Validation of a plasma-based miRNA PCR test for early detection of colorectal cancer.

Abstract

424 Background: Colorectal cancer (CRC) is a major cause of mortality in the western world. Early detection of CRC improves survival and screening for CRC has been clinically proven to lower CRC-related mortality in the screening population. However, although population screening programs have been implemented in a number of countries, screening rates among the 50-75 year olds are unsatisfactory. There is therefore a clear unmet need for a quick, sensitive, specific, and minimally invasive screening assay to select at risk individuals for definitive diagnosis by colonoscopy. Methods: In order to detect microRNA (miRNA) biomarkers for CRC in blood plasma, we developed an LNA-enhanced miRNA RT-qPCR platform with high sensitivity and linearity for optimal quantitation of miRNAs from limited plasma samples. A clinical reference- lab compatible workflow that allows for the entire procedure from sample preparation through data acquisition and QC to test result to be completed within one working day was established. A reference melting curve database has been implemented to ensure the integrity of each data point, and appropriate controls monitor plate-to-plate and day-to-day variation. State-of-the-art normalization protocols have been evaluated to ensure optimal normalization of datasets prior to data analysis. Results: We previously determined a miRNA signature in a multi hospital discovery cohort that is differentially expressed between healthy individuals and stage II CRC patients. Here we report on the validation of this miRNA signature in an independent set of plasma samples from CRC patients and healthy volunteers. We have counter-screened the miRNA signature in a set of patients with other prevalent diseases, including hypertension, diabetes, diverticulitis, and others. Conclusions: A plasma miRNA signature for early detection of CRC from patient plasma was developed and validated in an independent clinical sample set. The signature was specific with respect to other diseases prevalent in the screening population. A second large-scale validation project is on-going. We conclude that plasma miRNA biomarkers can constitute an effective minimally invasive approach to population-wide CRC screening.