Pediatrics International (2015) 57, 572–577
doi: 10.1111/ped.12682
Original Article
Influence of SLCO1B1 polymorphism on maintenance therapy for childhood leukemia Ryoko Suzuki,1 Hiroko Fukushima,2 Emiko Noguchi,3 Masahiro Tsuchida,7 Nobutaka Kiyokawa,8 Kazutoshi Koike,7 Enbo Ma,4 Hideto Takahashi,5,9 Chie Kobayashi,2 Ryoko Nakajima-Yamaguchi,6 Aiko Sakai,1 Makoto Saito,2 Atsushi Iwabuchi,2 Keisuke Kato,7 Tomohei Nakao,7 Ai Yoshimi,7 Ryo Sumazaki2 and Takashi Fukushima2 1 Department of Child Health, Graduate School of Comprehensive Human Sciences, Departments of 2Child Health, 3Genetics, 4Clinical Trial and Clinical Epidemiology and 5Epidemiology, Faculty of Medicine, University of Tsukuba, 6Department of Pediatrics, University of Tsukuba Hospital, Tsukuba, 7Department of Hematology and Oncology, Ibaraki Children’s Hospital, Mito, Ibaraki, 8 Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo and 9School of Medicine, Information Management and Statistics, Fukushima Medical University, Fukushima, Japan Abstract
Background: Management of the adverse effects of chemotherapy is essential to improve outcome of children with leukemia. Some genetic polymorphisms can predict treatment-related toxicity, and be used individually in dose modification of 6-mercaptopurine (6-MP) and methotrexate (MTX) in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). We investigated associations between clinical course and candidate gene polymorphisms less evaluated in Japanese patients. Methods: Fifty-three children who received maintenance chemotherapy were enrolled in this study. The scheduled dose of oral 6-MP was 40 mg/m2 daily and that of oral MTX was 25 mg/m2 weekly. The doses were adjusted according to white blood cell count (target range, 2.5–3.5 × 109/L) and aspartate aminotransferase and alanine aminotransferase level ( C than in the patients with wild homozygous genotype. The other analyzed polymorphisms were not associated with toxicity, 6-MP, or MTX dose. Conclusions: Polymorphism of SLCO1B1 c.521 T > C could be a strong predictor of 6-MP dose reduction in maintenance chemotherapy in childhood ALL.
Key words 6-mercaptopurine, childhood, leukemia, methotrexate, polymorphism, SLCO1B1. Childhood acute lymphoblastic leukemia (ALL) is the most common pediatric cancers, accounting for 30% of pediatric cancer,1 and the survival rate has improved up to 80% with the development of new drugs, intensified chemotherapy, supportive care, and the introduction of risk-adapted therapy.2–4 Some patients, however, have to reduce or discontinue treatment due to toxicity. Treatment of childhood ALL includes maintenance chemotherapy, which mainly consists of daily oral 6-mercaptopurine (6-MP) and weekly oral methotrexate (MTX). Part of the toxicity of these agents is known to be affected by genetic polymorphisms.5–8 We have previously reported that the polymorphism of MTHFR c.1298A > C could be a prognostic indicator in childhood lymphoid malignancy.9 6-Mercaptopurine is metabolized into an active and toxic metabolite, 6-thioguanine nucleotide (6-TGN), which is responsible for the elevated myelotoxicity of 6-MP.5 Thiopurine Correspondence: Takashi Fukushima, MD, PhD, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Email: [email protected] Received 29 September 2014; revised 26 November 2014; accepted 29 January 2015. © 2015 Japan Pediatric Society
methyltransferase (TPMT) is a key enzyme in the metabolism of 6-MP.10 Patients who carry variant alleles of TPMT and who have low TPMT activity experience high levels of 6-TGN in red blood cells and hematotoxicity.5 Inosine triphosphate pyrophosphatase (ITPA) is also an enzyme in the metabolism of 6-MP.10 Polymorphisms of ITPA have been associated with low ITPA activity, and result in hematotoxicity and hepatotoxicity.11,12 Multidrug-resistant protein (MRP) 4, which belongs to the superfamily of ATP-binding cassette (ABC) transporters, is an ATP-dependent efflux pump of 6-MP and 6-TGN.13 A polymorphism at MRP4 c.2269G > A reduces MRP4 function and results in myelosuppression.14 Methotrexate is a folate inhibitor widely used for chemotherapy of hematological malignancy.4,15,16 MTX is imported by the reduced folate carrier 1 (RFC1) or the solute carrier organic anion transporter 1B1 (SLCO1B1).17–19 In the cytoplasm, MTX inhibits dihydrofolate reductase (DHFR). DHFR catalyzes the conversion of folate into dihydrofolate (DHF) and tetrahydrofolate (THF), which are active forms of folate. THF is involved in the synthesis of DNA and RNA nucleotides. Inhibition of DHFR causes depletion of THF, which has a cytotoxic effect. MTX-polyglutamate
SLCO1B1 polymorphism in leukemia 573 inhibits thymidylate synthase (TS). Methylenetetrahydrofolate reductase (MTHFR) converts 5,10-methylenetetrahydrofolate (5,10-methylene-THF), which is a substrate of TS, to 5-methyletetrahtdrofolate (5-methyl-THF).20,21 Previous studies regarding maintenance chemotherapy for childhood ALL including a few on Japanese patients have noted an association between the variant allele of TPMT and hematological toxicity or lower 6-MP dose,5,22 and an association between the variant allele of ITPA and hematological toxicity or hepatotoxicity.11,23,24 Polymorphisms of MTHFR, RFC1, and SLCO1B1 involved in the metabolism and transport of folic acid have been shown to be associated with MTX concentration and toxicity under high-dose MTX.8,25,26 And patients with polymorphism of MTHFR c.677C > T more frequently had interruption in both 6-MP and MTX in maintenance chemotherapy for childhood ALL.7 There have been no studies, however, on the utility of MTX dose modification in maintenance chemotherapy for childhood ALL. In this study, we genotyped 8 polymorphisms in 6 candidate genes, TPMT, ITPA, MRP4, MTHFR, RFC1, and SLCO1B1, involved in the metabolism and transport of 6-MP and folic acid. Associations between polymorphisms and clinical course were analyzed in children treated with maintenance chemotherapy consisting of 6-MP and MTX.
Methods The objective was to examine associations between polymorphisms of TPMT*3C, ITPA c.94C > A, MRP4 c.2269G > A, MTHFR c.677C > T, MTHFR c.1298A>C, RFC1 c.80G > A, SLCO1B1 c.1865+4846 T > C, and SLCO1B1 c.521 T > C and clinical course in children with ALL who received maintenance chemotherapy consisting of 6-MP and MTX. Patients
Fifty-three children with ALL, whose germline genomic DNA samples were available, were enrolled in this study. All patients were treated at two institutions, University of Tsukuba Hospital and Ibaraki Children’s Hospital, between June 1998 and April 2013. Patients with Down syndrome and other congenital chromosome abnormalities were excluded. Informed consent was obtained from each parent or guardian, and informed assent was obtained from the patients according to age and level of comprehension. The study was approved by the ethics committees of each participating institution and conformed to the Ethical Guidelines for Human Genome/Gene Analysis Research of the Ministry of Health, Labor and Welfare of Japan and the Declaration of Helsinki. Treatment and toxicity evaluation
All patients received the same maintenance chemotherapy according to the Tokyo Children’s Cancer Study Group (TCCSG) protocol.3,27,28 The scheduled dose of oral 6-MP was 40 mg/m2 daily and that of oral MTX was 25 mg/m2 weekly. The drug dose was modified according to the following guideline: stepwise reduction by 25% according to white blood cell (WBC) count (target range, 2.5–3.5 × 109/L) in order of 6-MP, MTX, in turn. If WBC count is 750 IU/L, 6-MP and MTX are discontinued. And once AST and ALT improve to G, rs1142345), ITPA c.94C > A (rs1127354), MRP4 c.2269G > A (rs3765534), MTHFR c.677C > T (rs1801133), MTHFR c.1298 A > C (rs1801 131), SLCO1B1 c.1865+4846 T > C (rs11045879), and SLCO1B1 c.521 T > C (rs4149056) 7,9,24,29,30 were genotyped with the TaqMan PCR Genotyping Assays (ID: C_19567_20, C_27465 000_10, C_27478235_20, C_1202883_20, C_850486_20, C_311 06904_10, and C_30633906_10; Life Technologies, Foster City, CA, USA) following the manufacturer’s instructions. The polymorphism of RFC1 c.80G > A (rs1051266) was determined using the TaqMan PCR method as previously described.30 Automatic allele calling was performed using ABI PRISM 7900HT data collection and analysis software, version 2.2.2 (Life Technologies). Statistical analysis
Deviation from Hardy–Weinberg equilibrium was examined with the chi-squared test. Control genotype frequencies were obtained from the HapMap database (HapMap-JPT, http:// www.ncbi.nlm.nih.gov/). The genetic effects of associations between the case–control status and each individual SNP were assessed using the chi-squared test. For the analysis of associations between clinical course during maintenance chemotherapy and polymorphisms, the toxicity markers (lowest WBC count, highest ALT level, and the average dose of 6-MP and MTX) for each patient during maintenance chemotherapy and disease status and polymorphisms were assessed using the Mann-Whitney U-test. Analysis was conducted using SPSS, version 20.0 (SPSS, Chicago, IL, USA). First, P < 0.05 was considered statistically significant. For analysis of seven polymorphisms, P < 0.005 with Bonferroni correction was considered significant.
Results Patient characteristics
A total of 53 children were enrolled in the present study. There were 23 males and 30 females. Median age at diagnosis was 4.7 years (range, 1.4–15.8). At a median follow up of 6.5 years (range, © 2015 Japan Pediatric Society
574 R Suzuki et al. Table 1 Patient characteristics Total number patients 53 Sex Male 23 Female 30 Age at diagnosis (years) Median 4.7 (range, 1.4–15.8) Median 15.7 (range, 12.6–22.6) BMI (kg/m2) Immunophenotype B-cell precursor 46 T cell 2 Other 5 Follow up (years) Median 6.5 (range, 1.8–17.2) Disease status 1st CR 49 Relapse 4 BMI, body mass index; CR, complete remission.
1.8–17.2), 49 patients (92.5%) were in first complete remission and 4 (7.5%) had relapsed (Table 1). There was no association between the average doses of 6-MP or MTX and patient characteristics, such as age at diagnosis, sex, or body mass index (data not shown). Genotype
Table 2 lists the TPMT, ITPA, MRP4, MTHFR, RFC1, and SLCO1B1 genotype in patients and HapMap-JPT controls obtained from the NCBI database. Allele frequencies did not deviate significantly from the Hardy–Weinberg equilibrium and there were no differences with control data. As only one patient had variant allele G at TPMT*3C (c.719A > G), this polymorphism was excluded from further
analysis. SLCO1B1 c.1865 + 4846 T > C and SLCO1B1 c.521 T > C were in linkage disequilibrium (LD; D’ = 0.904, r2 = 0.308). Polymorphisms and toxicity
Table 3 lists toxicity data according to genotype. The average dose of 6-MP with at least one variant allele C at SLCO1B1 c.521 T > C (TC/CC) was 25.4 mg/m2/day and in patients with wild homozygous genotype (TT), it was 38.2 mg/m2/day (P = 0.003). There was no association between the average dose of 6-MP and the other six polymorphisms (ITPA c.94C > A, MRP4 c.2269G > A, MTHFR c.677C > T, MTHFR c.1298C > T RFC1 c.80G > A, and SLCO1B1 c.1865+4846 T > C). There was no association between 7 polymorphisms and the toxicity data (lowest WBC count, highest ALT level, and average dose of MTX). There was only one patient with variant allele G at TMPT*3C: for that patient, the average dose of 6-MP was 15.2 mg/m2, the average dose of MTX was 19.9 mg/m2, the lowest WBC count was 1800/μL, and the highest ALT was 768 IU/L. Relapse
All 4 patients who relapsed carried the wild homozygous genotype at MTHFR c.1298A > C (AA). The average dose of 6-MP and MTX in the patients who relapsed was 38.2 mg/m2/day and 23.0 mg/m2/week, respectively, whereas the average 6-MP and MTX doses in the patients who had not relapsed was 32.0 mg/m2/day and
Table 2 Allele frequency Gene *
TPMT 3C ITPA c.94C>A MRP4 c.2269G>A
MTHFR c.677C>T MTHFR c.1298A>C RFC1 c.80G>A
SLCO1B1 c.1865+4846T>C SLCO1B1 c.521T>C
†
Genotype
Present study n = 53
MAF
HWE P-value
HapMap-JPT†
P-value
AA AG GG CC CA AA GG GA AA UD CC CT TT AA AC CC GG GA AA UD TT TC CC TT TC CC
52 1 0 32 19 2 44 6 2 1 15 33 5 35 17 1 5 29 17 2 21 22 10 35 15 3
0.009
0.752
0.667
0.217
0.991
0.096
0.268
166 3 0 36 9 0 32 8 5
0.406
0.296
0.179
0.832
0.618
0.636
0.396
0.777
0.198
0.844
34 42 10 56 28 2 17 42 27 35 37 14 69 15 2
0.075 0.226
0.299 0.982 0.299
0.926 0.158
NCBI HapMap-JPT, database at National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/). MAF, minor allele frequency; HWE, Hardy–Weinberg equilibrium; UD, undetermined.
© 2015 Japan Pediatric Society
SLCO1B1 polymorphism in leukemia 575 Table 3 Clinical data vs genotype Gene ITPA c.94C>A MRP4 c.2269G>A MTHFR c.677C>T MTHFR c.1298A>C RFC1 c.80G>A SLCO1B1 c.1865+4846T>C SLCO1B1 c.521T>C
Genotype CC CA, AA CC CT, TT CC CT, TT AA AC, CC GG GA, AA TT TC, CC TT TC, CC
n 32 21 44 8 15 38 35 18 5 46 21 32 35 18
WBC
ALT
6-MP
MTX
/μL
P-value
U/L
P-value
mg/m2/day
P-value
mg/m2/week
P-value
1750 1800 1800 1600 1700 1800 1800 1700 1800 1700 1800 1750 1900 1700
0.964
308 284 260 346 325 263 284 308 524 237 340 212 329 108
0.920
31.2 34.4 31.2 35.1 31.6 32.7 35.1 25.9 25.2 31.9 38.4 30.1 38.2 25.4
0.383
21.1 19.9 21.1 16.9 18.7 21.5 21.1 18.9 18.7 21.1 20.2 21.6 21.1 17.7
0.617
0.558 0.384 0.332 0.940 0.716 0.332
0.872 0.953 0.918 0.078 0.606 0.098
0.681 0.953 0.049 0.842 0.122 0.003
0.108 0.333 0.599 0.724 0.971 0.145
6-MP; 6-mercaptopurine; ALT; alanine aminotransferase; MTXmethotrexate; WBC, white blood cells.
20.4 mg/m2/week, respectively, and no association was observed (P = 0.203, P = 0.351, respectively; data not shown).
Discussion We have shown that SLCO1B1 c.521 T > C TC/CC genotype was associated with a lower average dose of 6-MP in maintenance chemotherapy for children with ALL. Associations between polymorphisms of ITPA, MRP4, MTHFR, and RFC1 and adverse event and administered dose were not observed. SLCO1B1 is one of the main influx transporters expressed on the sinusoidal membrane of human hepatocyte, and extracts endogenous substrates (e.g. thyroid hormones, estradiol, bilirubin and bile acid) and drug substrates (e.g. 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, statins, and angiotensinconverting enzyme inhibitors) from the portal blood into hepatocytes.31 MTX is one of the substrates of SLCO1B1.18 Trevino et al. studied 640 children with ALL who underwent 2 or 5 g/m2 MTX, and reported that the C allele at SLCO1B1 c.1865+4846 T > C, which was in LD with SLCO1B1 c.521 T > C, was associated with low MTX clearance and gastrointestinal toxicity in a genome-wide association study.32 Radtke et al. studied 415 children with ALL who underwent 5 g/m2 MTX and reported that the C allele at SLCO1B1 c.521 T > C was associated with high MTX area under the concentration time curve (AUC)033 In addition, in other studies, 48H and low MTX clearance. polymorphisms of SLCO1B1 were confirmed to be associated with higher MTX concentration in Caucasian subjects.34,35 Fukushima et al. studied 103 Japanese children with lymphoid malignancy who underwent 3 or 5 g/m2 MTX, and reported that there was no association between the polymorphisms of SLCO1B1 and MTX concentration, toxicity, or disease outcome.9 Kato et al. studied 55 adult Japanese patients with rheumatoid arthritis who underwent MTX treatment (4-10 mg/body/week), and reported that there was no association between the polymorphisms of SLCO1B1 (c.521 T > C and c.388A > G ) and concentration of MTX-polyglutamate in red blood cells nor disease status.36 In the present study, there was an
association between the polymorphism of SLCO1B1 c.521 T > C, which was in LD with SLCO1B1 c.1865+4846 T > C, and a lower average dose of 6-MP rather than MTX, but no association between the polymorphism of SLCO1B1 c.521 T > C and the lowest WBC count and highest ALT level. In this study, the drug doses were reduced to avoid development of severe leukopenia and hepatotoxicity, and successful individualization of dose modification according to WBC count, AST, and ALT levels might result in a 6-MP dose reduction and the absence of severe leukopenia and hepatotoxicity. As patients who carried a variant allele of SLCO1B1 c.521 T > C might develop toxicity more frequently, the average dose of 6-MP was reduced. TPMT*3C (c.719A > G) is a known genetic factor of 6-MP toxicity,37 and planned dose modification for 6-MP is recommended on TPMT genotype.38 The polymorphisms of TPMT*3C (c.719A > G) is known to result in lower enzymatic activity of TPMT and to be associated with high 6-TGN concentration in red blood cells and hematologic toxicity.5,22 Stocco et al. studied 244 children with ALL who underwent maintenance chemotherapy and reported that 26.6% of patients with variant allele at ITPA c.94C > A had grade 3/4 febrile neutropenia after individualization by prospective dose adjustment of 6-MP according to TPMT genotype.23 Tanaka et al. studied 58 Japanese children with ALL who underwent maintenance chemotherapy consisting of 6-MP and MTX, and reported that patients with hepatotoxicity had lower erythrocyte ITPA activity.24 In Japanese inflammatory bowel disease (IBD) in adult patients treated with azathioprine or 6-MP, the 6-TGN level was higher and leukocyte count was lower in the patients with variant allele at MRP4 c.2269G > A than in the patients with wild homogenous genotype.39 In contrast, in the present study there was no association between the polymorphisms of ITPA and MRP4 and the dose of 6-MP and toxicity. In a Japanese childhood ALL cohort, 9 out of 37 patients without polymorphism of ITPA c.94C > A had lower ITPA activity, and some of the patients with high ITPA activity had hepatotoxicity.24 From these observations and our present results, other polymorphisms of ITPA or genes not yet examined could affect ITPA activity or 6-MP toxicity. © 2015 Japan Pediatric Society
576 R Suzuki et al. Since the variant allele frequency of TMPT*3C is very low (0.009), as previously reported,29 this polymorphism may be less valuable for Japanese patients. Several studies reported associations between 6-MP and MTX and polymorphisms of genes involved in the metabolism and transport of folic acid. Shimasaki et al. reported that children with lymphoid malignancy who underwent 6-MP and MTX treatment and who carried a variant allele at MTHFR c.677C > T, had interruptions in both 6-MP and MTX more frequently, and that children who carried a variant allele at RFC1 c.80G > A also had interruptions in 6-MP more frequently.7 Stocco et al. studied 63 adult patients with IBD who underwent 6-MP/azathioprine treatment, and reported that there was no association between the polymorphisms of MTHFR c.677C > T and c.1298 A > C and toxicity.40 Drozdzik et al. reported that 174 adult patients with rheumatoid arthritis who underwent MTX treatment (7.5-15 mg/ body/week) and carried a homozygous variant allele at RFC1 c.80G > A had increased aminotransferase activity, which resulted in discontinuation of treatment more frequently, but the difference was not significant.41 In the present study there was no association between the polymorphisms of MTHFR and RFC1, the average dose of 6-MP and MTX and toxicity (lowest WBC count and highest ALT level). A prospective study on maintenance chemotherapy for childhood ALL in a larger cohort is currently being organized. For that study, if the difference in average dose of 6-MP between the patients who carry at least one variant allele of SLCO1B1 and patients who carry wild homozygous genotype is 10 mg/m2/day, we will need 92 subjects for a type I error probability of 0.05 and a type II error probability of 0.1. Assuming that 10% will be excluded as ineligible for various reasons, an association between SLCO1B1 polymorphisms and 6-MP dose will be confirmed in a prospective study with more than 102 subjects. Additionally, we should investigate NUDT15 c.415C > T, which has been shown to be an important gene associated with leukopenia, especially in Asian patients.42 With regard to further studies, several investigations to clarify the definitive association between SLCO1B1 polymorphisms and clinical course of maintenance chemotherapy are needed. First, a replication cohort study is needed. Second, the concentration of metabolites of 6-MP and MTX or the activity levels of metabolizing enzymes, should be measured to clarify the direct correlations between polymorphisms, enzyme activity, and drug toxicity. And third, multivariate analysis should be done. Conclusions
This is the first report on ALL patients to find a definitive association between clinical course and SLCO1B1 polymorphism in maintenance chemotherapy. SLCO1B1 c.521 T > C TC/CC genotype was associated with a lower average dose of 6-MP. Associations of ITPA, MRP4, MTHFR, and RFC1 polymorphisms with toxicity and administered dose were not observed in maintenance chemotherapy for ALL. Polymorphism of SLCO1B1 c.521T>C could be a strong predictor of 6-MP dose reduction in maintenance chemotherapy in childhood ALL. © 2015 Japan Pediatric Society
Acknowledgments This work was supported by a grant from the National Center for Child Health and Development (25-2). The authors are grateful to National Center of Biotechnology Information for approval to use control genotype data. We would like to thank Mr Charles N. Jones for scientific writing assistance. The authors have no conflict of interest to declare with regards to this work.
References 1 Gatta G, Botta L, Rossi S. Childhood cancer survival in Europe 1999-2007: Results of EUROCARE-5 – a population-based study. Lancet Oncol. 2014; 15: 35–47. 2 Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet 2008; 371: 1030–43. 3 Tsuchida M, Ohara A, Manabe A. Long-term results of Tokyo Children’s Cancer Study Group trials for childhood acute lymphoblastic leukemia, 1984-1999. Leukemia 2010; 24: 383–96. 4 Moricke A, Reiter A, Zimmermann M. Risk-adjusted therapy of acute lymphoblastic leukemia can decrease treatment burden and improve survival: Treatment results of 2169 unselected pediatric and adolescent patients enrolled in the trial ALL-BFM 95. Blood 2008; 111: 4477–89. 5 Relling MV, Hancock ML, Rivera GK. Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus. J. Natl Cancer Inst. 1999; 91: 2001–8. 6 Stocco G, Crews KR, Evans WE. Genetic polymorphism of inosine-triphosphate-pyrophosphatase influences mercaptopurine metabolism and toxicity during treatment of acute lymphoblastic leukemia individualized for thiopurine-S-methyl-transferase status. Expert Opin. Drug Saf. 2010; 9: 23–37. 7 Shimasaki N, Mori T, Torii C. Influence of MTHFR and RFC1 polymorphisms on toxicities during maintenance chemotherapy for childhood acute lymphoblastic leukemia or lymphoma. J. Pediatr. Hematol. Oncol. 2008; 30: 347–52. 8 Laverdiere C, Chiasson S, Costea I. Polymorphism G80A in the reduced folate carrier gene and its relationship to methotrexate plasma levels and outcome of childhood acute lymphoblastic leukemia. Blood 2002; 100: 3832–4. 9 Fukushima H, Fukushima T, Sakai A. Polymorphisms of MTHFR associated with higher relapse/death ratio and delayed weekly MTX administration in pediatric lymphoid malignancies. Leuk. Res. Treat. 2013; 2013: 238528. 10 Adam de Beaumais T, Jacqz-Aigrain E. Pharmacogenetic determinants of mercaptopurine disposition in children with acute lymphoblastic leukemia. Eur. J. Clin. Pharmacol. 2012; 68: 1233–42. 11 Wan Rosalina WR, Teh LK, Mohamad N. Polymorphism of ITPA 94C > A and risk of adverse effects among patients with acute lymphoblastic leukaemia treated with 6-mercaptopurine. J. Clin. Pharm. Ther. 2012; 37: 237–41. 12 Hawwa AF, Millership JS, Collier PS. Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine. Br. J. Clin. Pharmacol. 2008; 66: 517–28. 13 Oevermann L, Scheitz J, Starke K. Hematopoietic stem cell differentiation affects expression and function of MRP4 (ABCC4), a transport protein for signaling molecules and drugs. Int. J. Cancer 2009; 124: 2303–11. 14 Krishnamurthy P, Schwab M, Takenaka K. Transporter-mediated protection against thiopurine-induced hematopoietic toxicity. Cancer Res. 2008; 68: 4983–9. 15 Pui CH, Sandlund JT, Pei D. Improved outcome for children with acute lymphoblastic leukemia: Results of Total Therapy Study XIIIB at St Jude Children’s Research Hospital. Blood 2004; 104: 2690–96.
SLCO1B1 polymorphism in leukemia 577 16 Woessmann W, Seidemann K, Mann G. The impact of the methotrexate administration schedule and dose in the treatment of children and adolescents with B-cell neoplasms: A report of the BFM Group Study NHL-BFM95. Blood 2005; 105: 948–58. 17 Zhao R, Diop-Bove N, Visentin M. Mechanisms of membrane transport of folates into cells and across epithelia. Annu. Rev. Nutr. 2011; 31: 177–201. 18 Niemi M, Pasanen MK, Neuvonen PJ. Organic anion transporting polypeptide 1B1: A genetically polymorphic transporter of major importance for hepatic drug uptake. Pharmacol. Rev. 2011; 63: 157–81. 19 Moscow JA. Methotrexate transport and resistance. Leuk. Lymphoma 1998; 30: 215–24. 20 Bagley PJ, Selhub J. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proc. Natl Acad. Sci. USA 1998; 95: 13217–20. 21 Gorlick R, Goker E, Trippett T. Intrinsic and acquired resistance to methotrexate in acute leukemia. N. Engl. J. Med. 1996; 335: 1041–8. 22 Adam de Beaumais T, Fakhoury M, Medard Y. Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy. Br. J. Clin. Pharmacol. 2011; 71: 575–84. 23 Stocco G, Cheok MH, Crews KR. Genetic polymorphism of inosine triphosphate pyrophosphatase is a determinant of mercaptopurine metabolism and toxicity during treatment for acute lymphoblastic leukemia. Clin. Pharmacol. Ther. 2009; 85: 164–72. 24 Tanaka Y, Manabe A, Nakadate H. The activity of the inosine triphosphate pyrophosphatase affects toxicity of 6-mercaptopurine during maintenance therapy for acute lymphoblastic leukemia in Japanese children. Leuk. Res. 2012; 36: 560–64. 25 Imanishi H, Okamura N, Yagi M. Genetic polymorphisms associated with adverse events and elimination of methotrexate in childhood acute lymphoblastic leukemia and malignant lymphoma. J. Hum. Genet. 2007; 52: 166–71. 26 de Deus DM, de Lima EL, Seabra Silva RM. Influence of methylenetetrahydrofolate reductase C677T, A1298C, and G80A polymorphisms on the survival of pediatric patients with acute lymphoblastic leukemia. Leuk. Res. Treat. 2012; 2012: 292043. 27 Manabe A, Ohara A, Hasegawa D. Significance of the complete clearance of peripheral blasts after 7 days of prednisolone treatment in children with acute lymphoblastic leukemia: The Tokyo Children’s Cancer Study Group Study L99-15. Haematologica 2008; 93: 1155–60. 28 Toyoda Y, Manabe A, Tsuchida M. Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood. J. Clin. Oncol. 2000; 18: 1508–16. 29 Osaki R, Imaeda H, Ban H. Accuracy of genotyping using the TaqMan PCR assay for single nucleotide polymorphisms
30
31 32
33
34
35 36
37 38
39
40 41 42
responsible for thiopurine sensitivity in Japanese patients with inflammatory bowel disease. Exp. Ther. Med. 2011; 2: 783–6. Stanislawska-Sachadyn A, Mitchell LE, Woodside JV. The reduced folate carrier (SLC19A1) c.80G > A polymorphism is associated with red cell folate concentrations among women. Ann. Hum. Genet. 2009; 73: 484–91. Kalliokoski A, Niemi M. Impact of OATP transporters on pharmacokinetics. Br. J. Pharmacol. 2009; 158: 693–705. Trevino LR, Shimasaki N, Yang W. Germline genetic variation in an organic anion transporter polypeptide associated with methotrexate pharmacokinetics and clinical effects. J. Clin. Oncol. 2009; 27: 5972–8. Radtke S, Zolk O, Renner B. Germline genetic variations in methotrexate candidate genes are associated with pharmacokinetics, toxicity, and outcome in childhood acute lymphoblastic leukemia. Blood 2013; 121: 5145–53. Csordas K, Lautner-Csorba O, Semsei AF. Associations of novel genetic variations in the folate-related and ARID5B genes with the pharmacokinetics and toxicity of high-dose methotrexate in paediatric acute lymphoblastic leukaemia. Br. J. Haematol. 2014; 166: 410–20. Ramsey LB, Panetta JC, Smith C. Genome-wide study of methotrexate clearance replicates SLCO1B1. Blood 2013; 121: 898–904. Kato T, Hamada A, Mori S. Genetic polymorphisms in metabolic and cellular transport pathway of methotrexate impact clinical outcome of methotrexate monotherapy in Japanese patients with rheumatoid arthritis. Drug Metab. Pharmacokinet. 2012; 27: 192–9. Stuart H, Orkin DEF, Look AT, Lux SE, Ginsburg D, Nathan DG (ed). Oncology of Infancy and Childhood, 1st edn. Saunders Elsevier, Philadelphia, 2009. Relling MV, Gardner EE, Sandborn WJ. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing. Clin. Pharmacol. Ther. 2011; 89: 387–91. Ban H, Andoh A, Imaeda H. The multidrug-resistance protein 4 polymorphism is a new factor accounting for thiopurine sensitivity in Japanese patients with inflammatory bowel disease. J. Gastroenterol. 2010; 45: 1014–21. Stocco G, Martelossi S, Sartor F. Prevalence of methylenetetrahydrofolate reductase polymorphisms in young patients with inflammatory bowel disease. Dig. Dis. Sci. 2006; 51: 474–9. Drozdzik M, Rudas T, Pawlik A. Reduced folate carrier-1 80G > A polymorphism affects methotrexate treatment outcome in rheumatoid arthritis. Pharmacogenomics J. 2007; 7: 404–7. Yang SK, Hong M, Baek J. A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia. Nat. Genet. 2014; 46: 1017–20.
© 2015 Japan Pediatric Society
doi: 10.1111/ped.12682
Original Article
Influence of SLCO1B1 polymorphism on maintenance therapy for childhood leukemia Ryoko Suzuki,1 Hiroko Fukushima,2 Emiko Noguchi,3 Masahiro Tsuchida,7 Nobutaka Kiyokawa,8 Kazutoshi Koike,7 Enbo Ma,4 Hideto Takahashi,5,9 Chie Kobayashi,2 Ryoko Nakajima-Yamaguchi,6 Aiko Sakai,1 Makoto Saito,2 Atsushi Iwabuchi,2 Keisuke Kato,7 Tomohei Nakao,7 Ai Yoshimi,7 Ryo Sumazaki2 and Takashi Fukushima2 1 Department of Child Health, Graduate School of Comprehensive Human Sciences, Departments of 2Child Health, 3Genetics, 4Clinical Trial and Clinical Epidemiology and 5Epidemiology, Faculty of Medicine, University of Tsukuba, 6Department of Pediatrics, University of Tsukuba Hospital, Tsukuba, 7Department of Hematology and Oncology, Ibaraki Children’s Hospital, Mito, Ibaraki, 8 Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo and 9School of Medicine, Information Management and Statistics, Fukushima Medical University, Fukushima, Japan Abstract
Background: Management of the adverse effects of chemotherapy is essential to improve outcome of children with leukemia. Some genetic polymorphisms can predict treatment-related toxicity, and be used individually in dose modification of 6-mercaptopurine (6-MP) and methotrexate (MTX) in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). We investigated associations between clinical course and candidate gene polymorphisms less evaluated in Japanese patients. Methods: Fifty-three children who received maintenance chemotherapy were enrolled in this study. The scheduled dose of oral 6-MP was 40 mg/m2 daily and that of oral MTX was 25 mg/m2 weekly. The doses were adjusted according to white blood cell count (target range, 2.5–3.5 × 109/L) and aspartate aminotransferase and alanine aminotransferase level ( C than in the patients with wild homozygous genotype. The other analyzed polymorphisms were not associated with toxicity, 6-MP, or MTX dose. Conclusions: Polymorphism of SLCO1B1 c.521 T > C could be a strong predictor of 6-MP dose reduction in maintenance chemotherapy in childhood ALL.
Key words 6-mercaptopurine, childhood, leukemia, methotrexate, polymorphism, SLCO1B1. Childhood acute lymphoblastic leukemia (ALL) is the most common pediatric cancers, accounting for 30% of pediatric cancer,1 and the survival rate has improved up to 80% with the development of new drugs, intensified chemotherapy, supportive care, and the introduction of risk-adapted therapy.2–4 Some patients, however, have to reduce or discontinue treatment due to toxicity. Treatment of childhood ALL includes maintenance chemotherapy, which mainly consists of daily oral 6-mercaptopurine (6-MP) and weekly oral methotrexate (MTX). Part of the toxicity of these agents is known to be affected by genetic polymorphisms.5–8 We have previously reported that the polymorphism of MTHFR c.1298A > C could be a prognostic indicator in childhood lymphoid malignancy.9 6-Mercaptopurine is metabolized into an active and toxic metabolite, 6-thioguanine nucleotide (6-TGN), which is responsible for the elevated myelotoxicity of 6-MP.5 Thiopurine Correspondence: Takashi Fukushima, MD, PhD, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Email: [email protected] Received 29 September 2014; revised 26 November 2014; accepted 29 January 2015. © 2015 Japan Pediatric Society
methyltransferase (TPMT) is a key enzyme in the metabolism of 6-MP.10 Patients who carry variant alleles of TPMT and who have low TPMT activity experience high levels of 6-TGN in red blood cells and hematotoxicity.5 Inosine triphosphate pyrophosphatase (ITPA) is also an enzyme in the metabolism of 6-MP.10 Polymorphisms of ITPA have been associated with low ITPA activity, and result in hematotoxicity and hepatotoxicity.11,12 Multidrug-resistant protein (MRP) 4, which belongs to the superfamily of ATP-binding cassette (ABC) transporters, is an ATP-dependent efflux pump of 6-MP and 6-TGN.13 A polymorphism at MRP4 c.2269G > A reduces MRP4 function and results in myelosuppression.14 Methotrexate is a folate inhibitor widely used for chemotherapy of hematological malignancy.4,15,16 MTX is imported by the reduced folate carrier 1 (RFC1) or the solute carrier organic anion transporter 1B1 (SLCO1B1).17–19 In the cytoplasm, MTX inhibits dihydrofolate reductase (DHFR). DHFR catalyzes the conversion of folate into dihydrofolate (DHF) and tetrahydrofolate (THF), which are active forms of folate. THF is involved in the synthesis of DNA and RNA nucleotides. Inhibition of DHFR causes depletion of THF, which has a cytotoxic effect. MTX-polyglutamate
SLCO1B1 polymorphism in leukemia 573 inhibits thymidylate synthase (TS). Methylenetetrahydrofolate reductase (MTHFR) converts 5,10-methylenetetrahydrofolate (5,10-methylene-THF), which is a substrate of TS, to 5-methyletetrahtdrofolate (5-methyl-THF).20,21 Previous studies regarding maintenance chemotherapy for childhood ALL including a few on Japanese patients have noted an association between the variant allele of TPMT and hematological toxicity or lower 6-MP dose,5,22 and an association between the variant allele of ITPA and hematological toxicity or hepatotoxicity.11,23,24 Polymorphisms of MTHFR, RFC1, and SLCO1B1 involved in the metabolism and transport of folic acid have been shown to be associated with MTX concentration and toxicity under high-dose MTX.8,25,26 And patients with polymorphism of MTHFR c.677C > T more frequently had interruption in both 6-MP and MTX in maintenance chemotherapy for childhood ALL.7 There have been no studies, however, on the utility of MTX dose modification in maintenance chemotherapy for childhood ALL. In this study, we genotyped 8 polymorphisms in 6 candidate genes, TPMT, ITPA, MRP4, MTHFR, RFC1, and SLCO1B1, involved in the metabolism and transport of 6-MP and folic acid. Associations between polymorphisms and clinical course were analyzed in children treated with maintenance chemotherapy consisting of 6-MP and MTX.
Methods The objective was to examine associations between polymorphisms of TPMT*3C, ITPA c.94C > A, MRP4 c.2269G > A, MTHFR c.677C > T, MTHFR c.1298A>C, RFC1 c.80G > A, SLCO1B1 c.1865+4846 T > C, and SLCO1B1 c.521 T > C and clinical course in children with ALL who received maintenance chemotherapy consisting of 6-MP and MTX. Patients
Fifty-three children with ALL, whose germline genomic DNA samples were available, were enrolled in this study. All patients were treated at two institutions, University of Tsukuba Hospital and Ibaraki Children’s Hospital, between June 1998 and April 2013. Patients with Down syndrome and other congenital chromosome abnormalities were excluded. Informed consent was obtained from each parent or guardian, and informed assent was obtained from the patients according to age and level of comprehension. The study was approved by the ethics committees of each participating institution and conformed to the Ethical Guidelines for Human Genome/Gene Analysis Research of the Ministry of Health, Labor and Welfare of Japan and the Declaration of Helsinki. Treatment and toxicity evaluation
All patients received the same maintenance chemotherapy according to the Tokyo Children’s Cancer Study Group (TCCSG) protocol.3,27,28 The scheduled dose of oral 6-MP was 40 mg/m2 daily and that of oral MTX was 25 mg/m2 weekly. The drug dose was modified according to the following guideline: stepwise reduction by 25% according to white blood cell (WBC) count (target range, 2.5–3.5 × 109/L) in order of 6-MP, MTX, in turn. If WBC count is 750 IU/L, 6-MP and MTX are discontinued. And once AST and ALT improve to G, rs1142345), ITPA c.94C > A (rs1127354), MRP4 c.2269G > A (rs3765534), MTHFR c.677C > T (rs1801133), MTHFR c.1298 A > C (rs1801 131), SLCO1B1 c.1865+4846 T > C (rs11045879), and SLCO1B1 c.521 T > C (rs4149056) 7,9,24,29,30 were genotyped with the TaqMan PCR Genotyping Assays (ID: C_19567_20, C_27465 000_10, C_27478235_20, C_1202883_20, C_850486_20, C_311 06904_10, and C_30633906_10; Life Technologies, Foster City, CA, USA) following the manufacturer’s instructions. The polymorphism of RFC1 c.80G > A (rs1051266) was determined using the TaqMan PCR method as previously described.30 Automatic allele calling was performed using ABI PRISM 7900HT data collection and analysis software, version 2.2.2 (Life Technologies). Statistical analysis
Deviation from Hardy–Weinberg equilibrium was examined with the chi-squared test. Control genotype frequencies were obtained from the HapMap database (HapMap-JPT, http:// www.ncbi.nlm.nih.gov/). The genetic effects of associations between the case–control status and each individual SNP were assessed using the chi-squared test. For the analysis of associations between clinical course during maintenance chemotherapy and polymorphisms, the toxicity markers (lowest WBC count, highest ALT level, and the average dose of 6-MP and MTX) for each patient during maintenance chemotherapy and disease status and polymorphisms were assessed using the Mann-Whitney U-test. Analysis was conducted using SPSS, version 20.0 (SPSS, Chicago, IL, USA). First, P < 0.05 was considered statistically significant. For analysis of seven polymorphisms, P < 0.005 with Bonferroni correction was considered significant.
Results Patient characteristics
A total of 53 children were enrolled in the present study. There were 23 males and 30 females. Median age at diagnosis was 4.7 years (range, 1.4–15.8). At a median follow up of 6.5 years (range, © 2015 Japan Pediatric Society
574 R Suzuki et al. Table 1 Patient characteristics Total number patients 53 Sex Male 23 Female 30 Age at diagnosis (years) Median 4.7 (range, 1.4–15.8) Median 15.7 (range, 12.6–22.6) BMI (kg/m2) Immunophenotype B-cell precursor 46 T cell 2 Other 5 Follow up (years) Median 6.5 (range, 1.8–17.2) Disease status 1st CR 49 Relapse 4 BMI, body mass index; CR, complete remission.
1.8–17.2), 49 patients (92.5%) were in first complete remission and 4 (7.5%) had relapsed (Table 1). There was no association between the average doses of 6-MP or MTX and patient characteristics, such as age at diagnosis, sex, or body mass index (data not shown). Genotype
Table 2 lists the TPMT, ITPA, MRP4, MTHFR, RFC1, and SLCO1B1 genotype in patients and HapMap-JPT controls obtained from the NCBI database. Allele frequencies did not deviate significantly from the Hardy–Weinberg equilibrium and there were no differences with control data. As only one patient had variant allele G at TPMT*3C (c.719A > G), this polymorphism was excluded from further
analysis. SLCO1B1 c.1865 + 4846 T > C and SLCO1B1 c.521 T > C were in linkage disequilibrium (LD; D’ = 0.904, r2 = 0.308). Polymorphisms and toxicity
Table 3 lists toxicity data according to genotype. The average dose of 6-MP with at least one variant allele C at SLCO1B1 c.521 T > C (TC/CC) was 25.4 mg/m2/day and in patients with wild homozygous genotype (TT), it was 38.2 mg/m2/day (P = 0.003). There was no association between the average dose of 6-MP and the other six polymorphisms (ITPA c.94C > A, MRP4 c.2269G > A, MTHFR c.677C > T, MTHFR c.1298C > T RFC1 c.80G > A, and SLCO1B1 c.1865+4846 T > C). There was no association between 7 polymorphisms and the toxicity data (lowest WBC count, highest ALT level, and average dose of MTX). There was only one patient with variant allele G at TMPT*3C: for that patient, the average dose of 6-MP was 15.2 mg/m2, the average dose of MTX was 19.9 mg/m2, the lowest WBC count was 1800/μL, and the highest ALT was 768 IU/L. Relapse
All 4 patients who relapsed carried the wild homozygous genotype at MTHFR c.1298A > C (AA). The average dose of 6-MP and MTX in the patients who relapsed was 38.2 mg/m2/day and 23.0 mg/m2/week, respectively, whereas the average 6-MP and MTX doses in the patients who had not relapsed was 32.0 mg/m2/day and
Table 2 Allele frequency Gene *
TPMT 3C ITPA c.94C>A MRP4 c.2269G>A
MTHFR c.677C>T MTHFR c.1298A>C RFC1 c.80G>A
SLCO1B1 c.1865+4846T>C SLCO1B1 c.521T>C
†
Genotype
Present study n = 53
MAF
HWE P-value
HapMap-JPT†
P-value
AA AG GG CC CA AA GG GA AA UD CC CT TT AA AC CC GG GA AA UD TT TC CC TT TC CC
52 1 0 32 19 2 44 6 2 1 15 33 5 35 17 1 5 29 17 2 21 22 10 35 15 3
0.009
0.752
0.667
0.217
0.991
0.096
0.268
166 3 0 36 9 0 32 8 5
0.406
0.296
0.179
0.832
0.618
0.636
0.396
0.777
0.198
0.844
34 42 10 56 28 2 17 42 27 35 37 14 69 15 2
0.075 0.226
0.299 0.982 0.299
0.926 0.158
NCBI HapMap-JPT, database at National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/). MAF, minor allele frequency; HWE, Hardy–Weinberg equilibrium; UD, undetermined.
© 2015 Japan Pediatric Society
SLCO1B1 polymorphism in leukemia 575 Table 3 Clinical data vs genotype Gene ITPA c.94C>A MRP4 c.2269G>A MTHFR c.677C>T MTHFR c.1298A>C RFC1 c.80G>A SLCO1B1 c.1865+4846T>C SLCO1B1 c.521T>C
Genotype CC CA, AA CC CT, TT CC CT, TT AA AC, CC GG GA, AA TT TC, CC TT TC, CC
n 32 21 44 8 15 38 35 18 5 46 21 32 35 18
WBC
ALT
6-MP
MTX
/μL
P-value
U/L
P-value
mg/m2/day
P-value
mg/m2/week
P-value
1750 1800 1800 1600 1700 1800 1800 1700 1800 1700 1800 1750 1900 1700
0.964
308 284 260 346 325 263 284 308 524 237 340 212 329 108
0.920
31.2 34.4 31.2 35.1 31.6 32.7 35.1 25.9 25.2 31.9 38.4 30.1 38.2 25.4
0.383
21.1 19.9 21.1 16.9 18.7 21.5 21.1 18.9 18.7 21.1 20.2 21.6 21.1 17.7
0.617
0.558 0.384 0.332 0.940 0.716 0.332
0.872 0.953 0.918 0.078 0.606 0.098
0.681 0.953 0.049 0.842 0.122 0.003
0.108 0.333 0.599 0.724 0.971 0.145
6-MP; 6-mercaptopurine; ALT; alanine aminotransferase; MTXmethotrexate; WBC, white blood cells.
20.4 mg/m2/week, respectively, and no association was observed (P = 0.203, P = 0.351, respectively; data not shown).
Discussion We have shown that SLCO1B1 c.521 T > C TC/CC genotype was associated with a lower average dose of 6-MP in maintenance chemotherapy for children with ALL. Associations between polymorphisms of ITPA, MRP4, MTHFR, and RFC1 and adverse event and administered dose were not observed. SLCO1B1 is one of the main influx transporters expressed on the sinusoidal membrane of human hepatocyte, and extracts endogenous substrates (e.g. thyroid hormones, estradiol, bilirubin and bile acid) and drug substrates (e.g. 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, statins, and angiotensinconverting enzyme inhibitors) from the portal blood into hepatocytes.31 MTX is one of the substrates of SLCO1B1.18 Trevino et al. studied 640 children with ALL who underwent 2 or 5 g/m2 MTX, and reported that the C allele at SLCO1B1 c.1865+4846 T > C, which was in LD with SLCO1B1 c.521 T > C, was associated with low MTX clearance and gastrointestinal toxicity in a genome-wide association study.32 Radtke et al. studied 415 children with ALL who underwent 5 g/m2 MTX and reported that the C allele at SLCO1B1 c.521 T > C was associated with high MTX area under the concentration time curve (AUC)033 In addition, in other studies, 48H and low MTX clearance. polymorphisms of SLCO1B1 were confirmed to be associated with higher MTX concentration in Caucasian subjects.34,35 Fukushima et al. studied 103 Japanese children with lymphoid malignancy who underwent 3 or 5 g/m2 MTX, and reported that there was no association between the polymorphisms of SLCO1B1 and MTX concentration, toxicity, or disease outcome.9 Kato et al. studied 55 adult Japanese patients with rheumatoid arthritis who underwent MTX treatment (4-10 mg/body/week), and reported that there was no association between the polymorphisms of SLCO1B1 (c.521 T > C and c.388A > G ) and concentration of MTX-polyglutamate in red blood cells nor disease status.36 In the present study, there was an
association between the polymorphism of SLCO1B1 c.521 T > C, which was in LD with SLCO1B1 c.1865+4846 T > C, and a lower average dose of 6-MP rather than MTX, but no association between the polymorphism of SLCO1B1 c.521 T > C and the lowest WBC count and highest ALT level. In this study, the drug doses were reduced to avoid development of severe leukopenia and hepatotoxicity, and successful individualization of dose modification according to WBC count, AST, and ALT levels might result in a 6-MP dose reduction and the absence of severe leukopenia and hepatotoxicity. As patients who carried a variant allele of SLCO1B1 c.521 T > C might develop toxicity more frequently, the average dose of 6-MP was reduced. TPMT*3C (c.719A > G) is a known genetic factor of 6-MP toxicity,37 and planned dose modification for 6-MP is recommended on TPMT genotype.38 The polymorphisms of TPMT*3C (c.719A > G) is known to result in lower enzymatic activity of TPMT and to be associated with high 6-TGN concentration in red blood cells and hematologic toxicity.5,22 Stocco et al. studied 244 children with ALL who underwent maintenance chemotherapy and reported that 26.6% of patients with variant allele at ITPA c.94C > A had grade 3/4 febrile neutropenia after individualization by prospective dose adjustment of 6-MP according to TPMT genotype.23 Tanaka et al. studied 58 Japanese children with ALL who underwent maintenance chemotherapy consisting of 6-MP and MTX, and reported that patients with hepatotoxicity had lower erythrocyte ITPA activity.24 In Japanese inflammatory bowel disease (IBD) in adult patients treated with azathioprine or 6-MP, the 6-TGN level was higher and leukocyte count was lower in the patients with variant allele at MRP4 c.2269G > A than in the patients with wild homogenous genotype.39 In contrast, in the present study there was no association between the polymorphisms of ITPA and MRP4 and the dose of 6-MP and toxicity. In a Japanese childhood ALL cohort, 9 out of 37 patients without polymorphism of ITPA c.94C > A had lower ITPA activity, and some of the patients with high ITPA activity had hepatotoxicity.24 From these observations and our present results, other polymorphisms of ITPA or genes not yet examined could affect ITPA activity or 6-MP toxicity. © 2015 Japan Pediatric Society
576 R Suzuki et al. Since the variant allele frequency of TMPT*3C is very low (0.009), as previously reported,29 this polymorphism may be less valuable for Japanese patients. Several studies reported associations between 6-MP and MTX and polymorphisms of genes involved in the metabolism and transport of folic acid. Shimasaki et al. reported that children with lymphoid malignancy who underwent 6-MP and MTX treatment and who carried a variant allele at MTHFR c.677C > T, had interruptions in both 6-MP and MTX more frequently, and that children who carried a variant allele at RFC1 c.80G > A also had interruptions in 6-MP more frequently.7 Stocco et al. studied 63 adult patients with IBD who underwent 6-MP/azathioprine treatment, and reported that there was no association between the polymorphisms of MTHFR c.677C > T and c.1298 A > C and toxicity.40 Drozdzik et al. reported that 174 adult patients with rheumatoid arthritis who underwent MTX treatment (7.5-15 mg/ body/week) and carried a homozygous variant allele at RFC1 c.80G > A had increased aminotransferase activity, which resulted in discontinuation of treatment more frequently, but the difference was not significant.41 In the present study there was no association between the polymorphisms of MTHFR and RFC1, the average dose of 6-MP and MTX and toxicity (lowest WBC count and highest ALT level). A prospective study on maintenance chemotherapy for childhood ALL in a larger cohort is currently being organized. For that study, if the difference in average dose of 6-MP between the patients who carry at least one variant allele of SLCO1B1 and patients who carry wild homozygous genotype is 10 mg/m2/day, we will need 92 subjects for a type I error probability of 0.05 and a type II error probability of 0.1. Assuming that 10% will be excluded as ineligible for various reasons, an association between SLCO1B1 polymorphisms and 6-MP dose will be confirmed in a prospective study with more than 102 subjects. Additionally, we should investigate NUDT15 c.415C > T, which has been shown to be an important gene associated with leukopenia, especially in Asian patients.42 With regard to further studies, several investigations to clarify the definitive association between SLCO1B1 polymorphisms and clinical course of maintenance chemotherapy are needed. First, a replication cohort study is needed. Second, the concentration of metabolites of 6-MP and MTX or the activity levels of metabolizing enzymes, should be measured to clarify the direct correlations between polymorphisms, enzyme activity, and drug toxicity. And third, multivariate analysis should be done. Conclusions
This is the first report on ALL patients to find a definitive association between clinical course and SLCO1B1 polymorphism in maintenance chemotherapy. SLCO1B1 c.521 T > C TC/CC genotype was associated with a lower average dose of 6-MP. Associations of ITPA, MRP4, MTHFR, and RFC1 polymorphisms with toxicity and administered dose were not observed in maintenance chemotherapy for ALL. Polymorphism of SLCO1B1 c.521T>C could be a strong predictor of 6-MP dose reduction in maintenance chemotherapy in childhood ALL. © 2015 Japan Pediatric Society
Acknowledgments This work was supported by a grant from the National Center for Child Health and Development (25-2). The authors are grateful to National Center of Biotechnology Information for approval to use control genotype data. We would like to thank Mr Charles N. Jones for scientific writing assistance. The authors have no conflict of interest to declare with regards to this work.
References 1 Gatta G, Botta L, Rossi S. Childhood cancer survival in Europe 1999-2007: Results of EUROCARE-5 – a population-based study. Lancet Oncol. 2014; 15: 35–47. 2 Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet 2008; 371: 1030–43. 3 Tsuchida M, Ohara A, Manabe A. Long-term results of Tokyo Children’s Cancer Study Group trials for childhood acute lymphoblastic leukemia, 1984-1999. Leukemia 2010; 24: 383–96. 4 Moricke A, Reiter A, Zimmermann M. Risk-adjusted therapy of acute lymphoblastic leukemia can decrease treatment burden and improve survival: Treatment results of 2169 unselected pediatric and adolescent patients enrolled in the trial ALL-BFM 95. Blood 2008; 111: 4477–89. 5 Relling MV, Hancock ML, Rivera GK. Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus. J. Natl Cancer Inst. 1999; 91: 2001–8. 6 Stocco G, Crews KR, Evans WE. Genetic polymorphism of inosine-triphosphate-pyrophosphatase influences mercaptopurine metabolism and toxicity during treatment of acute lymphoblastic leukemia individualized for thiopurine-S-methyl-transferase status. Expert Opin. Drug Saf. 2010; 9: 23–37. 7 Shimasaki N, Mori T, Torii C. Influence of MTHFR and RFC1 polymorphisms on toxicities during maintenance chemotherapy for childhood acute lymphoblastic leukemia or lymphoma. J. Pediatr. Hematol. Oncol. 2008; 30: 347–52. 8 Laverdiere C, Chiasson S, Costea I. Polymorphism G80A in the reduced folate carrier gene and its relationship to methotrexate plasma levels and outcome of childhood acute lymphoblastic leukemia. Blood 2002; 100: 3832–4. 9 Fukushima H, Fukushima T, Sakai A. Polymorphisms of MTHFR associated with higher relapse/death ratio and delayed weekly MTX administration in pediatric lymphoid malignancies. Leuk. Res. Treat. 2013; 2013: 238528. 10 Adam de Beaumais T, Jacqz-Aigrain E. Pharmacogenetic determinants of mercaptopurine disposition in children with acute lymphoblastic leukemia. Eur. J. Clin. Pharmacol. 2012; 68: 1233–42. 11 Wan Rosalina WR, Teh LK, Mohamad N. Polymorphism of ITPA 94C > A and risk of adverse effects among patients with acute lymphoblastic leukaemia treated with 6-mercaptopurine. J. Clin. Pharm. Ther. 2012; 37: 237–41. 12 Hawwa AF, Millership JS, Collier PS. Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine. Br. J. Clin. Pharmacol. 2008; 66: 517–28. 13 Oevermann L, Scheitz J, Starke K. Hematopoietic stem cell differentiation affects expression and function of MRP4 (ABCC4), a transport protein for signaling molecules and drugs. Int. J. Cancer 2009; 124: 2303–11. 14 Krishnamurthy P, Schwab M, Takenaka K. Transporter-mediated protection against thiopurine-induced hematopoietic toxicity. Cancer Res. 2008; 68: 4983–9. 15 Pui CH, Sandlund JT, Pei D. Improved outcome for children with acute lymphoblastic leukemia: Results of Total Therapy Study XIIIB at St Jude Children’s Research Hospital. Blood 2004; 104: 2690–96.
SLCO1B1 polymorphism in leukemia 577 16 Woessmann W, Seidemann K, Mann G. The impact of the methotrexate administration schedule and dose in the treatment of children and adolescents with B-cell neoplasms: A report of the BFM Group Study NHL-BFM95. Blood 2005; 105: 948–58. 17 Zhao R, Diop-Bove N, Visentin M. Mechanisms of membrane transport of folates into cells and across epithelia. Annu. Rev. Nutr. 2011; 31: 177–201. 18 Niemi M, Pasanen MK, Neuvonen PJ. Organic anion transporting polypeptide 1B1: A genetically polymorphic transporter of major importance for hepatic drug uptake. Pharmacol. Rev. 2011; 63: 157–81. 19 Moscow JA. Methotrexate transport and resistance. Leuk. Lymphoma 1998; 30: 215–24. 20 Bagley PJ, Selhub J. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proc. Natl Acad. Sci. USA 1998; 95: 13217–20. 21 Gorlick R, Goker E, Trippett T. Intrinsic and acquired resistance to methotrexate in acute leukemia. N. Engl. J. Med. 1996; 335: 1041–8. 22 Adam de Beaumais T, Fakhoury M, Medard Y. Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy. Br. J. Clin. Pharmacol. 2011; 71: 575–84. 23 Stocco G, Cheok MH, Crews KR. Genetic polymorphism of inosine triphosphate pyrophosphatase is a determinant of mercaptopurine metabolism and toxicity during treatment for acute lymphoblastic leukemia. Clin. Pharmacol. Ther. 2009; 85: 164–72. 24 Tanaka Y, Manabe A, Nakadate H. The activity of the inosine triphosphate pyrophosphatase affects toxicity of 6-mercaptopurine during maintenance therapy for acute lymphoblastic leukemia in Japanese children. Leuk. Res. 2012; 36: 560–64. 25 Imanishi H, Okamura N, Yagi M. Genetic polymorphisms associated with adverse events and elimination of methotrexate in childhood acute lymphoblastic leukemia and malignant lymphoma. J. Hum. Genet. 2007; 52: 166–71. 26 de Deus DM, de Lima EL, Seabra Silva RM. Influence of methylenetetrahydrofolate reductase C677T, A1298C, and G80A polymorphisms on the survival of pediatric patients with acute lymphoblastic leukemia. Leuk. Res. Treat. 2012; 2012: 292043. 27 Manabe A, Ohara A, Hasegawa D. Significance of the complete clearance of peripheral blasts after 7 days of prednisolone treatment in children with acute lymphoblastic leukemia: The Tokyo Children’s Cancer Study Group Study L99-15. Haematologica 2008; 93: 1155–60. 28 Toyoda Y, Manabe A, Tsuchida M. Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood. J. Clin. Oncol. 2000; 18: 1508–16. 29 Osaki R, Imaeda H, Ban H. Accuracy of genotyping using the TaqMan PCR assay for single nucleotide polymorphisms
30
31 32
33
34
35 36
37 38
39
40 41 42
responsible for thiopurine sensitivity in Japanese patients with inflammatory bowel disease. Exp. Ther. Med. 2011; 2: 783–6. Stanislawska-Sachadyn A, Mitchell LE, Woodside JV. The reduced folate carrier (SLC19A1) c.80G > A polymorphism is associated with red cell folate concentrations among women. Ann. Hum. Genet. 2009; 73: 484–91. Kalliokoski A, Niemi M. Impact of OATP transporters on pharmacokinetics. Br. J. Pharmacol. 2009; 158: 693–705. Trevino LR, Shimasaki N, Yang W. Germline genetic variation in an organic anion transporter polypeptide associated with methotrexate pharmacokinetics and clinical effects. J. Clin. Oncol. 2009; 27: 5972–8. Radtke S, Zolk O, Renner B. Germline genetic variations in methotrexate candidate genes are associated with pharmacokinetics, toxicity, and outcome in childhood acute lymphoblastic leukemia. Blood 2013; 121: 5145–53. Csordas K, Lautner-Csorba O, Semsei AF. Associations of novel genetic variations in the folate-related and ARID5B genes with the pharmacokinetics and toxicity of high-dose methotrexate in paediatric acute lymphoblastic leukaemia. Br. J. Haematol. 2014; 166: 410–20. Ramsey LB, Panetta JC, Smith C. Genome-wide study of methotrexate clearance replicates SLCO1B1. Blood 2013; 121: 898–904. Kato T, Hamada A, Mori S. Genetic polymorphisms in metabolic and cellular transport pathway of methotrexate impact clinical outcome of methotrexate monotherapy in Japanese patients with rheumatoid arthritis. Drug Metab. Pharmacokinet. 2012; 27: 192–9. Stuart H, Orkin DEF, Look AT, Lux SE, Ginsburg D, Nathan DG (ed). Oncology of Infancy and Childhood, 1st edn. Saunders Elsevier, Philadelphia, 2009. Relling MV, Gardner EE, Sandborn WJ. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing. Clin. Pharmacol. Ther. 2011; 89: 387–91. Ban H, Andoh A, Imaeda H. The multidrug-resistance protein 4 polymorphism is a new factor accounting for thiopurine sensitivity in Japanese patients with inflammatory bowel disease. J. Gastroenterol. 2010; 45: 1014–21. Stocco G, Martelossi S, Sartor F. Prevalence of methylenetetrahydrofolate reductase polymorphisms in young patients with inflammatory bowel disease. Dig. Dis. Sci. 2006; 51: 474–9. Drozdzik M, Rudas T, Pawlik A. Reduced folate carrier-1 80G > A polymorphism affects methotrexate treatment outcome in rheumatoid arthritis. Pharmacogenomics J. 2007; 7: 404–7. Yang SK, Hong M, Baek J. A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia. Nat. Genet. 2014; 46: 1017–20.
© 2015 Japan Pediatric Society
