Effects of ABCG2 C421A and ABCG2 G34A genetic polymorphisms on clinical outcome and response to imatinib mesylate, in Iranian chronic myeloid leukemia patients

Chronic myeloid leukemia (CML) is a multifactorial clonal myeloid neoplasm that mainly arises from the Philadelphia chromosome. Even though imatinib mesylate (IM) is considered the gold standard for first-line treatment, a number of CML patients have shown IM resistance that can be influenced by many factors, including pharmacogenetic variability. The present study examined whether two common single nucleotide polymorphisms (SNPs) of ABCG2 (G34A and C421A) contribute to IM resistance and/or good responses. A total of 72 CML patients were genotyped with high-resolution melting (HRM) and restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). We also determined the cytogenetic and hematological response, as evaluable factors for measuring response to imatinib. In the current study, we explored the relationship between the different variants of ABCG2 G34A and C421A and clinical response to imatinib among CML patients. There were no statistically significant differences between genotypes of C421A and G34A and allele frequencies among the resistant and responder groups, with response to IM (P > 0.05). Also, we found no statistically significant association between genotypes and cytogenetic and hematological responses. This is the first study to investigate the association between genotypes of the G34A and C421A SNPs and the outcome of IM treatment in Iranian population. As a whole, genotyping of these SNPs is unhelpful in predicting IM response in CML patients.

CML treatment prior to the 2000s was confined to nonspecific drugs such as busulfan, hydroxyurea, and interferon-alpha (IFN-α) [6]. Alternative therapies, including allogeneic stem cell transplantation (allo-SCT), are also available but come with health risks and mortality [4]. Imatinib mesylate (IM), used to treat a variety of cancers and is a selective tyrosine kinase inhibitor (TKI), is known to be the gold standard of first-line treatment for Philadelphia chromosome-positive CML [7,8]. The death rate in CML patients has been dropped since the introduction of IM in 2000 [9]. Inactive (closed) form of BCR-ABL is bound by IM, which prevents ATP binding [10]. Following this interaction, the subsequent phosphorylation and activation of downstream pathways are inhibited, resulting in apoptosis promotion of leukemic cells, and inhibiting leukemogenesis [11][12][13]. In line with the results of recent studies, IM significantly improved CML patients' clinical outcomes and prognoses. However, despite its remarkable efficacy, cancer treatment is hampered in some cases by primary or acquired resistance to IM and severe side effects [11,[14][15][16][17][18][19]. Increasing the dose of IM from 400 to 800 mg/day or switching to second-generation tyrosine kinase inhibitors such as dasatinib or nilotinib is recommended when there is no adequate response to IM or treatment fails [20].

Study subjects
The study involved 72 Ph-positive CML patients (37 females and 35 males) receiving 400 mg IM (Gleevec, STI571 (signal transduction inhibitor 571), and CGP57148B) daily for at least three months. Before participation in this study, these patients with chronic phase CML were diagnosed by the Department of Hematology, Omid Hospital (Isfahan, Iran). Written informed consent was obtained from all patients, and peripheral blood samples were collected in EDTA-containing tubes. Participants taking IM metabolism-interfering drugs, such as Phenobarbital and Phenytoin, were excluded. At the time of diagnosis, Philadelphia chromosome and BCR-ABL fusion mRNA presence were confirmed, respectively, by cytogenetic analysis and RT-PCR technique. Table 1 summarizes the demographic and clinical characteristics

Clinical assessment
The guidance for evaluation of clinical response and ordinary follow-up of patients were chased as given in "European Leukemia Net: guideline for managing CML patients". Bone marrow morphology and cytogenetic studies were utilized for early diagnosis of patients and definition of the phase of the disease at the time of clinical demonstration. The primary treatment prescribed was 400 mg of IM [46].
An indication of resistance can be determined by a lack of complete hematological response at 3 months, complete cytogenetic response (0% Ph-positive metaphases) at intervals of 12 months and (MMR) major molecular response (BCR-ABLIS ≤ 0.1%) at 18 months [47].
In this study, CML patients were followed up at an interval of 6 months. During this period, hematological, cytogenetic, and molecular responses were evaluated.

DNA extraction and SNP genotyping
Genomic DNA was extracted from peripheral blood lymphocytes using the PrimePrep Genomic DNA isolation kit (Genet Bio, Korea) based on the manufacturer's protocol. Qualitative and quantitative assessments of the isolated DNA were examined utilizing electrophoresis on 2% agarose gel and Thermo Scientific NanoDrop 2000 Spectrophotometer, respectively.
The G34A polymorphism was genotyped utilizing the allelic discrimination assay by High-Resolution Melting (HRM) technique (Rotor Gene-6000, Corbett Research, Sydney, Australia). PCR was performed in a final volume of 20 μL PCR mixture using HOT FIREPol ® EvaGreen ® HRM Mix (Solis BioDyne, Estonia). The HRM conditions were as follows: one cycle of 95 °C for 12 min to activate HOT FIREPOL DNA polymerase; 40 cycles of 95 °C for 15 s, 58 °C for 20 s, 72 °C for 20 s; and a HRM step from 76 to 86 °C rising at 0.2 °C with 2-s hold time after each step. The results were analyzed by Q 5plex HRM software V.2.3.4. Having found no feasible 80-100-bp primers, PCR-RFLP-based genotyping using the BseMI enzyme was designed to genotype the C421A polymorphism. After amplification DNA fragments by PCR, they were cut by 2 units of restriction enzyme BseMI for 16 h at 55 °C. Then, DNA fragments created after digestion run on a 3% agarose gel. Electrophoresis of these fragments could distinguish the genotypes based on fragments length. Primer sequences were designed using GeneRunner software. Primer sequences were as follows: Forward 5′AGG ATG ATG TTG TGA TGG GC3′, Reverse 5′TGA CCC TGT TAA TCC GTT CG3′ for C421A and Forward 5′ GTT GTG CCT GTC TTC CCA T 3′, Reverse 5′ TCG ACA AGG TAG AAA GCC AC 3′ for G34A. We randomly sequenced 10% of the samples using the ABI 3730XL automatic sequencer (Applied Biosystems, USA) to confirm results.

Statistical analysis
Data analysis was performed using SPSS software package version 26 (SPSS Inc., Chicago, IL, USA). The Chisquare test and independent samples t-test were used to assess the differences between demographics and response/resistance to IM. Then, the binary logistic model was exploited to calculate odds ratios (ORs) and 95% confidence intervals (CIs). We also performed Wilcoxon analysis and paired T-test to find out whether genotypes were associated with hematological responses. A P value lower than 0.05 was considered statistically significant. Table 1 lists the demographic and clinical characteristics of study participants. There was no statistically significant difference between the response to treatment and age (P = 0.179) or sex (P = 0.851). Furthermore, neither C421A (P = 0.146, P = 0.170) nor G34A (P = 0.235, P = 0.398) genotype and allele frequencies provided any statistically significant association with response to IM (Tables 2, 3).

Results
A hematologic index (Hb, WBC, and Plt count) analysis was also performed to find out how well the drug altered responder and resistant patients' hematologic profiles. Comparing the primary and secondary responses before and after drug intake, no significant relationship between drug use and hematological index was revealed, according to the data provided in Table 4. Moreover, genotypes did not significantly affect hematologic outcomes (WBC, Plt, and Hb count) ( Table 5) and cytogenetic response ( Table 6).

Discussion
Two types of ABCG2 variants, C421A and G34A, have been extensively studied in order to predict the IM response among CML patients. However, the results were noticeably contrasting. We compared the distribution of ABCG2 C421A and ABCG2 G34A polymorphism     Table 5 The comparison of hematological response between responder and resistant groups *WBC < 10 × 10 9 /L/ platelet < 450 × 109/L/ an absence of myelocytes, promyelocytes, or myeloblasts in peripheral blood **WBC and platelet counts have not returned to normal, there are immature cells seen in blood ***Before drug intake ****After drug intake  In the present study, no information on patients' molecular responses was available. Furthermore, we found that the frequencies of these alleles were insignificantly different between these two groups and were not found to be risk factors for resistance. Our results are in disagreement with some studies that the ABCG2 polymorphism is associated with response to IM [2,21,53].
The association of each SNP genotype of ABCG2 G34A and C421A with hematological responses (WBC, Hb, and Plt counts) was also examined. IM is effective in the hematological response since BCR-ABL protein is effective in proliferation of blood cells (myeloid), and BCR-ABL is IM's target, although according to Table 4, hematological indices did not vary significantly between the groups with different genotypes and drug therapy with IM did not significantly influence primary or secondary hematological response. In this regard, similarly results from study of CML patients in western of Iran showed no statistically significant correlation between ABCG2 C421A and hematological response (Hb, WBC, and Plt counts) [2]. The disconnection needs to be confirmed by further investigation.
Several studies have shown that the association between SNPs genotypes and cytogenetic response is more important for evaluating drug response than the relationship between SNPs genotypes and hematological response. According to our findings, no statistically significant relationship was found between G34A and C421A variants and cytogenetic response in both groups. Similarly, no significant correlation for ABCG2 C421A has been reported [21,54]. Also, no statistically significant association has been observed between cytogenetic response and SNP genotype of ABCG2 C421A and G34A, which is consistent with some previously published research [47].

Conclusion
To sum up, our study reveals that ABCG2/C421A and ABCG2/G34A polymorphisms are insignificantly associated with the optimal response rate to IM and cytogenetic/hematologic response and cannot be used as a predictive marker for optimal response/primary failure in CML patients receiving IM. Nevertheless, our study is the first that investigates the association between hematologic index among responder and resistant groups, as well as the association between these indexes and drug consumption. Our study had some restrictions such as not checking the molecular response test. In addition, the statistical population of the present study for assessing the relationship between response to treatment and combined SNPs was small. Therefore, additional studies with larger and different populations for evaluating the relationship between ABCB1, SLC22A1, CYP3A4, and CYP3A5 polymorphisms and response/resistance to IM are suggested.

Author contributions
NN contributed significantly to the preparation of this study by collected samples from Sayed Al-Shohada Hospital, designing the protocol of work, contributing to practical part of the study, and writing and editing the manuscript. VM, ZK, and MK contributed to collect samples from Sayed Al-Shohada Hospital and provide patients' data. Also, ZK contributed to practical part of the study, especially PCR-RFLP technique. EZ, EA, and FZ edited the manuscript, and analyzed and interpreted the patient data. SK was primarily responsible for the writing of the manuscript. All authors have read and approved the manuscript.

Funding
For this research, the author(s) did not receive any funding.

Availability of data and materials
The data that support the findings of this study are available on request from the corresponding author.

Declarations
Ethics approval and consent to participate