Abstract POSTER-TECH-1109: Robust gene expression and mutation analyses from RNA-sequencing of formalin-fixed diagnostic tumor samples
Authors: Graw Stefan , Meier Richard , Minn Kay , Godwin Andrew K , Beyerlein Peter , Chien Jeremy 
Published: August 2015

Abstract
Formalin-Fixed Paraffin Embedding (FFPE) has been the standard sample preparation for pathologists for decades. This created a mass of disease and normal tissue biospecimens with rich clinical annotation and patient follow-up data. If these FFPE archives proved useful for the next-generation sequence analyses, FFPE samples may be sequenced to investigate the complex genetic changes underlying tumor progression, therapy resistance and variability in disease outcome. Previous studies have successfully used FFPE-DNA for copy number analysis and mutation detection even though it is technically difficult to perform sequencing analyses on DNA isolated from FFPE samples. It is even more challenging to perform RNA sequence analysis because routine FFPE processing does not preserve high quality RNA.
Aim of this study is to determine the extent to which FFPE RNA expression is correlated with matched frozen tumor samples.
Six pairs of matched fresh frozen (FF) and FFPE ovarian tumor samples were processed for RNA sequence analysis. Multiple quality checks were performed before and after aligning the reads to the reference genome (hg19/GRCh37). Differential gene expression analysis was performed utilizing DESeq on aligned RNA-sequencing data, as well as data obtained by NanoString technology (250 selected genes). Both results were compared to each other. A pipeline called SNPiR was applied to detect mutation from the RNA sequencing data, and the mutation calls between the FF and FFPE samples were compared at the RNA and DNA level. For each analysis FF samples from the same cancer patients were used to verify the results from FFPE sample.
The quality checks indicate that FFPE samples show uniform coverage of 5’ and 3’ of the transcripts whereas FF samples show 3’ bias in transcript coverage. This difference is likely attributable to differences in library preparation steps for FF and FFPE samples. The sequencing libraries for FF samples were done by poly-A selection while the libraries for FFPE samples were prepared using Ribo- Minus RNA sequencing kit. The differential gene expression analysis showed a good correlation between each matching FF and FFPE samples with one exception. Analyzing the NanoString gene counts show not only a remarkable correlation between the matching FF and FFPE sample but also a correlation between FF and FFPE samples between the two techniques (RNA-sequencing and NanoString). Unfortunately the mutation calling show variable results with 3 paired samples showing high rates of concordant mutation calls between FF and FFPE samples while the remaining 3 paired samples showing low rates of concordant mutation calls. Ongoing studies are focusing on biological and technical variability that may account for the observed differences in concordant calls.
In summary, the results indicate that FFPE samples are a suitable replacement for FF samples in differential gene expression analysis. However, mutation analysis from FFPE samples is challenging and results are variable.
Citation Format: Stefan Graw, Richard Meier, Kay Minn, Andrew K Godwin, Peter Beyerlein, Jeremy Chien. Robust gene expression and mutation analyses from RNA-sequencing of formalin-fixed diagnostic tumor samples [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1109.