Biomedicine & Pharmacotherapy 68 (2014) 343–349

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Original article

Association of genotypes and haplotypes of multi-drug transporter genes ABCB1 and ABCG2 with clinical response to imatinib mesylate in chronic myeloid leukemia patients Anthony Au a,*, Abdul Aziz Baba b,c, Ai Sim Goh d, S. Abdul Wahid Fadilah e, Alan Teh f, Hassan Rosline g, Ravindran Ankathil a,* a

Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia Department of Internal Medicine and Clinical Haematology, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia c School of Medicine, International Medical University, 57000, Bukit Jalil, Kuala Lumpur, Malaysia d Department of Medicine, Hospital Pulau Pinang, 10990, Georgetown, Penang, Malaysia e Cell Therapy Centre, UKM Medical Centre, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia f Department of Haematology, Sime Darby Medical Center, 47500, Subang Jaya, Selangor, Malaysia g Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 December 2013 Accepted 20 January 2014

The introduction and success of imatinib mesylate (IM) has become a paradigm shift in chronic myeloid leukemia (CML) treatment. However, the high efficacy of IM has been hampered by the issue of clinical resistance that might due to pharmacogenetic variability. In the current study, the contribution of three common single nucleotide polymorphisms (SNPs) of ABCB1 (T1236C, G2677T/A and C3435T) and two SNPs of ABCG2 (G34A and C421A) genes in mediating resistance and/or good response among 215 CML patients on IM therapy were investigated. Among these patients, the frequency distribution of ABCG2 421 CC, CA and AA genotypes were significantly different between IM good response and resistant groups (P = 0.01). Resistance was significantly associated with patients who had homozygous ABCB1 1236 CC genotype with OR 2.79 (95%CI: 1.217–6.374, P = 0.01). For ABCB1 G2677T/A polymorphism, a better complete cytogenetic remission was observed for patients with variant TT/AT/AA genotype, compared to other genotype groups (OR = 0.48, 95%CI: 0.239–0.957, P = 0.03). Haplotype analysis revealed that ABCB1 haplotypes (C1236G2677C3435) was statistically linked to higher risk to IM resistance (25.8% vs. 17.4%, P = 0.04), while ABCG2 diplotype A34A421 was significantly correlated with IM good response (9.1% vs. 3.9%, P = 0.03). In addition, genotypic variant in ABCG2 421C>A was associated with a major molecular response (MMR) (OR = 2.20, 95%CI: 1.273–3.811, P = 0.004), whereas ABCB1 2677G>T/ A variant was associated with a significantly lower molecular response (OR = 0.49, 95%CI: 0.248–0.974, P = 0.04). However, there was no significant correlation of these SNPs with IM intolerance and IM induced hepatotoxicity. Our results suggest the usefulness of genotyping of these single nucleotide polymorphisms in predicting IM response among CML patients. ß 2014 Elsevier Masson SAS. All rights reserved.

Keywords: ABCB1 ABCG2 Chronic myeloid leukemia Imatinib mesylate Single nucleotide polymorphisms

1. Introduction Imatinib mesylate (IM), a competitive inhibitor of the BCR-ABL tyrosine kinase, has proven to be an extremely effective and generally well tolerated drug, producing durable responses in patients with chronic myeloid leukemia (CML). Despite the outstanding results obtained with IM for the treatment of CML, a significant proportion of patients show suboptimal response or develop resistance to IM. Mechanism of resistance to IM in CML

* Corresponding authors. Tel.: +6097676968. E-mail addresses: [email protected] (A. Au), [email protected] (R. Ankathil). 0753-3322/$ – see front matter ß 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2014.01.009

patients involve BCR-ABL dependent and BCR-ABL independent pathways. BCR-ABL dependent mechanism which mainly involve point mutations in the tyrosine kinase domain (TKD) and amplification of BCR-ABL gene, account for approximately 50% of patients who develop resistance [1]. For the CML patients who do not fit into the BCR-ABL dependent mechanisms of resistance, several other BCR-ABL independent mechanisms have been postulated. Pharmacogenetic variability, which influences the pharmacokinetics of IM, could be a possible BCR-ABL independent mechanism mediating resistance. IM is a substrate for the adenosine triphosphate binding cassette (ABC) transporters, ABCB1 and ABCG2. ATP Binding Cassette B1 (ABCB1) gene located at chromosome 7q21.1, consists

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of 28 introns and 28 exons and encodes for P-glycoprotein (P-gp), with 170 kDa or 1280 amino acids. ABCG2, also known as Breast Cancer Resistance Protein (BCRP) is the second member of the G family of ABC transporters. Located on chromosomal region 4q22, ABCG2 gene consists of 16 exons that span over 66 kb and encodes a 72-kDa membrane protein that is composed of 655 amino acids. Accordingly, ABCB1 and ABCG2 might be influencing the pharmacokinetics and intracellular or systemic level of IM. Both ABCB1 and ABCG2 display a high affinity for IM and have been demonstrated to confer resistance in vitro by extruding IM from haematopoietic cells [2]. Single nucleotide polymorphisms (SNPs) of these drug transporters could be potential determinants of variability in drug disposition and efficacy. ABCB1 is highly polymorphic with existing 50 and more single nucleotide polymorphisms (SNPs) yet to be identified. SNPs within ABCB1 gene have been associated with PGp over-expression. Three polymorphisms (T1236C, G2677T/A and C3435T) of ABCB1 have significant effect on P-gp expression, functionality and substrate distribution [3]. The 3435C>T located on exon 26, is in linkage disequilibrium with the other two SNPs, 2677G>T/A (exon 21) and 1236C>T (exon 12), of which 2677G>T/A is responsible for a substitution in the amino acid sequence (Ala893Ser/Thr). Genetic polymorphisms in ABC transporters have been associated with altered transporter functions of various drugs [4]. Moreover, the ABCB1 SNPs are highly polymorphic within different ethnic groups. BCRP expression and function can be altered by SNPs in ABCG2 gene. Two most common ABCG2 polymorphisms are 34 G>A, which codes for Val12Met and 421 C>A which codes for Glu141Lys (Zamber et al., 2003). Pharmacogenetic studies have been helpful in the evaluation of sensitivity profile of drugs. SNPs of ABCB1 and ABCG2 genes can cause inter-individual variations in the pharmacokinetics for IM. Therefore, we aimed to determine whether different genotype and haplotype pattern of SNPs ABCB1 (1236T>C, 2677G>T/A, and 3435C>T) and ABCG2 (34G>A and 421C>A) have any influence in mediating clinical response in CML patients undergoing IM treatment. IM pharmacogenetics may have an obvious correlation with the cytogenetic and molecular response of IM. Apart from IM response, the effects of pharmacogenetic covariates on hematological, non-hematological adverse side effects and hepatotoxicity among CML patients undergoing IM treatment were also examined. 2. Materials and methods 2.1. Sample collection This multi-centric study protocol was approved by Universiti Sains Malaysia Human Ethics Committee and registered under National Medical Research Register (NMRR), Ministry of Health Malaysia. A total of 215 Philadelphia (Ph) chromosome positive CML participants (age range between 11 to 78 years) undergoing 400 mg IM daily for at least 6 months in several local hospitals and medical centers in Malaysia were enrolled after obtaining written informed consent. Out of the 215 CML patients, 106 were males and 109 were females with a mean age of 41.5 years. When these CML patients were categorized based on IM treatment response, 107 were IM good responders and 108 IM were resistant Ph+ CML patients belonging to chronic and accelerated phase. Blood samples of these patients were collected after getting written informed consent and stored in EDTA vacutainer tubes until analysis. 2.2. Evaluating imatinib response and tolerance Hematologic, cytogenetic and molecular criteria were accessed in order to justify clinical response to IM by referring ‘‘European Leukemia Net: guideline for managing CML patients’’ [5].

Molecular response was classified based on BCR-ABL control gene transcript ratios, expressed on the International Scale, where major molecular response (MMR) and complete molecular response (CMR) were defined as ratios  0.1% and  0.0032% respectively. Cytogenetic response was classified as complete (0% Ph+ metaphases), partial (> 0 to 35% Ph+ cells), minor (> 35 to 65% Ph+ metaphases), minimal (> 65–95% Ph+ metaphases), and none (> 95–100% Ph+ metaphases) based on GTG banded analysis of a minimum 20 bone marrow metaphases. CML patients were categorized as in complete hematological response (CHR) when they achieved < 450  109/L platelet count, < 10  109/L WBC count and < 5% basophils. Those patients with CHR in 3 months, MCgR in 12 months and MMR in 18 months were considered as IM good responders. Those patients who did not achieve the above response criteria within the specified time frame were categorized under non responders/resistant group. In this study, common IM adverse side effects were non-hematological toxicity (nausea and/ or vomit, skin hypo-pigmentation and/or rash, join pain and/or muscle cramp, headache and/or fatigue) and hematological toxicity (anemia, thrombocytopenia and neutropenia) in all grades, after administration with 400 mg IM. Parameters of hepatic injury were assessed by biochemical parameters like levels of bilirubin, alkaline phosphatase (ALP) and alanine transaminase (ALT). 2.3. Genotyping DNA from the peripheral blood of the study subjects were extracted using QIAGEN amp DNA extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. DNA yield was standardized into 50 ng/ml after measurement with Infinite1 200 PRO NanoQuant. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was designed for amplification of the SNPs ABCB1 1236 T>C, 2677 G>T/A, 3435 C>T, and also the ABCG2 G34A and C421A SNPs. Forward and reverse primers were self-designed for 1236 T>C (F: 50 -CGAAGAGTGGGCACAAACCAG-30 and R: 50 -GCATGGGTCATCTCACCATC-30 ) and 2677 G>T/A (F: CCT TCA TCT ATG GTT GGC-30 and R: 50 - GCA TAG TAA GCA GTA GGG AG-30 ). Primers for 3435C>T was obtained from Ameyaw et al. [6] while 34G>A and 421C>A were referred to Kobayashi et al. [7]. Each PCR mixture consisted of 1 MyTaq Reaction Buffer (Bioline Ltd, London, UK), 1 unit MyTaq DNA Polymerase (Bioline Ltd, London, UK), 50 ng genomic DNA templates and ddH2O in a total volume of 20 ml. PCR conditions involved denaturation at 958 C for 1 min, and repeated 35 cycles consisting of 3 steps: denaturation at 958 C for 15 seconds; annealing at 638 C/568 C/618 C/618 C/608 C for 15 seconds and; extension at 728 C for 10 seconds, followed by 3 mins final extension at 728 C. PCR products were subsequently digested with Fermentas FastDigest (Fermentas, Lithuania) Eco0109 I for 30 mins, 378 C (1236T>C), Ban I 378 C for 30 mins (2677G>T), Rsa I for 30 mins, 378 C (2677G>A), Mbo I for 30 mins, 378 C (3435C>T), BseM I for 558 C, 20 mins (34G>A) and Taa I for 10 mins at 658 C (421C>A) respectively. Digested DNA fragments were electrophoresed on 3.5% Bioline Agarose HiRes gel (Bioline Ltd, London, UK) and stained with SyBr Green. As part of quality control checking, genotyping results were directly sequenced (First BASE Laboratories Sdn Bhd, Malaysia) in 10% of samples after the purification steps (GeneJET PCR Purification Kit, Fermentas). 2.4. Statistical analysis Hardy–Weinberg equilibrium was verified for all examined SNPs. Difference in genotype frequencies among the two groups of CML patients and the associations of the various genotypes with good response and resistance to IM were determined using Pearson X2 test. Odds ratios (OR) along with 95% confidence intervals (CI) and two-sided P-values were calculated by Epi Info

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patients with IM good response (20.4% vs. 8.4%) with OR 2.79 (95%CI: 1.217–6.374, P = 0.01). For the G2677T/A polymorphism, the frequency of genotype TT/AT/AA (29.9%) was higher among IM good response group compared to IM resistant group (13.9%) but statistically insignificant (P = 0.14). However, this genotype showed a significant lower risk for IM resistance with OR 0.38 (95%CI: 0.191–0.750, P = 0.004). The C3435T genotype was found not to influence IM response since the genotype frequencies of this SNP in both IM response and IM resistant groups were close to similar and without any significant difference (P > 0.05). Meanwhile for ABCG2 C421A, the homozygous major CC genotype with a frequency of 53.7%, was found to be significantly higher (P = 0.01) among IM resistant group compared to IM good response CML patients (38.3%). Risk calculation showed that CML patients with ABCG2 C421C genotype had a higher risk for development of resistance with OR 1.87 (95%CI: 1.085–3.214, P = 0.02). When the frequency of ABCG2 A421A was compared between CML patients showing good response and resistance to IM (16.8% vs. 5.6%), the frequency was significantly higher in CML patients with good response (P = 0.01) with OR 0.29 (95%CI: 0.111–0.765, P = 0.01). This indicates a significant lower risk of resistance development for CML patients with ABCG2 A421A variant genotype. Haplotypic frequencies of SNPs of ABCB1 and ABCG2 were determined among both IM responsive and resistance group CML patients. The most frequently observed ABCB1 haplotypes were 1236T/2677T/3435T (25%) and 1236C/2677G/3435C (21.7%). Among haplotypes, the haplotype T1236T2677C3435 with a frequency of 16.1% was significantly higher among IM responders with P value 0.04 (Table 1). Whereas the haplotype C1236G2677C3435 with a frequency of 25.8% was significantly higher among the IM

7 software. Chi-square or Fisher’s exact tests (if n < 5) was performed to associate the clinical response (cytogenetic and molecular response) and tolerance (hematological and nonhematological toxicity) with SNPs. Kruskal waillis test was used to compare the distribution of mean ALT, ALP and bilirubin values in the different genotype patterns. All these statistical tests were carried out by SPSS software version 20.0. P-values less than 0.05 were considered statistically significant. Haplotype frequencies were determined between IM response groups and haplotype with > 0.03% was presented. Corrected P-value was obtained after applying 1000 times per-mutation test, by using the Haploview 4.2 software. 3. Results 3.1. ABCB1 & ABCG2 SNPs in IM response The genotypic distribution of ABCB1 1236T>C, 2677G>T/A, and 3435C>T and ABCG2 34G>A and 421C>A polymorphisms among IM good response and resistant CML patients are shown in Table 1. With regard to ABCB1 C3435T and ABCG2 G34A, there was no significant difference in the genotype frequencies between CML patients showing good response and resistant to IM. However, the homozygous variants of ABCB1 G2677T/A and ABCG2 C421A genotypes were significantly higher in CML patients showing IM good response compared to IM resistant CML patients. The frequency of IM resistance was found to correlate with the number of C allele at locus ABCB1 1236, G allele at ABCB1 2677 locus and C alleles at ABCG2 allele. Resistance was higher among patients homozygous for the C1236C genotype when compared to Table 1 Association of ABCB1 and ABCG2 genotypes and haplotypes with IM response. SNPs

Genotype

n, frequency Responsive

Resistant

X2

P

OR

95% CI

X2

P

ABCB1 1236

TT CT CC

43 (40.2) 55 (51.4) 9 (8.4)

31 (28.7) 55 (50.9) 22 (20.4)

7.39

0.25

0.60 0.98 2.79

0.339–1.058 0.575–1.675 1.217–6.374

3.14 0.05 7.38

0.08 0.94 0.01*

ABCB1 2677

GG GT + GA TT + AA + TA

19 (17.8) 56 (52.3) 32 (29.9)

28 (25.9) 65 (60.2) 15 (13.9)

8.54

0.14

1.62 1.38 0.38

0.841–3.126 0.802–2.364 0.191–0.750

2.10 1.35 8.07

0.15 0.25 0.004*

ABCB1 3435

CC CT TT

26 (24.3) 64 (59.8) 17 (15.9)

31 (28.7) 58 (53.7) 19 (17.6)

0.84

0.65

1.25 0.78 1.13

0.683–2.303 0.454–1.338 0.552–2.315

0.54 0.82 0.11

0.46 0.37 0.74

ABCG2 34

GG GA AA

52 (48.6) 42 (39.3) 13 (12.1)

45 (41.7) 52 (48.1) 11 (10.2)

1.73

0.42

0.76 1.44 0.82

0.441–1.295 0.836–2.469 0.350–1.922

1.04 1.73 0.21

0.31 0.19 0.65

ABCG2 421

CC CA AA

41 (38.3) 48 (44.9) 18 (16.8)

58 (53.7) 44 (40.7) 6 (5.6)

9.09

0.01*

1.87 0.85 0.29

1.085–3.214 0.492–1.451 0.111–0.765

5.12 0.37 6.88

0.02* 0.54 0.01*

Gene

Haplotype

Frequency Responsive

Resistant

X2

P

ABCB1

TTT CGC TTC TGT CTC TGC CGT CTT

28.9 17.4 16.1 10.2 9.9 10.8 5.5 1.2

21.2 25.8 9.5 14.6 11.3 8.9 6.7 2.0

3.41 4.44 4.11 1.94 0.23 0.44 0.27 0.37

0.07 0.04* 0.04* 0.16 0.64 0.51 0.60 0.54

ABCG2

GC AC GA AA

38.1 22.6 30.1 9.1

43.7 30.3 22.0 3.9

1.4 3.3 3.7 4.8

0.24 0.07 0.06 0.03*

*P < 0.05 (Statistically significant).

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Table 2 Association of ABCB1 and ABCG2 genotypes with cytogenetic response. SNPs

Genotype

CCyR

Non-CyR

X2

P

OR

95% CI

X2

P

ABCB1 1236

TT CT CC

46 63 13

28 47 18

3.67

0.16

0.71 0.96 2.01

0.401–1.265 0.558–1.642 0.930–4.353

1.35 0.03 3.24

0.25 0.87 0.07

ABCB1 2677

GG GT + GA TT + AA + TA

24 65 33

23 56 14

4.54

0.10

1.34 1.33 0.48

0.701–2.567 0.768–2.293 0.239–0.957

0.79 1.03 4.45

0.37 0.31 0.03*

ABCB1 3435

CC CT TT

32 70 20

25 52 16

0.05

0.98

1.03 0.94 1.06

0.562–1.904 0.547–1.624 0.515–2.179

0.01 0.05 0.02

0.91 0.83 0.87

ABCG2 34

GG GA AA

52 53 17

45 41 7

2.34

0.31

1.26 1.03 0.51

0.734–2.171 0.596–1.768 0.199–1.268

0.71 0.01 2.18

0.40 0.92 0.14

ABCG2 421

CC CA AA

55 51 16

44 41 8

1.08

0.58

1.09 1.07 0.62

0.637–1.879 0.625–1.855 0.255–1.527

0.11 0.07 1.08

0.75 0.79 0.30

*P < 0.05 (Statistically significant).

resistance group (P = 0.04). For ABCG2 gene, only a single haplotype marker A34A421 was found to be significantly associated with IM good response (9.1% vs. 3.9%, P = 0.03). The distribution of cytogenetic response among CML patients with various ABCB1 and ABCG2 genotypes is summarized in Table 2. There was no statistically significant association between cytogenetic response and genotype frequency, except for ABCB1 2677. The variant genotypes (TT, AA and TA) were found to be associated with a significantly lower risk for CCyR with OR 0.48 (95%CI: 0.239–0.957, P = 0.03). For 1236 CC genotype also, a nearly significant lower risk association for cytogenetic response to IM (OR: 2.01, P = 0.07), was observed. The distribution of molecular response among CML patients with various ABCB1 and ABCG2 genotypes is summarized in Table 3. There were statistically significant differences in the molecular response of CML patients with 2677G>T/A and 421C>A genotypes. The presence of the variant genotype in ABCB1 2677 had a favorable impact on MMR achievement with OR 0.49 (95%CI: 0.248–0.974, P = 0.04). Nearly significant lower risk association was observed for ABCB1 1236 CC genotype with molecular response (P = 0.05). In ABCG2 421C>A, the frequency of MMR was 44/99 patients (44.44%) with the CC genotype, 57/92 (61.96%) with the CA genotype and 17/24 (70.83%) with the AA genotype. The odds ratio of CC over CA + AA was 2.20 (95%CI: 1.273–3.811, P = 0.004).

3.2. ABCB1 & ABCG2 SNPs in IM tolerance Table 4 shows association of ABCB1 and ABCG2 genotypes with IM induced hemato- and non hemato-toxicity. However, the incidence of severe neutropenia was higher in patients with 1236 CC genotype (13.3%, 2/15) than in patients with TC (11.9%, 8/67) or TT (2.1%, 1/47) genotypes but statistically insignificant (P = 0.14). No significant association of skin hypo-pigmentation or rash was found in patients with GG (30%, 9/30), GA + GT (28.9%, 22/76), and TT + TA + AA (8.7%, 2/23) genotypes (P = 0.12). 3.3. ABCB1 & ABCG2 SNPs in hepatotoxicity levels A comparison of ALT, ALP and bilirubin values among CML patients with different genotypes of each SNP is summarized in Fig. 1. No significant association were observed for these SNPs, as values of bilirubin, ALP and ALT did not differ significantly (P > 0.05) among patients with various genotypes. Even though, differences in the mean values of bilirubin were observed in ABCG2 421 C>A, with 11.7 mmol/L for CC, 9.7 mmol/L for CA and 14.2 mmol/L for AA genotype, it was statistically insignificant (P = 0.10). Also, the ALP values showed some differences in ABCB1 2677 G>T/A (a mean value of 102.2 U/l for GG, 78.6 U/I for GT + GA and 89.8 U/l for TT + TA + AA genotypes respectively) but still, the difference was not statistically significant (P = 0.28).

Table 3 Association of ABCB1 and ABCG2 genotypes with molecular response. SNPs

Genotype

MMR

Non-MMR

X2

P

OR

95% CI

X2

P

ABCB1 1236

TT CT CC

40 66 12

34 44 19

3.67

0.16

1.05 0.65 2.15

0.598–1.852 0.381–1.123 0.987–4.692

0.03 2.38 3.83

0.86 0.12 0.05

ABCB1 2677

GG GT+GA TT+AA+TA

24 62 32

23 59 15

4.54

0.10

1.22 1.40 0.49

0.637–2.327 0.813–2.418 0.248–0.974

0.35 1.48 4.23

0.55 0.22 0.04*

ABCB1 3435

CC CT TT

30 69 19

27 53 17

0.05

0.98

1.13 0.86 1.11

0.616–2.077 0.497–1.471 0.540–2.269

0.16 0.32 0.08

0.69 0.57 0.78

ABCG2 34

GG GA AA

56 47 15

41 41 15

2.34

0.31

0.81 1.11 1.26

0.472–1.393 0.641–1.909 0.580–2.719

0.58 0.13 0.34

0.45 0.72 0.56

ABCG2 421

CC CA AA

44 57 17

55 35 7

1.08

0.58

2.20 0.60 0.46

1.273–3.811 0.349–1.047 0.183–1.165

8.08 3.25 2.78

0.004* 0.07 0.10

*P < 0.05 (Statistically significant).

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Table 4 Association between ABCB1 and ABCG2 genotypes with hematological and non-hematological toxicities among CML patients undergoing IM treatment. SNPs

Genotype

Non Hematological Toxicities Nausea + Vomit

Hematological Toxicities

Hypopigmentation + Skin Rash

Joint Pain + Muscle Cramp

Fatigue + Headache + Fever

Anemia

Thrombocytopenia

Neutropenia

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

ABCB1 1236

TT CT CC P

14 24 5 0.82

33 44 11

11 16 6 0.39

36 51 9

8 3 1 0.71

39 64 14

4 9 3 0.47

43 57 12

4 14 2 0.19

43 53 13

7 17 0 0.53

40 50 15

1 8 2 0.14

46 59 13

ABCB1 2677

GG GT+GA TT+AA+TA P

7 31 5 0.96

24 46 18

9 22 2 0.12

21 54 21

4 6 2 0.68

26 70 21

2 10 4 0.48

28 65 19

7 10 3 0.40

23 66 20

7 13 4 0.75

23 63 19

4 6 1 0.49

26 70 22

ABCB1 3435

CC CT TT P

15 21 7 0.57

23 47 18

11 14 8 0.44

26 53 17

4 5 3 0.75

33 62 22

7 5 4 0.21

30 61 21

9 8 3 0.21

28 59 22

5 16 3 0.28

32 51 22

4 6 1 0.63

37 67 25

ABCG2 34

GG GA AA P

16 20 7 0.70

37 41 10

18 11 4 0.18

35 48 13

4 7 1 0.64

49 52 16

6 8 2 0.95

46 51 15

6 11 3 0.55

47 48 14

7 13 4 0.42

46 46 13

3 6 2 0.61

50 53 17

ABCG2 421

CC CA AA P

23 17 3 0.73

45 33 10

15 15 3 0.59

52 34 10

7 4 1 0.90

60 45 12

7 9 0 0.15

60 39 13

10 9 1 0.63

57 40 12

12 11 1 0.47

55 38 12

8 3 0 0.28

59 46 13

*P < 0.05 (Statistically significant).

4. Discussion Although a number of factors may contribute to interindividual variability in drug response, the genotype of a patient is increasingly implicated in influencing drug disposition and activity. SNPs in ABCB1 and ABCG2 have been demonstrated to display high affinity for IM and confer IM resistance in vitro by extruding IM from hematopoietic cells [8]. IM is a substrate of ABCG2 and ABCB1 in a narrow concentration range and act as inhibitor at higher concentrations due to its high affinity [9]. By contrast, the contribution of ABCB1 polymorphisms in modulating IM resistance has yet to be fully clarified, as the efflux of IM have not been able to correlate ABCB1 over-expression in IM resistant cell lines. RNAi silencing of ABCB1 gene was shown to restore IM sensitivity and increase IM intracellular levels in multi-drugresistant CML cell line [10,11]. Studies with CML patients have shown no association of 3435C>T polymorphism with the response to standard dose of IM (400 mg/OD). In conjunction with our earlier analysis, 3435 C>T SNP showed no risk association with IM response in smaller patients group [12]. When the polymorphism was evaluated separately either with the cytogenetic response or the molecular responses, interestingly, ABCB1 2677 variant was associated with MMR in CML patients. Furthermore, the wild type ABCB1 haplotype (C1236G2677C3435) was associated with IM resistance, which is in agreement with a report by Dulucq et al. in white population [13]. Although they found a higher frequency of MMR in patients with non-G genotypes at position 2677, they could not confirm these results in a larger patient cohort [14]. In contrast, our result is in accordance with the findings of Deenik et al. [15], in which patients with homozygous ABCB1 1236T showed a higher probability to obtain MMR. Similarly, a higher risk for IM resistance was reported for CML patients with homozygous ABCB1 3435T allele, whereas better CCyR was observed for patients with the AG/AT/AA genotype at position 2677 by Ni et al. [16]. This has been attributed as possibly be due to the fact that carriers of 2677 variant genotype have lower P-gp messenger RNA expression than

those who had 2677 wild type genotype [17]. Few other studies did not find an association between ABCB1 polymorphisms and IM response [14,18–20]. The ABCG2 SNPs G34A and C421A are the most frequent nonsynonymous polymorphisms in BCRP, which, like P-gp, causes removal of IM. ABCG2 gene is over-expressed in CD34+ CD38– human hematopoietic stem cells. In CML K-562 cell lines, higher levels of ABCG2 mRNA and BCRP protein were reported after longterm IM exposure and the levels decreased gradually at higher concentrations [21]. BCRP expression is lower in the livers of carriers of 421 AA genotype [22]. Experiments in vitro using IM substrates showed that the A421A variant of BCRP affects the IM accumulation [23]. Consistent with this, a lower IM clearance and a higher dose-adjusted IM trough concentration were found in CML patients with the ABCG2 A421A variant genotype [20,24], whereas the C421C genotype was associated with increased IM resistance [19]. Our results showed that the ABCG2 421 variant A allele is associated significantly with a higher rate of MMR in CML patients, and could be utilized as a predictive marker for IM response. Our results are in accordance with the study by Kim et al. [19] which indicated that the A421A genotype exhibited a higher MMR than the CA or CC genotype. Recently, Seong et al. [25] also reported that ABCG2 A421A genotype was significantly higher among CML patients with MMR but insignificantly associated with cytogenetic response, which is similar with to the present study. Few other previous reports failed to determine the effect of ABCG2 421C>A on the IM response and clearance [19,23,26,27]. Conversely, Takashi et al. [20] demonstrated that the dose-adjusted IM trough concentration was significantly higher in patients with ABCG2 A421A than in those with C421Cy. Therefore, the inter-individual genetic variation of the ABCG2 gene may contribute for the variability in the pharmacokinetics and clinical response of IM. The role of ABCB1 and ABCG2 polymorphisms in the development of IM intolerance and hepatotoxicity had not been documented in earlier studies. This study investigated the roles of these polymorphisms in patients’ hematological/non-hematological toxicity and liver function parameters. However, present

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Fig. 1. Mean values of bilirubin, ALP and ALT in different genotype groups of ABCB1 and ABCG2. The values are shown as U/l. ALP: alkaline phosphatase; ALT: alanine aminotransferase.

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study results showed no significant association between the studied polymorphisms and the incidence of severe toxicity induced by IM. These results suggest the exclusion of these drug transporter gene polymorphisms as risk factors for severe toxicity. The current study examined also the relationships between genetic variants and hepatotoxicity during IM therapy. But none of the SNPs showed any association with bilirubin, ALP and ALT levels. Many factors, other than the ABCB1 and ABCG2 genotypes (such as IM dosage, the pharmacokinetics of IM in plasma and interindividual variation of these parameters), could be influencing the liver toxicity. Further prospective studies are needed to elucidate these unresolved questions. 5. Conclusion Our results demonstrated a significant association of the SNPs ABCB1 T1236C, G2677T/A and ABCG2 C421A with IM efficacy. Haplotype pattern of C1236G2677C3435 seems to be associated with higher risk of IM resistance. Single genetic variation marker of ABCB1 G2677T/A and ABCG2 421C>A may influence the molecular response in CML with IM therapy. Pretreatment genotyping of these SNPs in CML patients appears to be useful for predicting IM resistance which may allow the best choice of drug treatment for CML patients. However, these findings needs to be validated with other pharmacogenetic variants and the plasma concentration of the drug administered through prospective studies with a larger patient population. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements The work was fully supported by Research University Grant (1001/PPSP/812067), Universiti Sains Malaysia. First author is a recipient of USM Fellowship 01/11. References [1] Jabbour E, Kantarjian H, Jones D, et al. Frequency and clinical significance of BCR-ABL mutations in patients with chronic myeloid leukemia treated with imatinib mesylate. Leukemia 2006;20(10):1767–73. [2] Nakanishi T, Shiozawa K, Hassel BA, et al. Complex interaction of BCRP/ABCG2 and imatinib in BCR-ABL-expressing cells: BCRP-mediated resistance to imatinib is attenuated by imatinib-induced reduction of BCRP expression. Blood 2006;108:678–84. [3] Cascorbi I, Gerloff T, Johne A, et al. Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Clin Pharmacol Ther 2001;69:169– 74. [4] Hughes T, Deininger M, Hochhaus A, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendation for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006;108:28–37. [5] Baccarani M, Cortes J, Pane F, et al. Chronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNet. J Clin Oncol 2009;27(35):6041–51.

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Association of genotypes and haplotypes of multi-drug transporter genes ABCB1 and ABCG2 with clinical response to imatinib mesylate in chronic myeloid leukemia patients.

The introduction and success of imatinib mesylate (IM) has become a paradigm shift in chronic myeloid leukemia (CML) treatment. However, the high effi...
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