Abstract 2816: Discovery of a miRNA-based RT-qPCR signature able to detect early stage colorectal cancer in blood plasma
Authors: Søren J. Nielsen, Hanni Willenbrock, Jacob U. Fog, Nana Jacobsen, Jan Stenvang, Thorarinn Blondal, Torben Ørntoft, Nils Brünner, Claus L. Andersen, Hans J. Nielsen, Adam Baker
Journal: Cancer Research (2011)

Abstract Colorectal cancer (CRC) ranks 4th in terms of prevalence and 2nd in number of deaths among cancers of the western world. Although early detection results in improved survival, and regular screening has been proven clinically to lower mortality from CRC, screening rates among the 50-75 year old population are unsatisfactory. There is therefore a clear unmet need for a quick, sensitive, specific, and convenient screening assay to select at risk individuals for definitive diagnosis by colonoscopy. Cellular miRNA profiles vary according to cell type and state, and cellular miRNAs are exported to extracellular fluids by both malignant cells and cells of the immune system. Blood plasma and serum miRNAs are stable under standard clinical sampling protocols and are therefore promising candidates for minimally invasive biomarkers for diverse pathological conditions. To screen for miRNAs in plasma, we developed an LNA-enhanced miRNA RT-qPCR platform with unprecedented sensitivity, selectivity and ease-of-use. An extensive informatics infrastructure has been put in place to allow semi-automated sample- and data-QC, and data analysis of large datasets. 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. Different normalization protocols are routinely evaluated to ensure correct normalization of the dataset prior to data analysis. Using this platform, we have performed a two-phased discovery program in plasma samples from stage II/III CRC patients and age- and gender-matched colonoscopy-verified healthy controls. A genome wide screen in blood plasma profiled 730 individual miRNAs from 50 stage II cancers and 50 matched controls. This allowed us to develop a candidate panel of miRNAs detectable in plasma and to calculate an appropriate design for the full discovery study. In the second phase of the program we profiled the candidate 378 individual miRNAs in 120 stage II CRC, 50 stage III, and 170 matched controls. The results of these screens have given us a list of miRNAs that are statistically significantly altered between cancers and healthy volunteers. Some of the differentially expressed miRNAs include miRNAs with known roles in cancer and/or inflammatory processes. From this list we have developed a diagnostic miRNA signature that we are moving into assay development. We will present our diagnostic miRNA signature and plans for our first validation study where we will test the signature in samples from a 5,000 patient retrospective clinical trial as well as future plans to test prospectively in an ongoing clinical trial recruiting ∼5,000 symptomatic individuals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2816. doi:10.1158/1538-7445.AM2011-2816