The Pharmacogenomics Journal (2015) 15, 226–234 © 2015 Macmillan Publishers Limited All rights reserved 1470-269X/15 www.nature.com/tpj ORIGINAL ARTICLE Integrin genetic variants and stage-specific tumor recurrence in patients with stage II and III colon cancer P Bohanes1, D Yang2, F Loupakis1, MJ LaBonte1, A Gerger1, Y Ning1, C Lenz1, F Lenz1, T Wakatsuki1, W Zhang1, L Benhaim1, A El-Khoueiry1, R El-Khoueiry1 and H-J Lenz1 Integrins (ITGs) are key elements in cancer biology, regulating tumor growth, angiogenesis and lymphangiogenesis through interactions of the tumor cells with the microenvironment. Moving from the hypothesis that ITGs could have different effects in stage II and III colon cancer, we tested whether a comprehensive panel of germline single-nucleotide polymorphisms (SNPs) in ITG genes could predict stage-specific time to tumor recurrence (TTR). A total of 234 patients treated with 5-fluorouracil-based chemotherapy at the University of Southern California were included in this study. Whole-blood samples were analyzed for germline SNPs in ITG genes using PCR–restriction fragment length polymorphism or direct DNA sequencing. In the multivariable analysis, stage II colon cancer patients with at least one G allele for ITGB3 rs4642 had higher risk of recurrence (hazard ratio (HR) = 4.027, 95% confidence interval (95% CI) 1.556–10.421, P = 0.004). This association was also significant in the combined stage II–III cohort (HR = 1.975, 95% CI 1.194–3.269, P = 0.008). The predominant role of ITGB3 rs4642 in stage II diseases was confirmed using recursive partitioning, showing that ITGB3 rs4642 was the most important factor in stage II diseases. In contrast, in stage III diseases the combined analysis of ITGB1 rs2298141 and ITGA4 rs7562325 allowed to identify three distinct prognostic subgroups (P = 0.009). The interaction between stage and the combined ITGB1 rs2298141 and ITGA4 rs7562325 on TTR was significant (P = 0.025). This study identifies germline polymorphisms in ITG genes as independent stage-specific prognostic markers for stage II and III colon cancer. These data may help to select subgroups of patients who may benefit from ITG-targeted treatments. The Pharmacogenomics Journal (2015) 15, 226–234; doi:10.1038/tpj.2014.66; published online 9 December 2014 INTRODUCTION attachment to the surrounding extracellular matrix and, for some Adjuvant chemotherapy has improved survival in patients with ITGs, also to other cells. ITGs are expressed on both tumor cells colon cancer. However, being active in patients with stage III colon and normal cells (for example, endothelial cells, platelets and cancer (and in a subgroup of stage II patients), adjuvant chemo- leucocytes). Therefore, they promote tumor growth not only by therapy fails to eradicate micro-metastatic disease in 30% of direct interaction of tumor cells with the microenvironment but patients.1,2 In contrast, as many as 30–40% of patients with stage also by regulating endothelial cell’s survival and migration during 6 III colon cancer are given unnecessary post-operative treatment angiogenesis and lymphangiogenesis. Recent evidence showing because their tumor has good intrinsic biologic behavior.3 For that ITGs are critical in cancer dormancy suggests that their stage II patients, there are currently no validated clinical param- differential expression or activity may be responsible for tumor eters or biomarkers that identify patients that should be given recurrence.7 post-operative chemotherapy. Therefore, both prognostic factors ITGs are heterodimeric cell surface receptors. Eighteen α- and and predictive factors for oxaliplatin and 5-fluorourcil efficacies are eight β-subunits associate to form 24 different non-covalently of critical importance. associated heterodimers with specific tissue distribution and Recent evidence indicates that molecular biomarkers may have unique ligand specificity.8 ITGs transmit signals in both direction a stage-specific distribution and influence in colon cancer,4,5 of the cell membrane: ITG binding to extracellular matrix changes which has led some authors to consider stage II and III colon the shape and composition of the cytoskeleton; intracellular signal cancer as distinct entities. This data has lead to active research to mechanisms activate the ITGs (conformational change) and identify key pathways that would be responsible for a different increase their affinity.9 These signals are integrated with those clinical behavior across colon cancer stages. A candidate group of originating from the growth factor receptors enabling a specific molecules is the adhesion cell receptors that interact with the cellular response in various biologic situations.10 Multiple ITG extracellular matrix, having a critical role in cell survival, prolifera- heterodimers have been shown to play a role in cancers, including tion, differentiation and migration. The major group of proteins colon cancer. However, most of the attention has been on αV promoting these interactions is the integrins (ITGs), a superfamily heterodimers, notably αVβ3, as it has been shown to be important of transmembrane-related adhesion receptors that regulate in tumor angiogenesis. Several drugs have thus been developed 1Sharon A. Carpenter Laboratory, Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA and 2Department of Preventive Medicine, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. Correspondence: Dr H-J Lenz, Sharon A. Carpenter Laboratory, Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA. E-mail: [email protected] Received 1 April 2014; revised 18 August 2014; accepted 28 August 2014; published online 9 December 2014 Integrin variants and stage II and III colon cancer P Bohanes et al 227 to target αV heterodimers.11 Another important heterodimer that of about 0.5. Deviation of allelic distribution of each ITG polymorphism has caught attention for drug development is α5β1 heterodimer, from Hardy–Weinberg equilibrium was tested using χ2-test in each ethnic as it has been involved in tumor angiogenesis and more recently group. The distribution of polymorphisms by baseline demographic, in cancer dormancy.12 However, recently, interest is growing on clinical and pathological characteristics was examined using Fisher’s exact other ITG heterodimers as many of them have been linked to test. The true mode of inheritance of all ITG polymorphisms tested is not known and this study assumed a codominant, additive, dominant or cancer prognosis. recessive genetic model wherever appropriate. The association between There is substantial genetic variability within the genes coding polymorphisms and TTR was analyzed using Kaplan–Meier curves and log- for the ITGs that may alter the gene expression and/or activity by rank test. In the multivariable Cox regression analysis, the model was affecting various steps in protein synthesis, including transcription, adjusted for stage and type of adjuvant chemotherapy, and stratified by translation or splicing. This, in turn, may alter the tumor behavior race. Other clinico-pathologic parameters were not entered in the model, and/or sensitivity to chemotherapy drugs, thereby causing inter- as they did not seem to be a significant prognostic impact in this cohort of 16 tumor stage or interindividual differences. Based on the hypoth- patients. Interactions between polymorphisms and stage on TTR were esis that specific germline single-nucleotide polymorphism (SNP) tested by comparing likelihood ratio statistics between the baseline and in ITG genes could have different effect across stage II and III colon nested Cox regression models that include the multiplicative product term. P-values for all polymorphisms were adjusted for multiple testing using a cancer patients, we investigated a comprehensive panel of ITG modified test of Conneely and Boehnke17 that was applied for the SNPs in patients with stage II and III colon cancer and linked them correlated tests due to linkage disequilibrium and the different modes of with outcome. Only ITG genes that have been previously asso- inheritance considered. We considered a pACT of o0.15 as being ciated with colon cancer were selected. potentially important given the candidate pathway approach. Recursive partitioning (RP), incorporating cross-validation, was used to explore and identify polymorphism profiles and interactions associated with TTR using MATERIALS AND METHODS the rPart-function in S-plus. Case-wise deletion for missing polymorphisms Eligible patients was used in univariate and multivariable analyses. In the RP analysis, all patients with at least one polymorphism result available were included. All A total of 234 patients with stage III and high-risk stage II colon cancer analyses were performed using the SAS statistical package version 9.2 (SAS were included in this study cohort. Whole blood was available from 206 Institute, Cary, NC, USA) and S-PLUS 7.0 (TIBCO, Palo Alto, CA, USA). patients. All patients were treated with 5-fluorouracil-based adjuvant chemotherapy at the Norris Comprehensive Cancer Center/University of Southern California or the Los Angeles County/University of Southern RESULTS California. At the completion of post-operative therapy, patients were followed clinically every 3 months for the first 2 years and then