Abstract 3631: Whole transcriptome and exome targeted RNA sequencing for FFPE tumor samples from clinical trials
Authors: Schleifman Erica B. , Kiialainen Anna , Roller Andreas , Bader Sabine , Motlhabi Maipelo , Hegde Priti , McCaffery Ian , Hampton Garret , Cannarile Michael , Cummings Craig , Spleiss Olivia , Peters Eric 
Published: July 2016

Abstract
Currently there are multiple approaches for measuring gene expression from human tissue samples on the market. These methods range from measuring the expression of a few genes using quantitative RT-PCR to measuring every gene in a sample by whole transcriptome RNA sequencing (RNA-Seq). Although many techniques and products for measuring gene expression are available, the highly degraded RNA isolated from formalin-fixed, paraffin embedded (FFPE) tumor tissues still poses a challenge for many of them. FFPE tumor samples are an abundant source of biomarker information and gene expression analysis in these samples has the potential to identify new signatures, biomarkers, or diagnostics that could predict patient response to treatment. Accurately measuring gene expression in a high throughput manner from this sample type has long been a struggle in the field.
To determine the ability of RNA-Seq to accurately measure gene expression from FFPE-derived RNA, we utilized a large collection of matched fresh frozen (FF) and FFPE samples from 12 different tumor indications. By comparing the results to the matching FF sample, we were able to determine the accuracy and sensitivity of each platform when using degraded FFPE-derived RNA. The FFPE samples in the collection had a wide range of RNA quality scores (RIN score: 0-6.8, DV200:0-78) representing what is typically isolated from clinical trial samples.
We further compared whole transcriptome RNA-Seq with a hybrid capture method, RNA Access, to determine the accuracy and feasibility of using this technology on degraded RNA. RNA Access selects for protein coding transcripts by hybridization, whereas whole transcriptome RNA-seq removes ribosomal RNA, but analyzes everything else i.e. the global transcriptome. All samples were sequenced to an average of 50 or 25 million paired-end reads for whole transcriptome RNA-Seq and RNA Access, respectively and 100 ng of RNA was used as input in both assays.
We conclude that both methods can be used for analyzing FFPE tumor samples. A similar dynamic range was observed for both methods and both show similar correlation between FF and FFPE samples within the method (0.76 vs 0.74). The overall concordance between methods was 0.69 and 0.63 for FF and FFPE, respectively. When looking specifically at the detection of 90 low expressing immune related genes, both assays were able to detect &gt;94% with an inter-platform correlation of 0.66 and 0.56 in FF and FFPE, respectively.
Whole transcriptome RNA-seq provides the maximum amount of information from clinical samples including anti-sense and non-coding RNA detection while RNA Access can be applied more cost-efficiently when one is mostly interested in the protein coding transcriptome.
Citation Format: Erica B. Schleifman, Anna Kiialainen, Andreas Roller, Sabine Bader, Maipelo Motlhabi, Priti Hegde, Ian McCaffery, Garret Hampton, Michael Cannarile, Craig Cummings, Olivia Spleiss, Eric Peters. Whole transcriptome and exome targeted RNA sequencing for FFPE tumor samples from clinical trials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3631.