Association of MDR1 gene polymorphism (G2677T) with imatinib response in Egyptian chronic myeloid leukemia patients

Abstract

Background

Despite the excellent efficacy results of imatinib treatment in CML patients, resistance to imatinib has emerged as a significant problem. Genetic variations in genes involved in drug transportation might influence the pharmacokinetic and metabolism of imatinib. The genotype of a patient is increasingly recognized in influencing the response to the treatment.

Aim

To investigate the genotype frequencies of single nucleotide polymorphisms (SNPs) G2677T in CML patients undergoing imatinib treatment to determine whether different genotype pattern of these SNPs have any influence in mediating response to imatinib.

Methods

A total of 96 CML and 90 control samples were analyzed for the human multidrug resistance gene 1 (MDR1) gene polymorphism (G2677T) using polymerase chain reaction-restriction fragment length polymorphism technique.

Results

Genotype distribution revealed a significant lower frequency of TT genotype in CML patients and non-significant difference in the GG, GT genotype frequencies between patients and controls (P = 0.004, 0.138, 0.210, respectively). GG genotype was significantly higher in chronic phase (P = 0.046), while GT genotype was significantly higher in Blastic crisis phase (P = 0.002). There was a significant difference in genotype frequency of G2677T among patients showing response and resistance to imatinib in chronic phase (P = 0.02). TT genotype was associated with complete hematological response (P = 0.01), complete cytogenetic response (P < 0.001), and better molecular response with a significant association (P < 0.001). GT genotype was associated with partial hematological response (P = 0.01) and minor cytogenetic response (P < 0.001). Optimal and suboptimal responses were observed for patients with TT genotype (P = 0.003). Failure of drug response was associated with GT genotype (P = 0.02); however, GG had no association with drug response. Multivariate analysis considered GT genotype as independent risk factor for resistance (P = 0.037), while TT genotype as protective factor against resistance to imatinib (P = 0.008).

Conclusion

Determination of MDR1 polymorphisms (G2677T) might be useful in response prediction to therapy with imatinib in patients with CML.

Keywords:

• Chronic myeloid leukemia
• Imatinib
• MDR1 gene
• Polymorphism

Introduction

Chronic myeloid leukemia (CML) is a myeloproliferative disorder that comprises 14% of all leukemias. Imatinib-mysylate, also known as Glivec or Gleevec, is frontline therapy for Philadelphia positive (Ph⁺) CML.¹ It specially targets tyrosine kinase domain of the BCR-ABL fusion protein, and blocks its phosphorylation, which is needed for kinase activation and signal transduction.² The treatment of CML patients with imatinib has resulted in complete cytogenetic response (CCR) rates of 65–85%.³ Despite of its striking efficacy, however, resistance develops over time in many patients. Resistance is more common in patients who start imatinib in late chronic phase and occurs in approximately 70% of patients treated in myeloid blast crisis and in >90% of those in lymphoid blast crisis.² Several molecular mechanisms leading to imatinib resistance have been proposed; amplification and overexpression of the BCR/ABL gene, point mutations in the ATP-binding site with kinase reactivation,⁴ or overexpression of the MDR1 gene.⁵ The human multidrug resistance gene (MDR1, ABCB1) is located on the long arm of seventh chromosome at q21.1 and consists of 28 exons,⁶ encodes for P-glycoprotein (P-gp) of 170KD, an ATP-binding cassette transmembrane transporter.⁷ P-gp is well known to be overexpressed in human tumor cells after cancer chemotherapy where it is associated with multidtug resistance and affect the pharmacokinetics of many drugs that are P-glycoprotein substrates.⁸ Imatinib is a substrate of P-gp-mediated efflux. The up-regulation of drug transporters (ABCB1) is one of specific causes of imatinib resistance, since it can be effluxed through MDR1 transporters,⁹ resulting in enhanced clearance of drug from the cell, reduced drug availability and drug resistance.² MDR1 gene is polymorphic, and more than 1279 single nucleotide polymorphisms (SNPs) have been identified.¹⁰ SNPs have the potential to affect the expression and function of the P-gp,^11,12 could also influence the efficiency of absorption or elimination of imatinib and could explain at least in part variable responses to this drug.^9,13 G2677T, is the most common variants in the coding region of ABCB1.¹¹ The G2677T polymorphism was significantly associated with increased or decreased plasma concentration of P-gp substrates.^14,15 Previous reports showed that individuals who had the 2677 TT genotype had lower P-gp messenger RNA expression than those who had 2677 GG genotype.¹⁶ On the contrary, some pharmacokinetic studies reported an opposite effect of the 2677T mutant allele, i.e. an increase in transport activity compared with that of 2677G allele,¹⁷ whereas Tanabe et al.¹⁸ reported a non-significant opposite trend for P-gp expression in placenta in relation to the G2677T polymorphism. These contradictions might be due to the presence of different amino acids at position 893, which might have different effects on different drugs. Identifying influential SNPs may allow to predict the drug disposition, response to imatinib, and may be helpful in dose adjustments of imatinib, which should be effective in case of imatinib resistant individuals.^9,19

Patients and methods

Patients

A total of 96 Ph⁺ CML patients admitted to Mansoura Oncology Center (OCMU) during 2012 were eligible in our study. They were treated regularly with (400–600 mg)imatinib. Sixty-six (68.75%) patients were in chronic phase, 18 (18.75%) in accelerated phase, and 12 (12.5%) patients in blastic crises. They were 54 (56.25%) males and 42 (43.75%) females with mean age of 44.44 ± 12.37 years. In addition, 90 healthy subjects with matched age and sex without history of cancer were randomly selected as a control group. A written informed consent was obtained from all subjects prior to their enrollment in this study.

Chronic-phase CML was defined by the presence of less than 10% blasts, less than 20% basophils, and less than 30% blasts plus promyelocytes in peripheral blood or bone marrow and a platelet count of at least 100 000/cmm, with no extramedullary involvement. Accelerated phase was characterized by a proportion of blasts of 10–30%, or blasts and promyelocytes < 30%, basophils > 20%, platelets >100 × 10⁹/l unrelated to therapy, or chromosomal abnormalities other than the Philadelphia chromosome, or progressive splenomegaly. Blast crisis was defined as the presence of >30% blasts in peripheral blood or bone marrow or as evidence for an extramedullary blast infiltration (except liver, spleen, or lymph nodes).³

The evaluation of the response is based on blood counts and differential (hematologic response, HR), on the examination of marrow cell metaphases (cytogenetic response, CgR) and on a quantitative assessment of BCR-ABL transcripts level (molecular response, MolR).²⁰ HR was defined as normalized peripheral blood cell counts (WBC <10 × 10⁹/l and platelet count <450 × 10⁹/l) without evidence of peripheral blasts, promyelocytes, or myelocytes, and without evidence of extramedullary disease including disappearance of palpable splenomegaly lasting for at least 4 weeks. Cytogenetic responses were categorized as complete (CCyR; 0% Ph⁺ cells in marrow by conventional cytogenetics or fluorescence in situ hybridization), partial (1–35% Ph⁺ cells in marrow), minor (36–65% Ph⁺ cells in marrow), minimal (66–95% Ph⁺ cells in marrow), and none (>95% Ph⁺ cells in marrow). A major cytogenetic response was defined as the sum of CCyR and partial cytogenetic response (0–35% Ph⁺ cells in marrow). Complete molecular response was defined by non-detectable, non-quantifiable BCR/ABL mRNA. Major MolR was defined as ratio of BCR-ABL to ABL (or other housekeeping genes) ≤0.1% on the international scale.^20,21

An optimal response to imatinib is defined by complete HR and at least minimal CgR (Ph⁺ <95%) at 3 months, at least partial CgR (Ph⁺ <35%) at 6 months, complete CgR at 12 months and major MolR (BCR-ABL: ABL ≤ 0.1%) at 18 months. Failure is defined by incomplete HR at 3 months, no CgR (Ph⁺ > 95%) at 6 months, less than partial CgR (Ph⁺ > 35%) at 12 months, less than complete CgR at 18 months and loss of a complete HR or a complete CgR. In any other situation, the response is defined suboptimal.²⁰

Patients were regularly monitored on an outpatient basis; biweekly physical examinations, blood counts, and biochemistry were obtained during the first month of imatinib therapy and then monthly until a cytogenetic response was achieved, and then every 3 months thereafter until a CCR was confirmed, bone marrow evaluation was performed every 3 months.

Methods

Cytogenetic analysis

Pretreatment cytogenetic analyses of bone marrow or peripheral blood for Philadelphia chromosome was done; metaphase chromosomes were banded by G-banding technique and Karyotyped according to the International System for Human Cytogenetic Nomenclature. A minimum of 20 metaphases was required to be examined for a patient to be classified as having normal cytogenetics.²²

Real-time quantitative polymerase chain reaction

To assess molecular responses, total RNA was extracted from peripheral or bone marrow blood cells. BCR-ABL and internal control transcript levels were quantified using real-time polymerase chain reaction (PCR) analysis (TaqMan) on an ABI prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). Specific PCR products were amplified and detected using dual-fluorescent non-extendable probes labeled with 6-carboxy-fluorescein (FAM), reporter and 6-carboxytetramethylrhodamine (TAMRA), quencher at 5′-end and 3′-end, respectively. The relative mRNA expression of BCR-ABL transcript was calculated using the comparative cycle threshold (Ct) method.²³

Analysis of G2677T polymorphism

DNA from the peripheral blood of the study subjects were extracted using QIAamp DNA blood mini kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The G2677T polymorphism was analyzed using a set of primers F5′-TGC AGG CTATAG GTT CCA GG-3′ and R5′-TTT AGTTTG ACT CAC CTT CCC G-3′ to amplify 224-bp fragment. PCRs were performed in a final volume of 25 µl with 0.5 µM of each of the primers (Sigma-Aldrich, St Louis, MO, USA), 400 µM of each dNTPs, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl (pH 8.3), and 1 U Taq polymerase (Roche Diagnostics, Mannheim, Germany). PCR amplification consisted of an initial denaturation for 5 minutes at 95°C followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute. The terminal elongation was performed at 72°C for 20 minutes. The amplified PCR products were digestedwith Ban I restriction enzyme (New England, Biolabs) for 1 hour at 37°C. After digestion, the products were electrophoresed on a 2% agarose gel for genotyping. 2677G allele creates site for Ban I enzyme and produces two fragments of 198 and 26 bp whereas 2677T allele was identified by single fragment of 224 bp (Fig. 1).

Figure 1. Electrophoretic pattern for MDR1 (G2677T) polymorphism by polymerase chain reaction-restriction fragment length polymorphism. Lane 1–50 bp ladder; lanes 2, 3, 4, 5, 8, 9, 11, 13-GG (198 bp); lanes 6, 7, 12-GT (198, 224 bp); lane 10-TT (224 bp).

Statistical analysis

All the statistical analyses were performed with Statistical Package for the Social Sciences (SPSS) 16.0. Mean (±SD) values were used for quantitative data, numbers and percentages were used for qualitative data. Chi-square test was calculated to test the significance of qualitative variables. One-way analysis of variance test were calculated to test for quantitative variables. Multivariate analysis was performed by unconditional logistic regression analysis. The risk associated with a genetic variant was expressed as odds ratio (with a 95% confidence interval). All the P values were two-sided and the level of significance was taken as P < 0.05.

Results

This study comprised 96 CML patients and 90 control subjects. The overall frequencies of the MDR-1 2677 GG, GT, TT, genotypes were 34.4, 46.9, and 18.8%, respectively. There was no significant difference in the GG, GT genotype frequencies, and a significant lower frequency of TT genotype between patients and controls (P = 0.13, 0.21, 0.004, respectively) (Table 1). No significant association of G2677T polymorphism was observed with respect to age and sex (P = 0.25, 0.55). When the association of G2677T polymorphisms with clinical phase was considered, GG genotype was significantly higher in chronic phase (27; 40.9%) compared to accelerated/blast crisis (6; 20%) (P = 0.046). Hetrozygous GT genotype was more frequent in blastic crisis (12; 100%) than other phases (P = 0.002). With respect to drug response status, complete hematological response was achieved in 49% of all cases, with significant association with TT genotype (77.8%), while partial hematological response was achieved in 51% of all cases, with significant association with GT genotype (64.4%) (P = 0.010).

Table 1. Distribution of MDR1 G2677T polymorphism in CML patients and control

Cytogenetic response varied significantly between GG, GT, and TT genotypes (P < 0.001). CCR was achieved in 45.8% of all cases, with significantly higher incidence of TT genotype (72.2%), minor cytogenetic response was achieved in 17.7% of all cases with higher incidence of GT genotype (31.1%).

Molecular response was achieved completely in 26%. A better molecular response was observed for patients with TT genotype with a significant association (P < 0.001) (Table 2).

Table 2. Patients’ characteristics according to MDR G2677T polymorphisms

Out of 96 CML patients, 50 (52.1%) were responsive (36 in chronic phase and 14 in accelerated/blast crisis) and 46 (47.9%) were resistant (30 in chronic phase and 16 in accelerated/blast crisis) to imatinib with a significant difference in the distribution of G2677T genotypes among sensitive and resistance groups in chronic phase (P = 0.002) (Table 3), where TT genotype was associated with optimal and suboptimal response to imatinib (P = 0.003), meanwhile GT was associated with response failure to imatinib (60%; P = 0.026). GG had no association with drug response (P = 0.93) (Table 4).

Table 3. Response to Imatinib according to MDR G2677T polymorphisms various in CML phases

Table 4. Response to imatinib according to MDR G2677T polymorphisms

The relationship between G2677T polymorphism and risk of imatinib resistance in CML patients was assessed by means of multivariate analysis that considered GT genotype as independent risk factor for resistance (P = 0.037, odds ratio, OR: 2.519, 95% confidence interval, CI: 1.059–5.990), meanwhile TT genotype as protective factor against resistance to imatinib (P = 0.008, OR: 0.166, 95% CI: 0.044–0.627). However, accelerated/blastic phases were not predictors for imatinib resistance (P = 0.777, OR: 1.139, 95% CI: 0.462–2.811).

Discussion

It is well known that different patients may respond differently to the same drug. The MDR1 polymorphisms might alter P-gp expression and activity toward specific anticancer agents, thereby influencing their therapeutic efficacy; the functional consequences of the changes at positions 2677 is still controversial.²⁴ The SNP G2677T in exon 21 (893 codon) results in the substitution of alanine to serine/threonine in such a manner that the lipophilic residue (Ala) is changed to hydrophilic residue (Ser, Thr) conferring higher resistance to various drugs such as adriamycin and vinblastine.^25,26 The frequency of G2677Tgenotypes in this study was similar to that previously reported by Dulucq et al.,¹⁹ and dissimilar to Ni et al.,²⁷ and Sailaja et al. ² Interethnic differences in the frequencies of several SNPs have been reported. In particular, G2677T SNPs have been found to differ significantly among different ethnic groups.²⁴ GG genotype was significantly higher in chronic phase. While GT was higher in blast crisis, likewise reported by Sailaja et al.², who reported that heterozygous 2677GT genotype frequency was increased in blast crisis compared to other phases. GT genotype frequency was associated with partial hematological and minor cytogenetic response, while the frequency of GG genotype was elevated in minimal cytogenetic response in consistent with Sailaja et al.² TT genotype was associated with CCR. However Ni et al.²⁷ reported a better CCR with GT genotype. A better molecular response was observed for patients with TT genotype with a significant association. Dulucq et al.¹⁹ reported a better major molecular response (MMR) for patients with TT genotype. Another study conducted by Kim et al.²⁸, on CML patients, did not find an association between G2677T polymorphism and MMR. We observed a significant difference in the distribution of G2677T genotypes among sensitive and resistance groups in chronic phase likewise reported by Ni et al.²⁷ TT was significantly associated optimal and suboptimal imatinib response that also confirmed by multivariate analysis. However, the study of Gurney et al.⁹, showed that patients with 2677T homozygotes had higher estimated imatinib clearance compared with other genotypes. Another study on AML patients reported that 2677T allele was associated significantly with shorter relapse time and survival rates compared to heterozygotes.²⁹ In some other studies, haplotype of 2677TT and 3435TT was associated with highest risk of drug resistance in lymphoproliferative diseases.³⁰ These results are surprising in light of impressive report demonstrating an association between decreased P-gp mRNA and 2677T allele.¹⁶ Failure of imatinib response was significantly higher with GT genotype that considered as independent risk factor for imatinib resistance by multivariate analysis. This may indicates that 2677 G allele carriers might be at risk for drug resistance. In consistent with our results, Dulucq et al.¹⁹ reported that the G allele at 2677 position was significantly associated with worse response in CML patients who were on imatinib therapy. The G allele was reported to have enhanced P-gp expression and was observed to be associated with increased efflux of P-gp substrates. Since imatinib is one of the P-gp substrate, hence G allele was associated with poor response to imatinib in CML patients.² In line with previous data, studies on G2677T polymorphism have yielded contradictory results, possibly due to small sample sizes and the different ethnic groups.^24,31 In conclusion, the results of our study demonstrated a significant association of the SNPs polymorphisms with imatinib efficacy. Hence, genotyping of MDR1 gene polymorphism (G2677T) might be helpful in planning the individualized therapy based on the genotype. Further genotype analysis of other ABCB1 polymorphisms in patients treated with imatinib may be used as a basis for studies on the relationship between ABCB1 genotypes and drug efficacy and may provide some insight into who is likely to respond optimally to imatinib.