Evaluation of the prognostic utility of miRNA and multiple genetic markers in stage IIA colon cancer.

Abstract

3630 Background: The variable prognoses of stage II colon cancer subjects have provoked a therapeutic dilemma of who should receive adjuvant therapy. Presently, prognosis is evaluated on often subjective histopathological features. Reliable, objective markers that can accurately predict recurrence risk are needed. Using retrospectively collected tumor samples with known clinical outcome, we evaluated the prognostic utility of miRNA together with clinicopathological features and molecular markers with suggestive prognostic value. Methods: We evaluated the expression of miRNA in 118 stage IIA FFPE tumors (73 non-recurrent, NR; 45 recurrent, R) using the Affymetrix Gene Chip V1.0 microarray, and performed RT-qPCR to verify select miRNAs. We also sequenced KRAS codons 12/13, and BRAF codon 600, and assessed MSI status using a fluorescent PCR-based assay. MMR protein expression was determined by IHC. An empirical approach was applied to screen miRNA using combinatorial criteria of microarray 2-way ANOVA analysis, Kaplan-Meier curve logrank, and single and multivariate Cox-regression. miRNA was the most significant predictor among all histopathological and molecular features (FDR<0.05). A miRNA classifier was built with nested two-layer cross-validation. Results: The top miRNA model was selected based on AUC estimates and Cox-regression p-value. This model included 30 miRNAs; 25 of these were common with the empirical approach, confirming the robustness of candidate selection. The miRNA classifier distinguished R from NR cases with an AUC of ~0.74. Among all histopathological and molecular features evaluated, grade was significantly associated with disease-free survival (logrank p<0.05). Both single variate and multivariate Cox analysis revealed that grade and PMS2 status were strongly associated with recurrence (p<0.05). Lastly, lack of expression of PMS2 and MLH1, BRAF V600E and MSI was highly correlated (Φ coefficient > 0.6), and associated with non-recurrent disease. Conclusions: These data provide strong support for miRNAs as independent prognostic biomarkers for stage IIA colon cancer.Validation work is ongoing to demonstrate the potential utility of these miRNAs in patient stratification.