Publication-only abstracts (abstract number preceded by an "e"), published in conjunction with the 2019 ASCO Annual Meeting but not presented at the Meeting, can be found online only.
SeqPlus sequencing methodology enables robust whole-genome sequencing, true variant detection, and novel genomic insights from archival esophageal carcinoma FFPE samples.
Cancer Prevention, Hereditary Genetics, and Epidemiology
2019 ASCO Annual Meeting
J Clin Oncol 37, 2019 (suppl; abstr e13016)
Author(s): Shannon Terrell Bailey, Belynda Hicks, Bin Zhu, Nan Hu, Phil R. Taylor, Jeffery R Gulcher, Hongye Sun, Jim Lund, Kelly Oliner, Richard T. Williams, Stephen J. Chanock, Alisa M. Goldstein; WuXi NextCODE Genomics, Cambridge, MA; Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD; National Cancer Institute DCEG, Biostatistics Branch, Bethesda, MD; National Cancer Institute, Bethesda, MD; National Cancer Institute, National Institutes of Health, Bethesda, MD; National Cancer Institute Division of Cancer Epidemiology & Genetics, Clinical Genetics Branch, Bethesda, MD
Background: Whole-genome sequencing (WGS) of formalin-fixed, paraffin-embedded (FFPE) samples could enable novel insights from archival sample collections, yet robust FFPE WGS is challenged by fragmented DNA, uneven genomic coverage & sequencing artifacts attributed to FFPE fixation. We report our proprietary extraction & library preparation methodology (SeqPlus) with high quality, uniform WGS sequencing performance comparable to that from fresh-frozen samples. Methods: We analyzed 20 paired esophageal carcinoma (EC) samples i.e., primary tumors & matched germline samples to assess SeqPlus performance on 10-15-year-old FFPE tissues, measure variant concordance between WGS and a high-depth sequencing panel (269 genes, 400x coverage) & identify novel genomic features. Results: At a targeted 70x WGS tumor sequencing depth, 93% of the genome was covered by ³ 20 reads, 99% of bases had 10x coverage & average duplicate reads were 31%. We noted similar transition/transversion ratios & mutational spectra as from fresh-frozen EC specimens, suggesting that extraction & library preparation contributes to prior FFPE artifacts. Concordance of tumor-specific SNVs & indels derived from WGS & targeted panel was high at 86%. All 76 targeted panel-detected variants above the WGS limit of detection (mutant allele frequency [MAF] > 10%) were detected by WGS, 2 variants (2 tumors) were detected only by WGS, and 12 variants at MAF ≤ 6% (9 tumors) were only detected by the targeted panel. Tumor WGS yielded SNV, indels & CNV findings beyond variants detected by targeted sequencing. WGS enabled detection of 10.4 putative cancer variants per tumor compared to 12 variants per patient from frozen specimens and a median of 7 (up to 16) cancer-associated variants in genes outside the targeted panel. WGS copy number analysis revealed CCND1, EGFR, TP63, and SOX2amplification, CDKN2A/B deletion and additional unrecognized genomic aberrations. Conclusions: Our study reinforces the utility of high-quality, uniform WGS sequencing of archival FFPE cancer samples with SeqPlus and unlocks the potential for massive-scale retrospective genomic analysis of archived pathology samples with associated clinical & outcomes data.