Personalized Medicine and Imaging DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer Sune Boris Nyga  rd 1 , Ben Vainer 2 , Signe Lykke Nielsen 1 , Fred Bosman 3 , Sabine Tejpar 4 , Arnaud Roth 5 , Mauro Delorenzi 6,7,8 , Nils Br € unner 1 , and Eva Budinska 9 Abstract Purpose: Prospective–retrospective assessment of the TOP1 gene copy number and TOP1 mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer. Experimental Design: Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant colon cancer trial (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinote- can. TOP1 copy number status was analyzed by fluorescence in situ hybridization (FISH) using a TOP1/CEN20 dual-probe combination. TOP1 mRNA data were available from previous analyses. Results: TOP1 FISH and follow-up data were obtained from 534 patients. TOP1 gain was identified in 27% using a single- probe enumeration strategy (4 TOP1 signals per cell) and in 31% when defined by a TOP1/CEN20 ratio  1.5. The effect of additional irinotecan was not dependent on TOP1 FISH status. TOP1 mRNA data were available from 580 patients with stage III disease. Benefit of irinotecan was restricted to patients character- ized by TOP1 mRNA expression  third quartile (RFS: HR adjusted , 0.59; P ¼ 0.09; OS: HR adjusted , 0.44; P ¼ 0.03). The treatment by TOP1 mRNA interaction was not statistically significant, but in exploratory multivariable fractional polynomial interaction anal- ysis, increasing TOP1 mRNA values appeared to be associated with increasing benefit of irinotecan. Conclusions: In contrast to the TOP1 copy number, a trend was demonstrated for a predictive property of TOP1 mRNA expres- sion. On the basis of TOP1 mRNA, it might be possible to identify a subgroup of patients where an irinotecan doublet is a clinically relevant option in the adjuvant setting of colon cancer. Clin Cancer Res; 22(7); 1621–31. Ó2015 AACR. Introduction Colorectal cancer is one of the leading causes for cancer related mortality in the world (1–3). Tumor stage at diagnosis remains the strongest prognostic factor, and treatment is guided according to the TNM staging system (4–8). Systemic treatment has improved progression-free survival (PFS) and overall survival (OS) for patients with advanced disease, but survival benefit of adjuvant systemic therapy is also evident for patients with high- risk localized disease (high-risk stage II) or regional disease (stage III; ref. 9). A limitation of systemic therapy is the great interpatient variability in drug efficacy and severity of adverse effects (10). In the pursuit of a more personalized treatment approach, it is clinically important to identify tumor characteristics that may serve as biomarkers which will accurately predict the likelihood of benefit in advance of therapy. The discovery and validation of predictive biomarkers are not only relevant in the development of new targeted drugs, but may be equally important for already implemented classic cytotoxic chemotherapy. The introduction of irinotecan in combination with 5-fluo- rouracil (5FU)/folinic acid (FA; e.g., FOLFIRI) has improved the clinical outcome of patients with metastatic colorectal cancer, and with efficacy equal to that of the oxaliplatin and 5FU/FA doublets, the FOLFIRI regimen is approved for first and second line therapy (9, 11–13). However, overall objective response rates following FOLFIRI remains below 50% and in combina- tion with noncomplete cross-resistance between FOLFIRI and the oxaliplatin doublets this emphasizes the importance of selecting the right treatment regimen in first line (12–14). Irinotecan is not recommended in the adjuvant setting of colon cancer because superiority of the 5FU/FA þ irinotecan combi- nations over 5FU/FA alone has not been demonstrated in any randomized controlled trials (RCT; refs. 15–18). However, the 1 University of Copenhagen, Faculty of Health and Medical Sciences, Copenhagen, Denmark. 2 Department of Pathology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark. 3 University of Lausanne, University Institute of Pathology, Lausanne, Switzerland. 4 Digestive Oncology Unit, University Hospital Gasthuisberg, Leuven, Belgium. 5 Oncosurgery Unit, University Hospital of Geneva, Geneva, Switzerland. 6 SIB Swiss Institute of Bioinformatics, Bioinformatics Core Facility, Lausanne, Switzerland. 7 University of Lausanne, Ludwig Center for Cancer Research, Lausanne, Switzerland. 8 Oncology Department, University of Lausanne, Lausanne, Switzerland. 9 Masaryk University, Institute of Biostatistics and Analyses, Brno, Czech Republic. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). N. Br€ unner and E. Budinska share senior authorship of this article. Corresponding Author: Nils Br€ unner, University of Copenhagen, Molecular Disease Biology Section, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, c/o Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. Phone: þ45 3533-3130; Fax: 45-3533-2755, E-mail: nbr@sund.ku.dk doi: 10.1158/1078-0432.CCR-15-0561 Ó2015 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 1621 on June 16, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst November 5, 2015; DOI: 10.1158/1078-0432.CCR-15-0561