Title: MicroRNA classifier and nomogram for metastasis prediction in colon cancer Running title: Prognostic microRNA classifier for colon cancer metastasis

Background: Colon cancer prognosis and treatment are currently based on a classification system still showing large heterogeneity in clinical outcome, especially in TNM-stages II-III. Prognostic biomarkers for metastasis risk are warranted as development of distant recurrent disease mainly accounts for the high lethality rates of colon cancer. MicroRNAs have been proposed as potential biomarkers for cancer. Furthermore, a verified standard for normalization of the amount of input material in PCR based relative quantification of miRNA expression is lacking. Methods: A selection of frozen tumor specimens from two independent patient cohorts with TNMstage II-III microsatellite stable primary adenocarcinomas were used for laser capture microdissection. Next generation sequencing was performed on small RNAs isolated from colorectal tumors from the Dutch cohort (N=50). Differential expression analysis, comparing in metastasizedand nonmetastasized tumors, identified prognostic microRNAs. Validation was performed on colon tumors from the German cohort (N=43) using qPCR. Results: MiR-25-3p and miR-339-5p were identified and validated as independent prognostic markers and used to construct a multivariate nomogram for metastasis risk prediction. The nomogram showed good probability prediction in validation. Additionally, we recommend combination of miR-16-5p and miR-26a-5p as standard for normalization in qPCR of colon cancer tissue-derived microRNA expression. Conclusions: In this international study we identified and validated a miRNA classifier in primary cancers, and propose a nomogram capable of predicting metastasis risk in microsatellite stable TNMstage II-III colon cancer. Impact: In conjunction with TNM-staging, by means of a nomogram, this microRNA classifier may allow for personalized treatment decisions based on individual tumor characteristics. on April 12, 2017. © 2014 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 14, 2014; DOI: 10.1158/1055-9965.EPI-14-0544-T