Relationship between SLCO1B3 and ABCA3 polymorphisms and imatinib response in chronic myeloid leukemia patients Luciene Terezina de Lima 1, Carolina Tosin Bueno1, Douglas Vivona1, Rosario Domiguez Crespo Hirata 1, Mario Hiroyuki Hirata 1, Vania Tiestsche de Moraes Hungria 2, Carlos Sérgio Chiattone 2, Maria Aparecida Zanichelli 3, Maria de Lourdes Lopes Ferrari Chauffaille 4, Elvira Maria Guerra-Shinohara 1 1
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil, 2Departamento de Hematologia e Hemoterapia, Santa Casa de Misericórdia de São Paulo, São Paulo, Brazil, 3Departamento de Hematologia, Hospital Brigadeiro, São Paulo, Brazil, 4 Departamento de Oncologia Clínica e Experimental, Universidade Federal de São Paulo, São Paulo, Brazil Background: Genetic variations in membrane transporters may contribute to imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML). Objective: To investigate the relationship between SLCO1B3, SLCO1A2, and ABCA3 polymorphisms and IM response in CML patients. Methods: Patients in chronic phase CML (N =118) were studied. All patients were treated with a standard dose of IM (400 mg/day) and classified into one of the two groups according to their responses. Major molecular response (MMR) and complete molecular response (CMR) were evaluated. Criteria for response failure were established according to European LeukemiaNet (2009). Analysis of the SLCO1B3 c.334T > G (rs4149117) and c.699G > A (rs7311358), SLCO1A2 c.516A > C (rs11568563) and c.-62361G > A (rs3764043), and ABCA3 c.1755C > G (rs323043) and c.4548-191C > A (rs150929) polymorphisms was carried out by real-time polymerase chain reaction. Results: SLCO1A2 and ABCA3 polymorphisms have similar frequencies between responders and nonresponders. SLCO1B3 699GG and 344TT genotypes were more frequent in the responder group (63.8%) than in the non-responder group (44.7%, P = 0.042). Furthermore, carriers of 699GA/AA and 334TG/GG genotypes presented a higher probability of not responding to the standard dose of IM (odds ratio: 2.17; 95% confidence interval: 1.02–4.64, P = 0.04). Poor CMR for ABCA3 4548-91C > A was observed in patients with the CC/CA genotype when compared to AA carriers in the responder group (P = 0.014). Conclusions: SLCO1B3 699GG and 344TT genotypes are associated with non-response to IM, while ABCA3 4548-91 CC/CA genotypes are related to poor CMR in CML patients treated with standard-dose imatinib. Keywords: Imatinib, Resistance, Chronic myeloid leukemia, Membrane transporters, ABCA3, SLCO1B3, Pharmacogenetics
Introduction Chronic myeloid leukemia (CML) is a clonal malignant disorder of pluripotent hematopoietic stem cells and it is characterized by the presence of the Philadelphia chromosome in over 90% of cases.1 The Philadelphia chromosome is a product of reciprocal translocation between the long groups of chromosomes 9 and 22 t(9;22)(q34;q11) that generates the BCR-ABL1 gene, an oncogene with high tyrosine kinase activity which interacts with a variety of signaling proteins leading to cellular transformation. Correspondence to: Elvira Maria Guerra-Shinohara, Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Av. Prof. Lineu Prestes, 580, CEP 05508-000 São Paulo, SP, Brazil. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845414Y.0000000181
Imatinib mesylate (IM) was specifically developed to inhibit the tyrosine kinase activity of the BCR-ABL1 protein and other tyrosine kinases by inactivating downstream signaling by binding to an active site of the tyrosine kinase, thus halting cell proliferation and inducing apoptosis.2 IM has been shown to induce cytogenetic and molecular remission in a majority of CML patients.3 However, IM resistance remains an issue and some patients fail to achieve optimal response. Several resistance mechanisms have been proposed4–7 and the main resistance mechanism is related to BCR-ABL1 dependency established by amplification and/or mutations of the kinase domain of BCR-ABL1. The influence
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of other factors, such as pharmacogenetics variability, the presence of polymorphisms or low intracellular uptake, and efflux activity, remain unclear.8 Several ABC and SLC transporters expressed in leukemia cells that not yet been associated with drug resistance could have prognostic relevance. ABCB1/ ABCG2 and SLC22A1 were extensively studied and associated with IM response.9 IM also interacts with other membrane transporters, such as ATP-binding cassette A3 (ABCA3), that participates in drug efflux, and organic anion transporter polypeptide 1A2 (SLCO1A2, OATP1A2), solute carrier B3 (SLCO1B3), responsible for drug uptake.10–12 Little is known about changes in the function of these transporters and the influence on IM response. Changes in the functionality of these transporters have been linked to single nucleotide polymorphisms (SNPs) and could be related with IM response variability.11,12 Due to the relevance of determining the factors that influence variability in response to CML treatment, the aim of this study was to investigate the relationship between SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) polymorphisms and IM response markers in CML patients.
Materials and methods Study population One hundred and eighteen patients with chronic phase CML, age 18- to 80-year-old, were enrolled at two health centers in São Paulo, Brazil from September 2009 to September 2010. CML patients with cytogenetic abnormalities other than Philadelphia chromosome and/or BCR-ABL1 mutations were excluded from this study. All patients were treated with a standard dose of IM (400 mg/day) with confirmed treatment adherence. They were subsequently classified into two groups according to their responses. The responder group consisted of 70 patients who had a complete cytogenetic response (CCyR) within 18 months of treatment. The non-responder group consisted of 48 patients who did not have a complete cytogenetic response with the initial dose (400 mg/day) of IM or who relapsed during treatment and were submitted to higher doses of 600 or 800 mg/day. Criteria for treatment failure were established according to European LeukemiaNet (2009).13 Samples from healthy blood donors (n = 119) were selected from a blood bank and used to evaluate the minor allele frequencies of SLCO1B3 c.334T > G, c.699G > A (rs4149117 and rs7311358, respectively), SLCO1A2 c.516A > C, c.-62-361G > A (rs11568563 and rs3764043, respectively), and ABCA3 c.1755C > G, c.4548-191C > A (rs323043 and rs150929, respectively) polymorphisms in a healthy sample population.
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Primary resistance was defined as lack of efficacy of IM from the start of therapy and secondary resistance was characterized by loss of complete cytogenetic response during IM treatment.14 The study protocol was approved by the Research Ethics Committees/Institutional Review Boards of the Universidade de São Paulo, Hospital Brigadeiro, and Santa Casa de Misericórdia de São Paulo, all in Brazil. Written informed consent was obtained from all participants.
Sample collection and DNA extraction Genomic DNA extraction was executed from peripheral blood collected in BD Vacutainer® System vacuum tubes containing K3EDTA anticoagulant using a QIAamp Blood Mini Kit DNA extraction kit (PreAnalytiX/Qiagen, Hilden, Germany), according to the manufacturer’s instructions. DNA samples were quantified by spectrophotometry (260 nm) and purity of resulting DNA was estimated at 260 and 280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Analysis of SLCO1B3, SLCO1A2, and ABCA3 polymorphisms Genotyping of the SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) polymorphisms was executed by real-time polymerase chain reaction (PCR) using Taqman™ SNP genotyping assays (C_25639181_40, C_25765587_40, C_25605827_10, C_27480030_10, C_3156784_10, C_3156769_10, respectively) (Applied Biosystems, Foster City, CA, USA), with the following amplification conditions: 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. The fluorescence of each sample allele was detected at 60°C on each PCR cycle. For quality control purposes, the realtime PCR plate samples were loaded with known genotypes, and about 15% of all genotyping was repeated.
BCR-ABL1 transcripts Total RNA was extracted from peripheral blood using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) and BCR-ABL1 transcripts were measured by realtime PCR as previously described.15 Major molecular response (MMR) and complete molecular response (CMR) were defined as a reduction of BCR-ABL1 transcript levels below 0.1 and 0.0032%, respectively, in peripheral blood samples standardized according to the international scale.15
Cytogenetic analysis Conventional G-banding on bone marrow metaphases was executed after short-term culturing (24 hours) using standard procedures, and the karyotypes were described according to ISCN 2009.16
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Statistical analysis Statistical analysis was carried out using the SPSS version 17.0 software program (SPSS Inc., Chicago, IL, USA). The standardized disequilibrium coefficient (D’) for pair-wise linkage disequilibrium was assessed with the expectation-maximization algorithm using the Haploview software program, and we considered linkage disequilibrium when D ′ ≥ 0.50. The averages of the numerical variables were compared using the student’s t-test. For categorical variables, the Chi-square, Fisher’s exact test, and likelihood ratio were used. The observed genotype frequencies were compared to the expected ones according to Hardy–Weinberg equilibrium. Univariate logistic regression was used to evaluate the association of genotypes and/or variant alleles of polymorphisms studied for CML risk. These models were also used to determine the probability of achieving MMR or CMR according to SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) polymorphisms. The significance level for statistical tests was 5% (i.e. P < 0.050).
Relationship between gene polymorphisms and IM response in CML patients
CCyR: 100.0 vs. 54.2% (P < 0.001), MMR: 82.9 vs. 54.1% (P < 0.001), and CMR: 21.4 vs. 6.3% (P = 0.021). Seventy-three percent of non-responders had primary resistance after 19.5 months of IM and 27% relapsed after 30.7 months of IM treatment (secondary resistance).
Allele and genotype frequencies of polymorphisms The distributions of genotypes for SLCO1B3, SLCO1A2, and ABCA3 polymorphisms are in the Hardy–Weinberg equilibrium, in both CML patients and healthy subject groups. The minor allele frequencies and genotypes of SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) were similar between CML patients and healthy subjects (P > 0.05) (data not shown), suggesting no association between these variants and CML risk. A strong linkage disequilibrium between SLCO1B3 c.699G > A and c.334T > G polymorphisms (D’ = 1.0) was observed.
Results Clinical data
SLCO1A2, SLCO1B3, ABCA3 polymorphisms and IM response
Patients had similar distributions in terms of age and number of blood cells (red blood cells, white blood cells, and platelets) (P > 0.050), according to the different response groups. About 60% of CML patients had received previous IFN-α treatment. Time of IM treatment was higher (P = 0.005) and time of diagnosis was lower (P = 0.007) in the responder group, compared to the non-responder group (Table 1). All patients were in complete hematological response and the CCyR, MMR, and CMR frequencies in the responder and non-responder groups were:
SLCO1A2 (c.516A > C, c.-62-361G > A) and ABCA3 (c.1755C > G, c.4548-191C > A) genotypes had similar frequencies between the responder and nonresponder groups (P > 0.05). The frequency of SLCO1B3 699GG and 344TT genotypes was higher in the responder group (63.8%) than in the nonresponder group (44.7%, P = 0.042). Furthermore, carriers of 699GA/AA and 334TG/GG genotypes presented a higher probability of not responding to the standard dose of IM (odds ratio (OR): 2.17; 95% confidence interval (CI): 1.02–4.64, P = 0.04) (Fig. 1).
Table 1 Distribution of age, number of blood cells (erythrocytes, leukocytes, and platelets), time of IM treatment, time of diagnoses, and preview treatment to IM of CML patients
Age Erythrocytes (0.106/mm3) Leukocytes* (0.103/mm3) Platelets* (0.103/mm3) Time of IM treatment* (months) Time of diagnoses* (months) Previous treatment** IFN-α use Yes No Time of IFN-α treatment >1 year A/c.334T > G) polymorphisms according to response to IM. OR, odds ratio; CI, confidence interval.
No association was found between SLCO1B3, SLCO1A2, and ABCA3 polymorphisms and MMR (data not shown). When CMR was analyzed, there was an association between ABCA3 4548-191CC + CA genotypes and poor CMR in the responder group (P = 0.014) (Table 2). Carriers of this genotype had a six times more probability of not achieving CMR in the responder group (OR: 6.37; 95% CI: 1.50–27.18, P = 0.012). High frequencies of SLCO1B3 699GG and 344TT genotypes were found in non-responders that achieved CMR with higher doses of IM (100 vs. 41.1%; P = 0.026), but only three individuals carrying these genotypes achieved CMR (Table 2).
Discussion Most CML patients have benefited with the introduction of IM treatment, however some patients do not respond satisfactorily. The investigation of pharmacogenetic factors related to SLCO1B3, SLCO1A2, ABCA3 genes could be associated with IM response Table 2
as well CML risk. As far as we know, this is the first study to identify that SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) polymorphisms are not associated with CML risk. SLCO1B3 is a polymorphic gene and the most common SNPs are c.344T > G (Ser112Ala) and c.699G > A (Met233Ile). These SNPs were associated with impaired transport activity in a single cell line (COS-7)17 and also with altered digoxin pharmacokinetics.18 In this study, we found high frequency for ancestral allele of SLCO1B3 c.334T > G and c.699G > A polymorphisms in the responder group, and a strong linkage disequilibrium between these two polymorphisms. It was demonstrated that CML patients with SLCO1B3 334GG genotype had renal clearance of IM 36% higher when compared to patients with TT and TG genotypes.19 In fact, another study showed higher intracellular concentrations of IM in CML patients who were carriers of the SLCO1B3 334TT genotype, but no difference in
Relationship of the SLCO1B3, SLCO1A2, and ABCA3 polymorphisms with CMR to IM in CML patients Responders
Non-responders
Genotypes
With CMR
Without CMR
SLCO1B3 c. 699G > A GG GA + AA SLCO1B3 c.334T > G TT TG + GG SLCO1A2 c.516A > C AA AC SLCO1A2 c.-62-361G > A GG GA ABCA3 c.4548-191C > A CC + CA AA ABCA3 c.1755C > G CC CG + GG
(N = 13) 6 (46.2) 7 (53.8) (N = 13) 6 (46.2) 7 (53.8) (N = 13) 13 (100.0) 0 (0.0) (N = 13) 12 (92.3) 1 (7.7) (N = 13) 8 (61.5) 5 (38.5) (N = 13) 9 (69.2) 4 (30.8)
(N = 56) 38 (67.9) 18 (32.1) (N = 56) 38 (67.9) 18 (32.1) (N = 56) 51 (91.1) 5 (8.9) (N = 56) 53 (94.6) 3(5.4) (N = 56) 51 (91.1) 5 (8.9) (N = 56) 40 (71.4) 16 (28.6)
P value 0.149*
0.149*
0.140*
0.745**
0.014*
0.876*
With CMR
Without CMR
(N = 3) 3 (100.0) 0 (0.0) (N = 3) 3 (100.0) 0 (0.0) (N = 3) 2 (66.7) 1 (33.3) (N = 3) 3 (100.0) 0 (0.0) (N = 4) 1 (25.0) 3 (75.0) (N = 4) 3 (75.0) 1 (25.0)
(N = 43) 18 (41.9) 25 (58.1) (N = 43) 18 (41.9) 25 (58.1) (N = 43) 40 (93.0) 3 (7.0) (N = 43) 41 (95.3) 2 (4.7) (N = 43) 13 (30.2) 30 (69.8) (N = 43) 34 (79.1) 9 (20.9)
The data are shown as number of subjects (percentage) and compared by *Chi-square testor **likelihood ratio. CMR, complete molecular response (BCR-ABL1 mRNA ≤0.0032%). Significant values (P < 0.05) are in bold.
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P value
0.026*
0.026*
0.206*
0.659**
0.824*
0.852*
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clinical response to IM among these patients was observed in those who were carriers of SLCO1B3 334TG/GG genotypes.20 The clinical data could have been skewed by the reduced number of patients enrolled in that study (N = 15).20 Our data suggests that the optimal response observed in SLCO1B3 334TT/699GG carriers could be associated with higher intracellular concentrations of IM. In vitro studies revealed that the SLCO1A2 c.516A > C variant reduced the capacity of cellular uptake of OATP1A2 substrates (estrone 3-sulfate, deltorphin II, and [D-penicillamine2,5]-enkephalin).21 The c.516A > C variant was also associated with reduced uptake of in vitro methotrexate and estrone3-sulfate.22 However, Eechoute et al. 23 demonstrated that the c.516A > C variant is not related to IM steady-state concentration in CML patients. Yamakawa et al. 24 observed that carriers of SLCO1A2 -62-361GG had 28% lower IM clearance compared to GA or AA carriers. In this study, the c.516A > C and -62-361G > A SNPs were not associated with IM response. Little is known about the biological action of ABCA3 c.4548-191C > A and c.1755C > G polymorphisms. ABCA3 c.4548-191C > A is an intronic SNP and accumulated data confirms that mutations located in introns could affect pre-mRNA processing through the inactivation of regulatory elements in this process.25 The overexpression of ABCA3 mRNA in CML progenitor cells was related to IM resistance due to high lysosomal sequestration measured by [(14)C]-labeled IM transportation.26 We observed higher frequency of patients with ABCA3 c.4548191CC/CA genotypes who did not achieve CMR in the responder group. These data suggest that ABCA3 c.4548-191C > A ancestral allele could influence ABCA3 expression or function impacting on CMR, but in this study we did not evaluate ABCA3 mRNA and protein expression to confirm these data. This is the first study to evaluate the influence of ABCA3 c.1755C > G polymorphism in drug resistance and we failed to find an association between genotype frequencies of ABCA3 c.1755C > G polymorphism and IM sensitivity in CML patients. In conclusion, SLCO1B3 699GG and 344TT genotypes are associated with failure clinical response and ABCA3 4548-91 CC/CA genotypes are related to poor CMR in responders to IM. These data could be useful for understanding IM resistance and response variability. Further studies are necessary to clarify the influence of these SNPs on the plasma and intracellular concentration of IM.
Acknowledgments LTL is recipient of fellowship from CNPq, Brazil. We thank all the patients who participated in this study.
Relationship between gene polymorphisms and IM response in CML patients
Disclaimer statements Contributors LTL, CTB and DV: were responsible for recruitment of participants and data collection; analysis; and drafting, critical revision, and final approval of the article. RDCH and MHH: were responsible for critical revision, drafting, and approval of the final report. VTMH, CSC, MAZ and MLLFC were part of the clinical team and were responsible for patient screening, recruitment, treatment, follow-up, data collection, critical revision, and approval of the final report. EMG-S was responsible for the study design, obtaining funding, coordination of the study, data analysis and critical revision, drafting and approval of the final report. Funding FAPESP, Brazil (#09/54184-0). Conflicts of interest None. Ethics approval This study protocol was approved by the Research Ethics Committees/Institutional Review Boards (REC/IRB) of the Universidade de São Paulo, Hospital Brigadeiro, and Santa Casa de Misericórdia de São Paulo, all in Brazil. Written informed consent was obtained from all participants.
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14 Quintás-Cardama A, Kantarjian HM, Cortes JE. Mechanisms of primary and secondary resistance to imatinib in chronic myeloid leukemia. Cancer Control 2009;16:122–31. 15 Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K, et al. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002;99:3472–5. 16 Shaffer LG, Slovak ML, Campbell LJ. ISCN 2009: An International System for Human Cytogenetic Nomenclature. Basel, S. Karger Publishing; 2009. 17 Hamada A, Miyano H, Watanabe H, Saito H. Interaction of imatinib mesilate with human P-glycoprotein. J Pharmacol Exp Ther. 2003;307:824–8. 18 Tsujimoto M, Hirata S, Dan Y, Ohtani H, Sawada Y. Polymorphisms and linkage disequilibrium of the OATP8 (OATP1B3) gene in Japanese subjects. Drug Metab Pharmacokinet. 2006;21:165–9. 19 Yamakawa Y, Hamada A, Nakashima R, Yuki M, Hirayama C, Kawaguchi T, et al. Association of genetic polymorphisms in the influx transporter SLCO1B3 and the efflux transporter ABCB1 with imatinib pharmacokinetics in patients with chronic myeloid leukemia. Ther Drug Monit. 2011;33:244–50. 20 Nambu T, Hamada A, Nakashima R, Yuki M, Kawaguchi T, Mitsuya H, et al. Association of SLCO1B3 polymorphism
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with intracellular accumulation of imatinib in leukocytes in patients with chronic myeloid leukemia. Biol Pharm Bull. 2011;34:114–9. Lee W, Glaeser H, Smith LH, Roberts RL, Moeckel GW, Gervasini G, et al. Polymorphisms in human organic aniontransporting polypeptide 1A2 (OATP1A2): implications for altered drug disposition and central nervous system drug entry. J Biol Chem. 2005;280:9610–7. Badagnani I, Castro RA, Taylor TR, Brett CM, Huang CC, Stryke D, et al. Interaction of methotrexate with organic-anion transporting polypeptide 1A2 and its genetic variants. J Pharmacol Exp Ther. 2006;318:521–9. Eechoute K, Franke RM, Loos WJ, Scherkenbach LA, Boere I, Verweij J, et al. Environmental and genetic factors affecting transport of imatinib by OATP1A2. Clin Pharmacol Ther. 2011;89:816–20. Yamakawa Y, Hamada A, Shuto T, Yuki M, Uchida T, Kai H, et al. Pharmacokinetic impact of SLCO1A2 polymorphisms on imatinib disposition in patients with chronic myeloid leukemia. Clin Pharmacol Ther. 2011;90:157–63. Baralle D, Baralle M. Splicing in action: assessing disease causing sequence changes. J Med Genet. 2005;42:737–48. Chapuy B, Panse M, Radunski U, Koch R, Wenzel D, Inagaki N, et al. ABC transporter A3 facilitates lysosomal sequestration of imatinib and modulates susceptibility of chronic myeloid leukemia cell lines to this drug. Haematologica 2009;94:1528–36.
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil, 2Departamento de Hematologia e Hemoterapia, Santa Casa de Misericórdia de São Paulo, São Paulo, Brazil, 3Departamento de Hematologia, Hospital Brigadeiro, São Paulo, Brazil, 4 Departamento de Oncologia Clínica e Experimental, Universidade Federal de São Paulo, São Paulo, Brazil Background: Genetic variations in membrane transporters may contribute to imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML). Objective: To investigate the relationship between SLCO1B3, SLCO1A2, and ABCA3 polymorphisms and IM response in CML patients. Methods: Patients in chronic phase CML (N =118) were studied. All patients were treated with a standard dose of IM (400 mg/day) and classified into one of the two groups according to their responses. Major molecular response (MMR) and complete molecular response (CMR) were evaluated. Criteria for response failure were established according to European LeukemiaNet (2009). Analysis of the SLCO1B3 c.334T > G (rs4149117) and c.699G > A (rs7311358), SLCO1A2 c.516A > C (rs11568563) and c.-62361G > A (rs3764043), and ABCA3 c.1755C > G (rs323043) and c.4548-191C > A (rs150929) polymorphisms was carried out by real-time polymerase chain reaction. Results: SLCO1A2 and ABCA3 polymorphisms have similar frequencies between responders and nonresponders. SLCO1B3 699GG and 344TT genotypes were more frequent in the responder group (63.8%) than in the non-responder group (44.7%, P = 0.042). Furthermore, carriers of 699GA/AA and 334TG/GG genotypes presented a higher probability of not responding to the standard dose of IM (odds ratio: 2.17; 95% confidence interval: 1.02–4.64, P = 0.04). Poor CMR for ABCA3 4548-91C > A was observed in patients with the CC/CA genotype when compared to AA carriers in the responder group (P = 0.014). Conclusions: SLCO1B3 699GG and 344TT genotypes are associated with non-response to IM, while ABCA3 4548-91 CC/CA genotypes are related to poor CMR in CML patients treated with standard-dose imatinib. Keywords: Imatinib, Resistance, Chronic myeloid leukemia, Membrane transporters, ABCA3, SLCO1B3, Pharmacogenetics
Introduction Chronic myeloid leukemia (CML) is a clonal malignant disorder of pluripotent hematopoietic stem cells and it is characterized by the presence of the Philadelphia chromosome in over 90% of cases.1 The Philadelphia chromosome is a product of reciprocal translocation between the long groups of chromosomes 9 and 22 t(9;22)(q34;q11) that generates the BCR-ABL1 gene, an oncogene with high tyrosine kinase activity which interacts with a variety of signaling proteins leading to cellular transformation. Correspondence to: Elvira Maria Guerra-Shinohara, Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Av. Prof. Lineu Prestes, 580, CEP 05508-000 São Paulo, SP, Brazil. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845414Y.0000000181
Imatinib mesylate (IM) was specifically developed to inhibit the tyrosine kinase activity of the BCR-ABL1 protein and other tyrosine kinases by inactivating downstream signaling by binding to an active site of the tyrosine kinase, thus halting cell proliferation and inducing apoptosis.2 IM has been shown to induce cytogenetic and molecular remission in a majority of CML patients.3 However, IM resistance remains an issue and some patients fail to achieve optimal response. Several resistance mechanisms have been proposed4–7 and the main resistance mechanism is related to BCR-ABL1 dependency established by amplification and/or mutations of the kinase domain of BCR-ABL1. The influence
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Relationship between gene polymorphisms and IM response in CML patients
of other factors, such as pharmacogenetics variability, the presence of polymorphisms or low intracellular uptake, and efflux activity, remain unclear.8 Several ABC and SLC transporters expressed in leukemia cells that not yet been associated with drug resistance could have prognostic relevance. ABCB1/ ABCG2 and SLC22A1 were extensively studied and associated with IM response.9 IM also interacts with other membrane transporters, such as ATP-binding cassette A3 (ABCA3), that participates in drug efflux, and organic anion transporter polypeptide 1A2 (SLCO1A2, OATP1A2), solute carrier B3 (SLCO1B3), responsible for drug uptake.10–12 Little is known about changes in the function of these transporters and the influence on IM response. Changes in the functionality of these transporters have been linked to single nucleotide polymorphisms (SNPs) and could be related with IM response variability.11,12 Due to the relevance of determining the factors that influence variability in response to CML treatment, the aim of this study was to investigate the relationship between SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) polymorphisms and IM response markers in CML patients.
Materials and methods Study population One hundred and eighteen patients with chronic phase CML, age 18- to 80-year-old, were enrolled at two health centers in São Paulo, Brazil from September 2009 to September 2010. CML patients with cytogenetic abnormalities other than Philadelphia chromosome and/or BCR-ABL1 mutations were excluded from this study. All patients were treated with a standard dose of IM (400 mg/day) with confirmed treatment adherence. They were subsequently classified into two groups according to their responses. The responder group consisted of 70 patients who had a complete cytogenetic response (CCyR) within 18 months of treatment. The non-responder group consisted of 48 patients who did not have a complete cytogenetic response with the initial dose (400 mg/day) of IM or who relapsed during treatment and were submitted to higher doses of 600 or 800 mg/day. Criteria for treatment failure were established according to European LeukemiaNet (2009).13 Samples from healthy blood donors (n = 119) were selected from a blood bank and used to evaluate the minor allele frequencies of SLCO1B3 c.334T > G, c.699G > A (rs4149117 and rs7311358, respectively), SLCO1A2 c.516A > C, c.-62-361G > A (rs11568563 and rs3764043, respectively), and ABCA3 c.1755C > G, c.4548-191C > A (rs323043 and rs150929, respectively) polymorphisms in a healthy sample population.
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Primary resistance was defined as lack of efficacy of IM from the start of therapy and secondary resistance was characterized by loss of complete cytogenetic response during IM treatment.14 The study protocol was approved by the Research Ethics Committees/Institutional Review Boards of the Universidade de São Paulo, Hospital Brigadeiro, and Santa Casa de Misericórdia de São Paulo, all in Brazil. Written informed consent was obtained from all participants.
Sample collection and DNA extraction Genomic DNA extraction was executed from peripheral blood collected in BD Vacutainer® System vacuum tubes containing K3EDTA anticoagulant using a QIAamp Blood Mini Kit DNA extraction kit (PreAnalytiX/Qiagen, Hilden, Germany), according to the manufacturer’s instructions. DNA samples were quantified by spectrophotometry (260 nm) and purity of resulting DNA was estimated at 260 and 280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Analysis of SLCO1B3, SLCO1A2, and ABCA3 polymorphisms Genotyping of the SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) polymorphisms was executed by real-time polymerase chain reaction (PCR) using Taqman™ SNP genotyping assays (C_25639181_40, C_25765587_40, C_25605827_10, C_27480030_10, C_3156784_10, C_3156769_10, respectively) (Applied Biosystems, Foster City, CA, USA), with the following amplification conditions: 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. The fluorescence of each sample allele was detected at 60°C on each PCR cycle. For quality control purposes, the realtime PCR plate samples were loaded with known genotypes, and about 15% of all genotyping was repeated.
BCR-ABL1 transcripts Total RNA was extracted from peripheral blood using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) and BCR-ABL1 transcripts were measured by realtime PCR as previously described.15 Major molecular response (MMR) and complete molecular response (CMR) were defined as a reduction of BCR-ABL1 transcript levels below 0.1 and 0.0032%, respectively, in peripheral blood samples standardized according to the international scale.15
Cytogenetic analysis Conventional G-banding on bone marrow metaphases was executed after short-term culturing (24 hours) using standard procedures, and the karyotypes were described according to ISCN 2009.16
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Statistical analysis Statistical analysis was carried out using the SPSS version 17.0 software program (SPSS Inc., Chicago, IL, USA). The standardized disequilibrium coefficient (D’) for pair-wise linkage disequilibrium was assessed with the expectation-maximization algorithm using the Haploview software program, and we considered linkage disequilibrium when D ′ ≥ 0.50. The averages of the numerical variables were compared using the student’s t-test. For categorical variables, the Chi-square, Fisher’s exact test, and likelihood ratio were used. The observed genotype frequencies were compared to the expected ones according to Hardy–Weinberg equilibrium. Univariate logistic regression was used to evaluate the association of genotypes and/or variant alleles of polymorphisms studied for CML risk. These models were also used to determine the probability of achieving MMR or CMR according to SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) polymorphisms. The significance level for statistical tests was 5% (i.e. P < 0.050).
Relationship between gene polymorphisms and IM response in CML patients
CCyR: 100.0 vs. 54.2% (P < 0.001), MMR: 82.9 vs. 54.1% (P < 0.001), and CMR: 21.4 vs. 6.3% (P = 0.021). Seventy-three percent of non-responders had primary resistance after 19.5 months of IM and 27% relapsed after 30.7 months of IM treatment (secondary resistance).
Allele and genotype frequencies of polymorphisms The distributions of genotypes for SLCO1B3, SLCO1A2, and ABCA3 polymorphisms are in the Hardy–Weinberg equilibrium, in both CML patients and healthy subject groups. The minor allele frequencies and genotypes of SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) were similar between CML patients and healthy subjects (P > 0.05) (data not shown), suggesting no association between these variants and CML risk. A strong linkage disequilibrium between SLCO1B3 c.699G > A and c.334T > G polymorphisms (D’ = 1.0) was observed.
Results Clinical data
SLCO1A2, SLCO1B3, ABCA3 polymorphisms and IM response
Patients had similar distributions in terms of age and number of blood cells (red blood cells, white blood cells, and platelets) (P > 0.050), according to the different response groups. About 60% of CML patients had received previous IFN-α treatment. Time of IM treatment was higher (P = 0.005) and time of diagnosis was lower (P = 0.007) in the responder group, compared to the non-responder group (Table 1). All patients were in complete hematological response and the CCyR, MMR, and CMR frequencies in the responder and non-responder groups were:
SLCO1A2 (c.516A > C, c.-62-361G > A) and ABCA3 (c.1755C > G, c.4548-191C > A) genotypes had similar frequencies between the responder and nonresponder groups (P > 0.05). The frequency of SLCO1B3 699GG and 344TT genotypes was higher in the responder group (63.8%) than in the nonresponder group (44.7%, P = 0.042). Furthermore, carriers of 699GA/AA and 334TG/GG genotypes presented a higher probability of not responding to the standard dose of IM (odds ratio (OR): 2.17; 95% confidence interval (CI): 1.02–4.64, P = 0.04) (Fig. 1).
Table 1 Distribution of age, number of blood cells (erythrocytes, leukocytes, and platelets), time of IM treatment, time of diagnoses, and preview treatment to IM of CML patients
Age Erythrocytes (0.106/mm3) Leukocytes* (0.103/mm3) Platelets* (0.103/mm3) Time of IM treatment* (months) Time of diagnoses* (months) Previous treatment** IFN-α use Yes No Time of IFN-α treatment >1 year A/c.334T > G) polymorphisms according to response to IM. OR, odds ratio; CI, confidence interval.
No association was found between SLCO1B3, SLCO1A2, and ABCA3 polymorphisms and MMR (data not shown). When CMR was analyzed, there was an association between ABCA3 4548-191CC + CA genotypes and poor CMR in the responder group (P = 0.014) (Table 2). Carriers of this genotype had a six times more probability of not achieving CMR in the responder group (OR: 6.37; 95% CI: 1.50–27.18, P = 0.012). High frequencies of SLCO1B3 699GG and 344TT genotypes were found in non-responders that achieved CMR with higher doses of IM (100 vs. 41.1%; P = 0.026), but only three individuals carrying these genotypes achieved CMR (Table 2).
Discussion Most CML patients have benefited with the introduction of IM treatment, however some patients do not respond satisfactorily. The investigation of pharmacogenetic factors related to SLCO1B3, SLCO1A2, ABCA3 genes could be associated with IM response Table 2
as well CML risk. As far as we know, this is the first study to identify that SLCO1B3 (c.334T > G, c.699G > A), SLCO1A2 (c.516A > C, c.-62-361G > A), and ABCA3 (c.1755C > G, c.4548-191C > A) polymorphisms are not associated with CML risk. SLCO1B3 is a polymorphic gene and the most common SNPs are c.344T > G (Ser112Ala) and c.699G > A (Met233Ile). These SNPs were associated with impaired transport activity in a single cell line (COS-7)17 and also with altered digoxin pharmacokinetics.18 In this study, we found high frequency for ancestral allele of SLCO1B3 c.334T > G and c.699G > A polymorphisms in the responder group, and a strong linkage disequilibrium between these two polymorphisms. It was demonstrated that CML patients with SLCO1B3 334GG genotype had renal clearance of IM 36% higher when compared to patients with TT and TG genotypes.19 In fact, another study showed higher intracellular concentrations of IM in CML patients who were carriers of the SLCO1B3 334TT genotype, but no difference in
Relationship of the SLCO1B3, SLCO1A2, and ABCA3 polymorphisms with CMR to IM in CML patients Responders
Non-responders
Genotypes
With CMR
Without CMR
SLCO1B3 c. 699G > A GG GA + AA SLCO1B3 c.334T > G TT TG + GG SLCO1A2 c.516A > C AA AC SLCO1A2 c.-62-361G > A GG GA ABCA3 c.4548-191C > A CC + CA AA ABCA3 c.1755C > G CC CG + GG
(N = 13) 6 (46.2) 7 (53.8) (N = 13) 6 (46.2) 7 (53.8) (N = 13) 13 (100.0) 0 (0.0) (N = 13) 12 (92.3) 1 (7.7) (N = 13) 8 (61.5) 5 (38.5) (N = 13) 9 (69.2) 4 (30.8)
(N = 56) 38 (67.9) 18 (32.1) (N = 56) 38 (67.9) 18 (32.1) (N = 56) 51 (91.1) 5 (8.9) (N = 56) 53 (94.6) 3(5.4) (N = 56) 51 (91.1) 5 (8.9) (N = 56) 40 (71.4) 16 (28.6)
P value 0.149*
0.149*
0.140*
0.745**
0.014*
0.876*
With CMR
Without CMR
(N = 3) 3 (100.0) 0 (0.0) (N = 3) 3 (100.0) 0 (0.0) (N = 3) 2 (66.7) 1 (33.3) (N = 3) 3 (100.0) 0 (0.0) (N = 4) 1 (25.0) 3 (75.0) (N = 4) 3 (75.0) 1 (25.0)
(N = 43) 18 (41.9) 25 (58.1) (N = 43) 18 (41.9) 25 (58.1) (N = 43) 40 (93.0) 3 (7.0) (N = 43) 41 (95.3) 2 (4.7) (N = 43) 13 (30.2) 30 (69.8) (N = 43) 34 (79.1) 9 (20.9)
The data are shown as number of subjects (percentage) and compared by *Chi-square testor **likelihood ratio. CMR, complete molecular response (BCR-ABL1 mRNA ≤0.0032%). Significant values (P < 0.05) are in bold.
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P value
0.026*
0.026*
0.206*
0.659**
0.824*
0.852*
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clinical response to IM among these patients was observed in those who were carriers of SLCO1B3 334TG/GG genotypes.20 The clinical data could have been skewed by the reduced number of patients enrolled in that study (N = 15).20 Our data suggests that the optimal response observed in SLCO1B3 334TT/699GG carriers could be associated with higher intracellular concentrations of IM. In vitro studies revealed that the SLCO1A2 c.516A > C variant reduced the capacity of cellular uptake of OATP1A2 substrates (estrone 3-sulfate, deltorphin II, and [D-penicillamine2,5]-enkephalin).21 The c.516A > C variant was also associated with reduced uptake of in vitro methotrexate and estrone3-sulfate.22 However, Eechoute et al. 23 demonstrated that the c.516A > C variant is not related to IM steady-state concentration in CML patients. Yamakawa et al. 24 observed that carriers of SLCO1A2 -62-361GG had 28% lower IM clearance compared to GA or AA carriers. In this study, the c.516A > C and -62-361G > A SNPs were not associated with IM response. Little is known about the biological action of ABCA3 c.4548-191C > A and c.1755C > G polymorphisms. ABCA3 c.4548-191C > A is an intronic SNP and accumulated data confirms that mutations located in introns could affect pre-mRNA processing through the inactivation of regulatory elements in this process.25 The overexpression of ABCA3 mRNA in CML progenitor cells was related to IM resistance due to high lysosomal sequestration measured by [(14)C]-labeled IM transportation.26 We observed higher frequency of patients with ABCA3 c.4548191CC/CA genotypes who did not achieve CMR in the responder group. These data suggest that ABCA3 c.4548-191C > A ancestral allele could influence ABCA3 expression or function impacting on CMR, but in this study we did not evaluate ABCA3 mRNA and protein expression to confirm these data. This is the first study to evaluate the influence of ABCA3 c.1755C > G polymorphism in drug resistance and we failed to find an association between genotype frequencies of ABCA3 c.1755C > G polymorphism and IM sensitivity in CML patients. In conclusion, SLCO1B3 699GG and 344TT genotypes are associated with failure clinical response and ABCA3 4548-91 CC/CA genotypes are related to poor CMR in responders to IM. These data could be useful for understanding IM resistance and response variability. Further studies are necessary to clarify the influence of these SNPs on the plasma and intracellular concentration of IM.
Acknowledgments LTL is recipient of fellowship from CNPq, Brazil. We thank all the patients who participated in this study.
Relationship between gene polymorphisms and IM response in CML patients
Disclaimer statements Contributors LTL, CTB and DV: were responsible for recruitment of participants and data collection; analysis; and drafting, critical revision, and final approval of the article. RDCH and MHH: were responsible for critical revision, drafting, and approval of the final report. VTMH, CSC, MAZ and MLLFC were part of the clinical team and were responsible for patient screening, recruitment, treatment, follow-up, data collection, critical revision, and approval of the final report. EMG-S was responsible for the study design, obtaining funding, coordination of the study, data analysis and critical revision, drafting and approval of the final report. Funding FAPESP, Brazil (#09/54184-0). Conflicts of interest None. Ethics approval This study protocol was approved by the Research Ethics Committees/Institutional Review Boards (REC/IRB) of the Universidade de São Paulo, Hospital Brigadeiro, and Santa Casa de Misericórdia de São Paulo, all in Brazil. Written informed consent was obtained from all participants.
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