Abstract 4276: Target enrichment to enable RNA-sequencing on FFPE samples: Application to a patient selection biomarkers in colorectal cancer

Abstract

Abstract In proof of concept clinical studies, selecting for patients that are the most likely to respond to a given therapy will increase the chance of demonstrating clinical benefit for a new investigational agent. Archived Formalin-Fixed, Paraffin-Embedded (FFPE) tissue taken at the time of first biopsy and cancer diagnosis are a ready source of material for such patient selection assays; however if the selection assay is RNA-based sequencing based assay, FFPE tumor tissues are typically refractory to standard methods. Here we describe Target Enrichment, an RNA-sequencing technique that enabled analysis of FFPE tumor tissue. The Target Enrichment technique provides the necessary, high quality gene expression data required for identifying the mesenchymal subtype of CRC. In this experiment, RNA was extracted from a set of 192 histologically characterized, archived colorectal cancer (CRC) FFPE tumor tissues with sufficient representation of all Consensus Molecular Subtypes of CRC. A major issue with FFPE tissue is that nucleic acids are not well preserved in the fixation process. The RNA extraction method was carefully optimized in advance toward improving yield and integrity of the total RNA. In order to accommodate the low FFPE RNA integrity, we used Target Enrichment, a technique derived from whole-exome sequencing that isolates and amplifies library fragments corresponding to only the transcribed regions of interest. After testing several protocols, we established a Target Enrichment technique to remove low-quality library fragments that would otherwise interfere and impair sequencing quality. This method allowed us to generate high-quality sequencing data for samples that proved challenging using traditional RNA-seq methods. Fresh-frozen tissue samples were used as a benchmark and our Target Enrichment technique showed comparable performance across multiple sequencing quality metrics including mapping rate, expression profiling efficiency, number of genes detected, also the pass rate. Furthermore, data generated using our Target Enrichment technique aligned with the known frequencies of the Consensus Molecular Subtypes of CRC. Overall, our Target Enrichment technique provides a protocol for generating high quality RNA-seq libraries from highly degraded FFPE RNA. Citation Format: Maximilian Rogers-Grazado, Joachim Theilhaber, Jack Pollard, Alexei Protopopov. Target enrichment to enable RNA-sequencing on FFPE samples: Application to a patient selection biomarkers in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4276.