Background: Human organic cationic transporter1 (Hoct1) is a plasma membrane transporter responsible for the main influx of Imatinib into chronic myeloid leukemia (CML) cells. Single nucleotide polymorphisms (SNPs) in the gene coding for hOCT1 are important factors causing Imatinib resistance. We investigated the frequency of hOCT1 SNP C480G among Egyptian CML patients and its relation to early molecular response as an indicator of treatment outcome. Materials and Methods: Two groups of CML patients were included in this study. Group I consisted of 25 patients responding to Imatinib treatment (Imatinib responsive) and group II consisted of 25 patients resistant to Imatinib (Imatinib resistant). Response criteria were assessed according to the NCCN (National Comprehensive Cancer Network) guidelines 2017. Twenty healthy controls of matched age and sex were also included (group III). For all patients, we studied hOCT1 C480G at initial presentation using Taqman drug metabolism genotyping as well as BCR-ABL percent at diagnosis and after 3 months interval. Results: hOCT1 C480G was present in 32% of studied CML patients. CC (wild) was detected in 68% of group I and 64% of group II. CG (mutant heterozygous) was present in 28% of group I and 36% of group II while GG (mutant homozygous) was detected in only one case in group I. CG was also detected in 15% of control subjects There was no significant difference between hOCT1 C480G polymorphism and Early Molecular Response (χ2 = 0.089, p = 0.765). Conclusions: hOCT1 C480G polymorphism has no association with Imatinib resistance in Egyptian population. However, further studies on a larger number of patients are still needed to confirm this finding.
CML (Chronic Myeloid Leukemia) is a myeloproliferative neoplasm with an incidence of 1 - 2:100,000 people in developed nations and it is caused by a balanced translocation t(9;22) (q34;q11.2) [
Imatinib was the first Tyrosine kinase inhibitor (TKI) licensed and called as “magical bullet”, when it revolutionized the treatment of CML [
The frequency of Imatinib resistance and intolerance is estimated at 30% - 40% [
The human Organic Cation Transporter 1 (HOCT1), encoded by the solute carrier 22 family (SLC22A1) gene, is a plasma membrane transporter protein responsible for the translocation of a broad range of drugs [
Over 1000 SNPs (Single Nucleotide polymorphisms) have been identified, of which 22 have been linked to the treatment outcome of the drugs transported by HOCT1 [
Among the commonest SNPs is hOCT1 C480G (rs683369) in which nucleotide 480 C is changed to G leading to Leu160Phe replacement [
hOCT1 shows genetic heterogeneity in different ethnic groups. Caucasian, African and American (Puerto Ricans, Colombian and Mexican) populations
present higher variability than Asians and Pacific Islanders [
This study is the first one in Egypt designed to investigate the frequency of hoct1 C480G SNP and its effect on EMR to Imatinib in Egyptian CML patients while there were other studies on hOCT1 were presented by Vine et al. and Paolo et al. [
This study included 50 CML patients treated at the Hematology Clinic, Alexandria Main University Hospital and the Medical Research Institute Alexandria University, over 10 months duration between May 2016 and March 2017.The patients were divided according to the early molecular response(EMR) defined as BCR-ABL transcript ≤10% at 3 months of treatment into two groups: group I (Imatinib responsive) consisted of 25 CML patients who achieved EMR and group II (Imatinib resistant) consisted of 25 CML patients who failed to achieve EMR .Response criteria were assessed according to the NCCN guidelines [
20 healthy matched subjects were recruited as control group. Risk stratification of CML patients was done using Sokal risk score into low, intermediate and high risk. Patients with hepatic or renal failure, concomitant chronic illness and pregnant females were excluded from the study.
All patients participating in this study were subjected to history taking, clinical examination with special emphasis on spleen size, Complete blood count (CBC), measurement of BCR-ABL1 transcripts at diagnosis and after 3 months of Imatinib treatment by quantitative Reverse transcriptase polymerase chain reaction (RT-PCR) and assessment of the polymorphism of hOCT1 C480G on peripheral blood samples by real time PCR using Taqman drug metabolism genotyping assay. Some data were present in the patients’ records at the time of the study while other data were conducted at the same time of the study. This study was approved by the local ethics committee at Alexandria University Hospitals, Egypt which goes in accordance with the Helsinki Declaration.
At first genomic DNA extraction was done using Thermo Scientific Gene JET Genomic DNA Purification Kit. The concentration of DNA was estimated by measuring the absorbance at 260nm (A260) using a spectrophotometer (Nanodrop® ND-1000 spectrophotometer). DNA purity was estimated from the A260/A280 ratio. An A260/A280 ratio between 1.7 and 2.0 generally represents a high-quality DNA sample.
Samples were stored at −20˚C. The following primer was used AGTCCTGTT TGAATGCGGGCTTCTT[C/G]TTTGGCTCTCTCGGTGTTGGCTACT. Primers were validated through NCBI blast. PCR reaction mix was performed using Taqman drug metabolism genotyping assay. Analysis of the SNP for hOCT1 C480G (rs683369) was done by Real time PCR. Thermocycling conditions were 10 min at 95˚C, followed by 40 cycles of denaturation (95˚C, 15 sec). Results were divided into CC (wild type) CG (mutant heterozygous) and GG (mutant homozygous).
Total RNA was extracted from peripheral blood mononuclear cells using RNeasy Midi Kit (Qiagen) and was converted into cDNA using ipsogen RT kit (Qiagen). RQ-PCR was analysed through Rotor-gene Q instrument using ipsogen BCR-ABL1 kit (Qiagen). The absolute quantities of BCR-ABL and ABL transcripts in patient specimens were determined by reference to standard curves [
Data were analyzed using IBM Statistical Package for the Social Sciences (SPSS) (version 24.0; SPSS Inc., Chicago, IL, USA). Data were tested for normality using Kolmogorov-Smirnov test, Shapiro-Wilk test. Mean and standard deviation were used to describe the scale data while frequency was used to describe the categorical data. Parametric data were analyzed using t test to compare 2 means and ANOVA(f) test to compare more than 2 means. Non-parametric data were analyzed using Mann Whitney and Kruskal Wallis tests. Qualitative data such as sex, Sokal score, genotype distribution and frequencies were presented by percentages and tested by Chi Square χ2 and Fisher Exact Test according to the categories and cells’ estimation percent when more than 20% of the cells have expected count <5. A difference was considered significant if p value was less than 0.05 in all analyses.
Baseline characteristics including age, sex, spleen size, Sokal score and Bcr-ABL1 transcripts at 3 months are presented in
The distribution of hOCT1 C480G genotypes in both CML and control groups is shown in
Parameter | Groups (n = 70) | Test of sig. | P | |||||
---|---|---|---|---|---|---|---|---|
Group I (n = 25) | Group II (n = 25) | Control (n = 20) | ||||||
No. | % | No. | % | No. | % | |||
Sex | χ2 = 0.373 | 0.830 | ||||||
Male | 12 | 48 | 14 | 56 | 11 | 55 | ||
Female | 13 | 52 | 11 | 44 | 9 | 45 | ||
Age (years) | 43.7 ± 13.4 | 48.64 ± 16.77 | 39.1 ± 12.03 | F = 2.463 | 0.093 | |||
Genotype CC CG GG | 17 (68%) 7 (28%) 1 (4%) | 16 (64%)) 9 (36%) 0 (0%) | 17 (85%) 3 (5%) 0 (0%) | χ2 = 4.21 | 0.312 |
Group I: Imatinib responsive, Group II: Imatinib resistant; CC (wild), CG (mutant heterozygous), GG (mutant homozygous).
Parameter | Group I (n = 25) | Group II (n = 25) | Test of sig | P |
---|---|---|---|---|
Spleen size (cm) | 20.78 ± 4.2 | 25.10 ± 3.6 | t = 3.770 | 0.001* |
Sokal score | 1.21 ± 0.342 | 1.5 ± 0. 36 | t = 2.819 | 0.002* |
Sokal risk category Low risk Intermediate High | 4 (16%) 10 (40%) 11 (44%) | - 5 (20.8%) 19 (79.2%) | χ2 = 7.402 | 0.023* |
BCR-ABL1 at diagnosis (%) | 70.37 ± 26.25 | 73.88 ± 22.85 | u = 295.000 | 0.731 |
BCR-ABL1 transcript at 3 months (%) | 4.53 ± 5.23 | 35.54 ± 8.13 | u = 14.500 | 0.000* |
C480G genotype CC CG+GG | 17 (51.5%) 8 (47%) | 16 (48.5%) 9 (53.0%) | χ2 = 0.089 | 0.765 |
Group I (Imatinib responsive), group II (Imatinib resistant); CC wild, CG mutant heterozygous, GG mutant homozygous; *P is significant when ≤0.05.
When categorizing CML patients according to the Sokal risk score and C480G polymorphism, it was found that in group I, patients having the CC genotype were 23.5% (four patients) low risk Sokal, 47.1% (8 patients) were intermediate risk and 29.5% (five patients) were high risk whereas patients with the CG or GG genotype were 25% (two patients) intermediate risk and 75% (six patients) were high risk. In group II, patients carrying the CC genotype were 26.7% (four patients) intermediate risk and 73.3% (11 patients) were high risk whereas patients who had the CG genotype, 11.1% (one patient) was intermediate risk and 88.9% (eight patients) were high risk. Sokal score was significantly higher among group II especially patients carrying the CG genotype (χ2 = 11.954, p = 0.026).
There is no significant difference in age (p = 0.463), sex (p = 0.709), BCR-ABL1 at diagnosis (p = 0.902), EMR (p = 0.765), Hb level (p = 0.726), WBCs (p = 0.197) and platelets count (p = 0.990) among the CC, CG and GG. There was significant difference among hOCT1 C480G genotypes and spleen size (p = 0.001) in the CML studied groups (
This study aimed at investigating the frequency of the polymorphism of Hoct1 transporter C480G and EMR. The present study couldn’t find a statistically significant effect of hOCT1 polymorphism on the EMR. In accordance with our results, Takahashi et al. studied hOCT1 C480G polymorphism among other polymorphisms and their effects on Imatinib drug concentration and clinical response. They found no association between hOCT1 C480G polymorphism and molecular response [
Parameters | Group I | Group II | Test of Sig. | P | ||
---|---|---|---|---|---|---|
CC (n = 17) | CG + GG (n = 8) | CC (n = 16) | CG (n = 9) | |||
Age (years) | 41.82 ± 12.17 | 47.88 ± 15.90 | 50.25 ± 17.92 | 45.78 ± 15.1 | F = 0.871 | 0.463 |
Sex Male Female | 8 (47.1%) 9 (52.9%) | 5 (62.5%) 3 (37.5%) | 6 (37.5%) 10 (32.5%) | 5 (55.6%) 4 (44.4%) | χ2 = 1.645 | 0.709 |
Spleen size (cm) | 20.28 ± 4.08 | 22.13 ± 4.49 | 24.03 ± 3.20 | 26.89 ± 3.72 | F = 6.437 | 0.001* |
Sokal score Low Intermediate High | 4 (23.5%) 8 (47.1%) 5 (29.4) | 0 2 (25%) 6 (75%) | 0 4 (26.7%) 11 (73.3%) | 0 1 (11.1%) 8 (88.9%) | χ2 = 11.954 | 0.026* |
Hemoglobin (g/dl) | 10.61 ± 1.46 | 9.74 ± 2.56 | 10.28 ± 2.04 | 9.93 ± 2.18 | F = 0.440 | 0.726 |
Platelets (×109/L) | 351.8 ± 177.2 | 384.3 ± 226.5 | 372.3 ± 264.5 | 373.6 ± 233.4 | H = 0.116 | 0.990 |
WBCs (×109/L) | 187.6 ± 163.9 | 249.6 ± 178.1 | 114.1 ± 109.4 | 155.2 ± 125.6 | H = 4.676 | 0.197 |
BCR-ABL1 at diagnosis (%) | 69.96 ± 25.14 | 73.29 ± 32.12 | 72.12 ± 21.16 | 77.0 ± 26.63 | H = 0.484 | 0.902 |
EMR** | ≤10% | >10% | χ2 = 0.089 | 0.765 |
Group I (Imatinib responsive), group II (Imatinib resistant); CC (wild), CG (mutant heterozygous), GG (mutant homozygous); *p significant if ≤0.05; **EMR: Early molecular response.
Similarly, Vine et al. investigated the relation between hOCT1 SNPs (C480G, Met408Val) and multidrug resistance gene (MDR1) SNP and Imatinib response using restriction enzyme analysis and correlated them with Imatinib response and Imatinib drug level. hOCT1 polymorphisms did not predict treatment failure when correlated with Imatinib levels. Patients with GG C480G genotype needed a slightly higher Imatinib level to achieve molecular response but this wasn’t statistically significant [
Contradictory results were reported by Paolo et al. who found that hOCT1 C480G may be associated with a reduced intracellular uptake of Imatinib with the following increased risk of treatment failure. Paolo et al. correlated Imatinib drug concentration with the presence of the hOCT1 C480G and Imatinib response [
In the present study, Sokal score was significantly advanced in group II patients especially those with the CG genotype (p = 0.026).This results are more or less similar to Chhikara et al. who reported that patients with high OCT1 expression had significant higher Sokal scores than those with low OCT1 expression [
Although the present work couldn’t find a significance impact of C490G polymorphism on EMR, BCR-ABL1 transcript level at 3 months were significantly higher in group II especially those having the heterozygous form (CG). CG genotype may be associated with a higher risk evidenced in this study by the statistically more advanced Sokal score and higher BCR-ABL transcripts at 3 months.
From this study, it was concluded that C480G polymorphism is present in Egyptian CML patients. EMR was not affected by the presence of C480G polymorphism in CML patients. Spleen size and Sokal score were significantly higher in patients carrying the mutant (CG) than those with normal wild (CC). So, C480G polymorphism may be associated with higher risk. Testing these findings in a larger patient population with newly diagnosed CML is recommended.
Hamed, N.A.M., Neanea, H., Ghanem, A.M., Elgammal, M.M.A. and Samir, Y. (2018) Polymorphism of Human Organic Cationic Transporter1 (C480G) in Egyptian Chronic Myeloid Leukemia Patients on Imatinib. American Journal of Molecular Biology, 8, 83-91. https://doi.org/10.4236/ajmb.2018.82007