Editor’s Highlight: Dose–Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded Samples

Abstract

Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here, we evaluated transcriptomic dose responses using RNA-sequencing in paired FFPE and frozen (FROZ) samples from 2 archival studies in mice, one <2 years old and the other >20 years old. Experimental treatments included 3 different doses of di(2-ethylhexyl)phthalate or dichloroacetic acid for the recently archived and older studies, respectively. Total RNA was ribo-depleted and sequenced using the Illumina HiSeq platform. In the recently archived study, FFPE samples had 35% lower total counts compared to FROZ samples but high concordance in fold-change values of differentially expressed genes (DEGs) (r =0.99), highly enriched pathways (90% overlap with FROZ), and benchmark dose estimates for preselected target genes (<5% difference vs FROZ). In contrast, older FFPE samples had markedly lower total counts (3% of FROZ) and poor concordance in global DEGs and pathways. However, counts from FFPE and FROZ samples still positively correlated (r = 0.84 across all transcripts) and showed comparable dose responses for more highly expressed target genes. These findings highlight potential applications and issues in using RNA-sequencing data from FFPE samples. Recently archived FFPE samples were highly similar to FROZ samples in sequencing quality metrics, DEG profiles, and dose-response parameters, while further methods development is needed for older lower-quality FFPE samples. This work should help advance the use of archival resources in chemical safety and translational science.

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“…Finally, in our study we used FFPE samples: this treatment is known to produce bias, as we directly observe in some of our libraries and probably affecting low expressed genes due to high rate of RNA degradation. However, studies have been conducted 76 proving that results on FFPE samples correlated with the one obtained with fresh tissue with some caveats.…”

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“…All of the samples were formalin-fixed, paraffin-embedded tissue samples from either the Small Animal Hospital of Zurich or external cases sent in by veterinarians practising in Switzerland. Cases were selected with the help of a board-certified veterinary pathologist (A.M.) using the following criteria; female dogs, simple mammary carcinomas, appropriate tumour stroma content, FFPE samples not older than two years [58], areas with no obvious or only negligible inflammation, and samples were paraffin-embedded on their arrival day at the Pathology, (i.e., no prolonged storage in formalin). During our initial screening for suitable cases, those which contained only highly inflamed stroma were excluded.…”

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“…Capturing and sequencing intronic DNA to identify gene fusions may be problematic because genomic breakpoints are broadly distributed within corresponding introns that frequently contain multiple repeat sequences . Although NGS analysis of transcriptomes (RNA‐seq) is an alternative approach, RNA‐seq using FFPE samples is challenging because the RNA recovered from FFPE is severely fragmented and modified . Although other approaches such as the cDNA capture method or anchored multiplex PCR‐based method were recently reported to be successful methods for RNA‐seq with FFPE samples, large‐scale validation for their clinical use is required.…”

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