Abstract 1156: Applying targeted Next-Gen Sequencing and miRNA expression profiling for cancer biomarker discovery

The Biomarker Discovery Center in Heidelberg, Germany develops new approaches to cancer biomarker identification and validation using high throughput targeted next-generation sequencing and miRNA assays to characterize patient samples from the German Cancer Research Center (DKFZ) and other leading research institutions. Using HybSelect™, febit9s product for sequence capture, we demonstrated high throughput processing of patient samples upstream of targeted Next-Gen Sequencing. Overlapping probes were designed to capture the genomic regions of interest (i.e., targets), and then the Geniom Biochips were synthesized for the HybSelect procedure. DNA libraries were prepared from clinical specimens using bar-coded sequencing adaptors from Life Technologies, and the targets were enriched using the HybSelect Sequence Capture Protocol. Bar-coded targets from hundreds of patient samples were sequenced using a SOLiD System with a febit Geniom RT Analyzer. The variants were compared to the reference sequences from healthy individuals. In a related study, changes in miRNA expression profiles were examined in certain cancers. The relative stability of miRNA and the relatively accessible nature of bodily fluids such as blood make miRNA a promising molecule for cancer biomarker discovery. For evaluation of the miRNA expression profiles of cancer patients, we utilized a Microfluidic Primer Extension Assay (MPEA), which starts with the hybridization of unlabeled total RNA samples to Geniom miRNA Biochips. After hybridization, selective enzymatic elongation of the correctly hybridized miRNAs was carried out with biotinylated nucleotides, and then detected with SAPE (streptavidin-conjugated phycoerythrin). The content included on Geniom miRNA Biochips was based on the latest Sanger miRBase update, thus guaranteeing that all human miRNAs, including the most recently validated miRNAs were addressed. With only 130 ng of total RNA required, this method dramatically reduced the amount of sample needed to reliably measure miRNAs and to develop predictive miRNA signatures. Analyses of complex miRNA expression patterns in blood of cancer patients compared to those of healthy donors allowed for distinct classification of the samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1156.