated induced hypomethylation along with a poor prognosis. The remaining nine male patients did not exhibit any methylation changes induced by chemotherapy.

In the female subgroup, one M1 subtype patient (Ara-C + ACLA + G-CSF) exhibited induced hypermethylation with a poor prognosis, one M3 subtype patient (IDA + Ara-C) exhibited induced hypomethylation with a poor prognosis, and one M3 subtype patient (Ara-C) demonstrated induced hypermethylation along with remission. However, the remaining 14 female patients did not show any methylation changes following chemotherapy.