As formalin-fixed, paraffin-embedded (FFPE) tissue is being utilized for next-generation sequencing (NGS) in research and clinical settings, we conducted a study through the Surveillance, Epidemiology and End Results (SEER)-Linked Virtual Tissue Repository (VTR) Pilot Program to determine the quality of sequencing data obtained using FFPE-derived DNA and RNA. Forty-eight pancreatic ductal adenocarcinoma (PDAC) patients, comprising 24 case-control pairs based on survival time (≥5 years [cases] vs <24 months [controls]), were selected. Participating SEER registries obtained selected diagnostic tissue blocks collected clinically and stored for 4-18 years. DNA and RNA were extracted from the FFPE specimens for 36 patients (18 pairs). Whole genome (WGS) and whole exome sequencing (WES) were performed on tumor and normal DNA from 16 patients, and a methylation array was conducted on tumor DNA from 6 of these patients. RNA-Seq was conducted on tumor RNA from 36 patients. The median coverage depths for tumor were above 300x for WES and 60x for WGS. However, the majority of sequencing reads (>60%) were duplicates. Concordant mutations (SNVs, MNVs and indels) were >50% by WGS and WES from the majority of samples (n=11, 69%), and the most common discordant mutations were C>T. On average, mutant allele frequencies (MAFs) were 20% in coding regions and 15% across the whole genome, consistent with tumor content as measured by methylation analysis for five tumor samples (18%, 22%, 28%, 42%, and 50%). WES and/or WGS revealed that specimens for five of 27 PDAC subjects tested had a high fraction of variants overlapping with germline variants in dbSNP (≥20%), indicating that tumor cellularity was low among these samples. TP53, KRAS, CDKN2A, SMAD4, and RNF43 were the most frequently mutated genes from these specimens, consistent with genes reported in studies using fresh frozen tissue. Point mutations comprised most of the gene variants, and indels were found in CDKN2A, SMAD4, and RNF43. Most of the mutation status (e.g. missense, nonsense or indels) were concordantly called by WES and WGS (e.g., 81% for TP53, 100% CDKN2A, 94% SMAD4, and 94% RNF43). Most discordant calls were mutations identified by WES but not WGS (e.g., 8 [50%] for KRAS and 3 [19%] TP53). All samples yielded RNA-Seq reads with <30% exonic mapping, 39% (14) of which had <10% exonic mapping. Our study provided important evidence for NGS applications on DNA and RNA from archival PDAC FFPE tissue specimens stored for up to 18 years. These findings demonstrate that, with sufficient tumor content and coverage depth, FFPE-derived DNA is adequate for identifying somatic driver gene mutations in PDAC patients and that the it is feasible to utilize the population-based, SEER-Linked VTR as an infrastructure for obtaining diagnostic tissue for molecular studies.
Citation Format: Yao Yuan, Alison Van Dyke, Valentina Petkov, Alyssa Tuan, Aatur Singhi, Lynn Matrisian, Lola Rahib, John Pearson, Katia Nones, Nicola Waddell, Yongmei Zhao, Tsai-wei Shen, Bao Tran, Jyoti Shetty, Elizabeth Gillanders, Danielle Carrick, Rosemary Cress, Lloyd Mueller, Brenda Hernandez, Charles Lynch, Thomas Tucker, Xiao-Cheng Wu, Lynne Penberthy. Evaluation of next generation sequencing of DNA and RNA from archival formalin-fixed, paraffin-embedded pancreatic cancer tissue: A pilot study of the SEER-linked virtual tissue repository [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 224.