The Pharmacogenomics Journal (2013) 13, 417 -- 422 & 2013 Macmillan Publishers Limited All rights reserved 1470-269X/13 www.nature.com/tpj ORIGINAL ARTICLE A genome-wide association study for irinotecan-related severe toxicities in patients with advanced non-small-cell lung cancer J-Y Han1, ES Shin2, Y-S Lee3, HY Ghang4, S-Y Kim3, J-A Hwang3, JY Kim1 and JS Lee1 The identification of patients who are at high risk for irinotecan-related severe diarrhea and neutropenia is clinically important. We conducted the first genome-wide association study (GWAS) to search for novel susceptibility genes for irinotecan-related severe toxicities, such as diarrhea and neutropenia, in non-small-cell lung cancer (NSCLC) patients treated with irinotecan chemotherapy. The GWAS putatively identified 49 single-nucleotide polymorphisms (SNPs) associated with grade 3 diarrhea (G3D) and 32 SNPs associated with grade 4 neutropenia (G4N). In the replication series, the SNPs rs1517114 (C8orf34), rs1661167 (FLJ41856) and rs2745761 (PLCB1) were confirmed as being associated with G3D, whereas rs11128347 (PDZRN3) and rs11979430 and rs7779029 (SEMAC3) were confirmed as being associated with G4N. The final imputation analysis of our GWAS and replication study showed significant overlaps of association signals within these novel variants. This GWAS screen, along with subsequent validation and imputation analysis, identified novel SNPs associated with irinotecan-related severe toxicities. The Pharmacogenomics Journal (2013) 13, 417--422; doi:10.1038/tpj.2012.24; published online 5 June 2012 Keywords: C8orf34; FLJ41856; PLCB1; PDZRN3; SEMA3C; irinotecan INTRODUCTION technologies has paved the way for a genome-wide association 13 Irinotecan is one of the most active chemotherapeutic agents for study (GWAS) to identify novel susceptibility genes. Therefore, patients with colorectal and lung cancers. However, some patients we conducted the first GWAS to search for novel genes involved suffer from irinotecan-related severe toxicities, such as diarrhea in susceptibility to irinotecan-related severe toxicity, such as and neutropenia.1 Although comprehensive data supporting the diarrhea and neutropenia, in NSCLC patients treated with association between homozygous UGT1A1*28 and grade 4 irinotecan chemotherapy. neutropenia exist, this association does not account for all 2--4 toxicities observed with irinotecan chemotherapy. Furthermore, MATERIALS AND METHODS genetic variations in other UGT1A genes and drug transporters Study population have been suggested to contribute to the variability of irinotecan To construct a relatively homogenous treatment group, we restricted pharmacokinetics and toxicities.5--8 Previously, we investigated the patients who participated in clinical trials and received irinotecan-based associations of variants in other UGT1A genes and transporters chemotherapy as first-line therapy for their advanced NSCLC. In addition, with irinotecan pharmacokinetics and toxicity in Korean non- we collected toxicity data prospectively through clinical trials. For the initial small-cell lung cancer (NSCLC) patients treated with irinotecan- 9--11 GWAS discovery stage, cases and controls were obtained from two phase II based chemotherapy. We found that the UGT1A1*6/*6 and studies of irinotecan and cisplatin chemotherapy, which enrolled chemo- SLCO1B1 521TC or CC genotypes were associated with a naive patients with advanced NSCLC. A total of 81 patients received significantly higher area under the curve (AUC) of SN-38, an irinotecan 80 mg mÀ2 intravenously (i.v.) on days 1 and 8 plus cisplatin active metabolite of irinotecan, and were independently pre- 60 mg mÀ2 i.v. on day 1 every 3 weeks, whereas 22 patients were treated dictive for grade 4 neutropenia (G4N). Although no significant with irinotecan 65 mg mÀ2 and cisplatin 30 mg mÀ2 i.v. on days 1 and 8 pharmacokinetic parameters for severe diarrhea were found, the every 3 weeks. Full details of the studies have been reported UGT1A9*22 9/9, ABCC2 3972CC and ABCG2 34GA or AA genotypes previously.14,15 The significance of the single-nucleotide polymorphisms 12 were independently predictive for grade 3 diarrhea (G3D). (SNPs) identified at the discovery stage was subsequently confirmed Nevertheless, much of the variation in irinotecan pharmaco- through a replication study in independent subjects enrolled in another kinetics and toxicity remains unexplained. randomized phase II study of irinotecan-based chemotherapy, which also Owing to the severity of irinotecan-related toxicity, identifica- recruited chemo-naive patients with advanced NSCLC. In this study, 73 tion of patients who are at high risk for severe diarrhea and patients received irinotecan 80 mg mÀ2 i.v. on days 1 and 8 plus cisplatin neutropenia is clinically essential. To date, irinotecan pharmaco- 60 mg mÀ2 i.v. on day 1 every 3 weeks, and 73 patients received irinotecan genetic studies have mainly consisted of association analyses of 90 mg mÀ2 i.v. on days 1 and 8 plus oral capecitabine 1000 mg mÀ2 twice candidate genes. However, the toxicity phenotypes of cancer daily on days 1 through 14 every 3 weeks. These studies were conducted patients are complicated by many factors; thus, a more compre- under the approval of the ethical review boards and the guidelines hensive and systemic approach is needed. The recent develop- for good clinical practice. All subjects gave informed consent for the ment of new-generation and cost-effective high-throughput genetic testing. 1Lung Cancer Branch, Research Institute and Hospital, National Cancer Center, Goyang, Gyeonggi, Republic of Korea; 2DNALink, Seoul, Republic of Korea; 3Functional Genomic Branch, Research Institute and Hospital, National Cancer Center, Goyang, Gyeonggi, Republic of Korea and 4Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. Correspondence: Dr J-Y Han, Center for Lung Cancer, Research Institute and Hospital, National Cancer Center, 111 Jungbalsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 410-769, Republic of Korea. E-mail: [email protected] Received 23 February 2012; revised 26 April 2012; accepted 30 April 2012; published online 5 June 2012 GWAS for irinotecan toxicities J-Y Han et al 418 Definition of case and control for irinotecan-related severe Table 1. Characteristics of study subjects toxicities Our goal was to find novel genetic variants associated with irinotecan- GWAS sample (%) Replication sample (%) related severe diarrhea and neutropenia through a GWAS. Cases were required to have G3D or G4N during the course of irinotecan-based Number 101 146 chemotherapy. Controls had not experienced G3D or G4N during Age, years irinotecan-based chemotherapy. Toxicity was assessed throughout treat- Median (range) 60 (29--76) 58 (31--77) ment and was graded using the National Cancer Institute’s Common Terminology Criteria (NCI-CTC version 2.0). Gender Male 77 (76) 134 (92) Female 24 (24) 12 (8) Genotyping and quality control Peripheral blood samples were obtained at baseline, and genomic DNA Grade 4 neutropenia was extracted using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, Yes 24 (24) 21 (14) No 77 (76) 125 (86) USA) in accordance with the manufacturer’s instructions. A genome-wide scan was performed using an Affymetrix Genome-Wide Human SNP array Grade 3 diarrhea 5.0 (Affymetrix, Santa Clara, CA, USA) comprising 440 094 genome-wide Yes 11 (11) 13 (9) SNPs. Genotype calls were determined from the fluorescence intensities No 90 (89) 133 (91) using the DM algorithm with a 0.33 P-value setting, as well as the B-RLMM algorithm. Abbreviation: GWAS, genome-wide association study. Quality control procedures included the following steps. First, only samples with at least 95% call rate were included. SNPs with a call rate o95% in each panel of case or control were discarded. Second, SNPs deviating from Hardy--Weinberg equilibrium in controls (Po0.001) were in the cases (G3D or G4N) was o0.15, we identified 48 SNPs for not included in the w2 test. Finally, those with a minor allele frequency of G3D and 31 SNPs for G4N. Next, we proceeded to replicate these 40.05 were included for analysis. associations in an independent NSCLC patient population receiv- Based on the initial GWAS results, we selected the most promising SNPs ing an irinotecan-based chemotherapy. The replication analysis for subsequent genotyping in the replication sample according to the revealed the three most significantly associated SNPs for each following inclusion criteria: (1) Po1 Â 10À4 in the GWAS allelic association toxicity. The significant associations observed in the GWAS and analyses and (2) minor allele frequency in the case population (G3D or the replication study are shown in Table 2. rs1517114 in intron 3 G4N) of X0.15. Genotyping was performed using an iPLEX Gold assay on of C8orf34 (chromosome 8 open reading frame 34), rs1661167 in 0 the MassARRAY platform (Sequenom, San Diego, CA, USA) based on the 3 untranslated region of FLJ41856 (CEACAM22P (carcinoem- matrix-assisted laser desorption/ionization time-of-flight spectrometry, bryonic antigen-related cell adhesion molecule 2, pseudogene)) and according to the manufacturer’s instructions.16 For quality control of the rs2745761 in intron 2 of PLCB1 (phospholipase C, b1) showed genotyping assay, 10% of the samples were run as duplicates and tested strong associations with G3D. Regarding G4N, rs11128347 in for concordance. Additionally, genotype clusters were examined manually intron 3 of PDZRN3 (PDZ domain--containing ring finger 3) and two to determine their fitness.