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1Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, South Korea; .... call rate
Original Article

Polymorphic Markers Associated With Severe Oxaliplatin-Induced, Chronic Peripheral Neuropathy in Colon Cancer Patients Hong-Hee Won, PhD1,2; Jeeyun Lee, MD, PhD3; Joon Oh Park, MD, PhD3; Young Suk Park, MD, PhD3; Ho Yeong Lim, MD, PhD3; Won Ki Kang, MD, PhD3; Jong-Won Kim, MD, PhD4; Soo-Youn Lee, MD, PhD4,5; and Se Hoon Park, MD, PhD3

BACKGROUND: To identify potential genetic markers for severe oxaliplatin-induced chronic peripheral neuropathy (OXCPN), the authors performed a genome-wide association analysis of patients with colon cancer who received oxaliplatin-based chemotherapy. METHODS: This was a prospective study in which DNA was purified in peripheral blood from patients with colon cancer who received oxaliplatin. The primary endpoint was the development of severe (grade 2 lasting for >7 days or grade 3) OXCPN. For the discovery set, genotyping was done for 96 patients who received adjuvant fluorouracil and oxaliplatin using the a genome-wide human single-nucleotide polymorphism (SNP) array. An association between polymorphisms and severe OXCPN was investigated. At the same time, 247 patients who received oxaliplatin-based, first-line chemotherapy for advanced disease were enrolled as a validation set. RESULTS: Among the 32 genotyped candidate SNPs selected from the discovery set, 9 SNPs in 8 genes (tachykinin, precursor 1[TAC1]; forkhead box C1 [FOXC1]; integrin, alpha 1 [ITGA1]; acylphosphatase 2, muscle type [ACYP2]; deleted in lymphocytic leukemia, 7 [DLEU7]; B-cell translocation gene 4 [BTG4]; calcium/calmodulin-dependent protein kinase II inhibitor 1 [CAMK2N1]; and phenylalanyl-tRNA synthase 2 [FARS2]) had nominal replication (P < .05). The most significant association was observed at reference SNP number (rs)10486003 in TAC1 (P ¼ 4.84  107) in combined data from 2 sets. Five SNPs (rs10486003, rs2338, rs830884, rs843748, and rs797519) were significant in a multiple regression analysis (P < .05). Overall prediction accuracy calculated by the regression model was 72.8% (95% confidence interval, 65.8%-79.9%) in the model development and 75.9% (95% confidence interval, 66.9%-84.9%) in the model evaluation. CONCLUSIONS: The current results indicated that a genome-wide pharmacogenomic approach is useful for identifying novel polymorphism predictors of severe OXCPN that may be used in personalized C 2011 American Cancer Society. chemotherapy. Cancer 2012;118:2828-36. V KEYWORDS: colon cancer, genome-wide association, neuropathy, neurotoxicity, oxaliplatin, pharmacogenomics.

INTRODUCTION The mainstay of first-line treatment of advanced colorectal cancer (CRC) is a chemotherapy regimen combining oxaliplatin or irinotecan with fluoropyrimidine with or without leucovorin (FOLFOX or FOLFIRI, respectively).1 In addition to the activity and safety reported with first-line FOLFOX,2,3 the regimen has been approved in the adjuvant setting.4 The most common and problematic toxicity of oxaliplatin is cumulative peripheral neuropathy.5 The risk factors for Presented in part at the 46th Annual Meeting of the American Society of Clinical Oncology; June 4-8, 2010; Chicago, IL. We thank Dr Seonwoo Kim for statistical analyses and Ms Jung-Ae Kim for blood sample preparation. Jeeyun Lee, Soo-Youn Lee, and Se Hoon Park designed the study. Jeeyun Lee, Joon Oh Park, Young Suk Park, Ho Yeong Lim, and Se Hoon Park followed the patients and advised in the creation of the protocol. Hong-Hee Won, Jeeyun Lee, Jong-Won Kim, Soo-Youn Lee, and Se Hoon Park collected the data and performed analyses. Hong-Hee Won and Se Hoon Park drafted the article. Jong-Won Kim provided advice on the study design. All authors critically reviewed and approved the final article. Corresponding authors: Soo-Youn Lee, MD, PhD, Department of Laboratory Medicine and Genetics, Samsung Medical Center, Seoul 135-710, South Korea; Fax: (011) 82-2-3410-1754; [email protected]; Se Hoon Park, MD, PhD, Department of Medicine, Samsung Medical Center, Seoul 135-710, South Korea; Fax: (011) 82-2-3410-1754; [email protected] 1 Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, South Korea; 2Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology, Daejeon, South Korea; 3Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 4Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 5Division of Clinical Pharmacology, Clinical Trial Center, Samsung Medical Center, Seoul, South Korea

DOI: 10.1002/cncr.26614, Received: July 29, 2011; Revised: August 29, 2011; Accepted: September 7, 2011, Published online October 21, 2011 in Wiley Online Library (wileyonlinelibrary.com)

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SNP Markers for Oxaliplatin Neuropathy/Won et al

developing oxaliplatin-induced chronic peripheral neurotoxicity (OXCPN) includes treatment schedule, time of infusion, dose of oxaliplatin, and pre-existing peripheral neuropathy.6 In addition to efforts to identify clinical characteristics predictive for OXCPN, studies have been done to establish the role of various genetic markers for OXCPN. Optimization of oxaliplatin-based chemotherapy depends on patient selection, and the exploration of predictive genetic markers for the incidence or severity is important. Lecomte et al. indicated a significant correlation between the glutathione S-transferase p1 (GSTP1) polymorphism and oxaliplatin-related cumulative neurotoxicity.7 However, other researchers reported contradicting results with regard to the association of GSTP1 with neurotoxicity.8,9 Previous studies have focused on a limited number of candidate genes that are involved in specific biologic functions or processes to identify genetic polymorphisms associated with platinum drug sensitivity or resistance. The genes involved in several mechanisms, such as mismatch-repair genes (human mutL homolog 1 [hMLH1], hMLH2, human postmeiotic segregation 2 [hPMS2], human mutH 2 [hMSH2], hMSH3, and hMSH6), replicative bypass, base excision repair (XRCC1), nucleotide excision repair (ERCC1 and ERCC2), GSTP1, and epidermal growth factor receptor (EGFR), were the main candidates in previous studies.9,10 However, an association between SNPs of the genes and platinum drug response was not consistently replicated.11 With the recent advance in molecular genotyping technology, the genotyping of hundreds of SNPs enables us to examine genetic differences in individual patients with regard to drug response or side effects. In the current study, we performed a genome-wide association (GWA) analysis in a cohort of patients with colon cancer who received oxaliplatin-based combination chemotherapy to identify the SNPs associated with severe OXCPN. After validating the significant SNPs selected from the GWA analysis, we evaluated those polymorphisms as genetic predictors for predicting severe OXCPN.

MATERIALS AND METHODS Patients In the current prospective study, we recorded the incidence and severity of OXCPN in all consecutive patients with stage II through IV colon cancer who received oxaliplatin-based combination chemotherapy (National Clinical Trial NCT00977717; available at: http://www.clinicaltrials.gov;

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accessed September 30, 2011). Full details of the current study have been published previously.12 This study was approved by the institutional review board, and all patients provided written informed consent before registration. In an adjuvant setting, we used a modified FOLFOX regimen (oxaliplatin 85 mg/m2 intravenously on day 1, leucovorin 200 mg/m2 intravenously on day 1, and fluorouracil 400 mg/m2 as an intravenous bolus on day 1 followed by 2400 mg/m2 intravenously over 46 hours). In the first-line setting, the treating physician determined chemotherapy (ie, either oxaliplatin 130 mg/m2 intravenously on day 1 plus capecitabine 2000 mg/m2/d on days 1-14 [XELOX] repeated every 3 weeks or FOLFOX) for each patient. Adjuvant FOLFOX was repeated every 2 weeks until recurrence, unacceptable toxicity, a patient chose to discontinue treatment, or a maximum of 12 cycles. Clinical Evaluation of Neurotoxicity Patients were followed on day 1 of each chemotherapy cycle. OXCPN was evaluated and rated in accordance with National Cancer Institute (NCI) criteria (Common Toxicity Criteria for Adverse Events, version 3; NCI, Bethesda, Md). In addition to subjective assessment of OXCPN, we performed a short, standardized neurologic examination that included the testing of exteroceptive sensation in hands and feet and testing of proprioceptive sensation. Dose modifications were performed based on the laboratory test results and the degree of nonhematologic adverse events observed during the preceding cycle. In particular, oxaliplatin was reduced for episodes of prolonged (7 days) grade 2 OXCPN or temporary (1. The remaining 5 SNPs had the opposite MAF distribution, ie, low MAF in the

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affected group. The 7 previously reported SNPs (rs11615 and rs3212986 in ERCC1, rs13181 and rs179993 in ERCC2, rs1695 in GSTP1, rs2298771 in SCN1A, and rs25487 in XRCC1) had no association with severe OXCPN in the validation set. Prediction Model for Severe Neuropathy Occurrence The prediction probabilities were calculated by multiple logistic regression analysis using severe OXCPN occurrence as the dependent variable and the replicated SNPs as the independent variables. Clinical variables were not included in the model, because there was no significant association between baseline clinical characteristics and the incidence of severe OXCPN. Moreover, because 2 SNPs, rs17140129 and rs6924717, were in complete linkage disequilibrium (r2 ¼ 1), only rs17140129 was

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Original Article

Figure 1. This is a Manhattan plot of genome-wide association results. P values were calculated using an allelic test with the Fisher exact test. Blue boxes represent candidate single-nucleotide polymorphisms (SNPs) for validation. The SNP identifiers (reference SNP [rs] numbers) are shown for the replicated SNPs in the validation set, and the significant SNPs in the multiple regression analysis are indicated in red.

included in the regression model. Five SNPs (rs10486003, rs2338, rs830884, rs843748, and rs797519) were significant in the multiple logistic regression analysis (P < .05). Among the 206 tested samples, excluding the 41 samples that were missing genotypes, 55 samples with a probability between .4 and .6 were considered unpredictive. The model yielded an overall prediction accuracy of 72.8% (110 of 151 samples; 95% CI, 65.8%-79.9%) for the predicted 151 samples (Table 3). The positive predictive value of the model was 70.6% (24 of 34 samples; 95% CI, 55.3%-85.9%), and the negative predictive value was 73.5% (86 of 117 samples; 95% CI, 65.5%-81.5%). Sensitivity and specificity were 43.6% (24 of 55 samples; 95% CI, 30.5%-56.7%) and 89.6% (86 of 96 samples; 95% CI, 83.5%-95.7%), respectively. When the model was tested for the whole population, including the unpredictive 55 samples, the area under the receiver characteristics curve of the model was 0.72. Detailed genotype combinations of 5 polymorphisms of the prediction model are summarized in Table 4. To evaluate the model, we tested prediction performance using the discovery set (n ¼ 96), which was not included in the model development. The model could predict severe OXCPN occurrence for 87 of 96 samples (90.6%; the remaining 9 samples had a probability between .4 and .6) and also had good prediction performance (Table 3). The overall accuracy was 75.9% (66 of 87

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samples; 95% CI, 66.9%-84.9%). The positive and negative predictive values were 86.4% (19 of 22 samples; 95% CI, 72%-100%) and 85.5% (47 of 55 samples; 95% CI, 76.1%-94.8%), respectively. Sensitivity was 70.4% (19 of 27 samples; 95% CI, 53.1%-87.6%), and specificity was 94% (47 of 50 samples; 95% CI, 87.4%-100%). The area under the receiver characteristics curve for the model that included the 9 unpredictive samples was 0.88.

DISCUSSION In the current study, we identified several polymorphisms associated with severe OXCPN in 2 independent data sets, and we also evaluated these polymorphisms as potential predictive markers. Five SNPs (rs10486003, rs2338, rs830884, rs843748, and rs797519) were significant in the multiple regression analysis (P < .05). The overall predictive accuracy calculated by the regression model was 72.8% (95% CI, 65.8%-79.9%) in the model development and 75.9% (95% CI, 66.9%-84.9%) in the model evaluation. Our samples have strength in terms of an ethnically homogeneous population and a well controlled phenotype, because they were collected prospectively in a single institute. Most patients did not have chemotherapy-related symptoms before study entry. To the best of our knowledge, this is the first study to examine a genetic association with severe OXCPN on a genome-wide scale, Cancer

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SNP Markers for Oxaliplatin Neuropathy/Won et al

Table 2. Single-Nucleotide Polymorphisms Associated With Severe Neuropathy

SNP

Chr

Position

Genea

Study

Pb

Minor/ Major Allelec

MAF in Samples With SN

MAF in Samples Without SN

OR (95% CI)

rs10486003

7

97067714

TAC1

6

1518612

FOXC1, GMDS

rs830884

5

52056153

ITGA1, PELO

rs843748

2

54356416

ACYP2, TSPYL6

rs797519

13

50129133

DLEU7

rs4936453

11

110805992

BTG4, POU2AF1

rs12023000

1

20662232

CAMK2N1

rs17140129

6

5243361

FARS2, LYRM4

rs6924717

6

5249850

FARS2, LYRM4

2.04105 2.46103 4.84107 4.36105 4.25103 4.63106 2.36105 5.83103 1.74106 2.59105 1.07102 1.01105 6.37105 3.61102 8.21105 7.84105 3.80102 9.86105 7.84105 4.01102 8.81105 1.37105 4.74102 3.23105 1.37105 4.74102 3.23105

T/C

rs2338

Discovery Validation Combined Discovery Validation Combined Discovery Validation Combined Discovery Validation Combined Discovery Validation Combined Discovery Validation Combined Discovery Validation Combined Discovery Validation Combined Discovery Validation Combined

0.08 0.12 0.11 0.42 0.38 0.39 0.06 0.10 0.09 0.33 0.26 0.28 0.19 0.33 0.29 0.10 0.25 0.21 0.10 0.22 0.19 0.21 0.11 0.14 0.21 0.11 0.14

0.33 0.24 0.27 0.15 0.25 0.22 0.32 0.20 0.23 0.09 0.16 0.14 0.47 0.43 0.44 0.35 0.35 0.35 0.35 0.32 0.33 0.02 0.06 0.05 0.02 0.06 0.05

0.17 0.43 0.32 4.18 1.83 2.27 0.15 0.45 0.32 5.20 1.85 2.43 0.26 0.65 0.50 0.21 0.64 0.49 0.21 0.62 0.47 14.45 2.04 3.34 14.45 2.04 3.34

A/G

C/T

A/G

C/G

G/T

G/A

G/A

T/C

(0.07-0.42) (0.25-0.74) (0.19-0.52) (2.11-8.29) (1.22-2.75) (1.58-3.26) (0.06-0.40) (0.25-0.80) (0.19-0.54) (2.33-11.59) (1.16-2.94) (1.61-3.68) (0.14-0.52) (0.44-0.97) (0.35-0.72) (0.09-0.48) (0.42-0.98) (0.33-0.71) (0.09-0.48) (0.40-0.97) (0.31-0.70) (3.22-64.92) (1.03-4.03) (1.81-6.35) (3.22-64.92) (1.03-4.03) (1.81-6.35)

Abbreviations: A, adenine; ACYP2, acylphosphatase 2, muscle type; BTG4, B-cell translocation gene 4; C, cytosine; CAMK2N1, calcium/calmodulin-dependent protein kinase II inhibitor 1; Chr, chromosome; CI, confidence interval; DLEU7, deleted in lymphocytic leukemia, 7; FARS2, phenylalanyl-tRNA synthetase 2, mitochondrial; FOXC1, forkhead box C1; G, guanine; GMDS, guanosine diphosphate-mannose 4,6-dehydratase; ITGA1, integrin, alpha 1; LYRM4, LYR motif containing 4; MAF, minor allele frequency; OR, odds ratio; PELO, petola homolog (Drosophila); POU2AF1, Pit-Oct-Unc (POU) class 2-associating factor 1; rs, reference SNP; SN, severe neuropathy; SNP, single-nucleotide polymorphism; T, thymine; TAC1, tachykinin, precursor 1; TSPYL6, testis-specific Y-encodedlike protein 6. a Genes within 100 Kb of the SNP are listed, and the nearest gene of each SNP is underlined; rs10486003 is located 130 Kb upstream of TAC1. b P values were calculated using an allelic test with the Fisher exact test. c Alleles were determined as a plus strand in the combined set.

and our findings suggest many plausible candidate genes with regard to biologic relevance in the development of neurotoxicity. The patients in our study received a remarkable amount of oxaliplatin. Given the large doses, it is understandable than clinicians and patients are concerned about the bothersome side effects of oxaliplatin.12 OXCPN is recognized as a complaint that can particularly affect quality of life. It is noteworthy that some patients discontinued treatment because of severe OXCPN rather than tumor progression. Once developed, the treatment for severe OXCPN includes only conservative measures, including dose reduction and/or delay, analgesics, or the avoidance of cold. Patients who receive anticancer chemotherapy exhibit variation in terms of both treatment outcome and

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Table 3. Prediction of Severe Neuropathy Occurrence

Predicted Outcomea

Observed Outcome: No. of Patients Without SN

With SN

Total

86 10 96

31 24 55

117 34 151

47 3 50

8 19 27

55 22 87

Model development, n ¼ 206 Without SN (P.6) Total

Model evaluation, n ¼ 96 Without SN (P.6) Total

Abbreviations: SN, severe neuropathy. a Predicted outcomes were based on probability calculated from multiple logistic regression analysis.

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Original Article Table 4. Genotype Combinations of 5 Polymorphisms of Prediction Model

Prediction

rs10486003

rs2338

rs830884

rs843748

rs797519

Predicted Probability of Severe Neuropathy, %a

Predicted severe neuropathy will occur

CC CC CC CC CC CC CC CC CC CC CC TC CC CC CC CC CC CC CC CC CC CC CC CC CC TC TC TC TC TC TC TC TC TC TC TC TC TC TC TT TT TT TT TT TT TT TT

AA AA AA AA AA AG AG AG AG GG GG AG AG AG AG AG AG GG GG GG GG GG GG GG GG AG AG AG AG AG GG GG GG GG GG GG GG GG GG AA AG AG GG GG GG GG GG

TT TT TT TT TT TC TT TT TT TT TT TT TC TC TC TC TT CC TC TC TC TC TT TT TT TC TC TC TT TT CC TC TC TC TC TC TT TT TT TT TC TT TC TC TC TT TT

AA AG AG AG GG AA AA AG AG AA AG AG AG GG GG GG GG AG AG GG GG GG AG GG GG AG AG GG GG GG GG AG AG GG GG GG AG GG GG GG GG GG GG GG GG AG GG

GG CC CG GG GG GG CG CG GG GG GG GG CC CC CG GG CC CG CG CC CG GG CC CC CG CG GG GG CC CG CC CG GG CC CG GG CG CG GG CG GG GG CC CG GG CG CG

>60

Predicted severe neuropathy will not occur