Abstract 2801: Detection of biallelic loss of DNA repair genes in formalin-fixed, paraffin embedded (FFPE) tumor samples using a novel tumor-only sequencing panel with error correction
Authors: Glodzik Dominik , Selencia Pier , Rogge Ryan , Silverman Ian M. , Zinda Michael , Koehler Maria , Daber Robert D. , Johnson Verity , Reis-Filho Jorge S. , Rimkunas Victoria 
Published: June 2022

Abstract
Background: Loss-of-function (LOF) mutations in DNA damage response (DDR) tumor suppressor genes are compensated for by functional redundancies, exposing synthetic lethal (SL) interactions and opportunities for targeted therapy. Patient selection for SL-based therapy may be improved by assessment of gene-specific loss of heterozygosity (LOH) and biallelic LOF, neither of which is routinely reported by existing targeted sequencing panels. The Synthetic Lethal Interactions for Precision Diagnostics (SNiPDx) targeted sequencing panel features a novel bioinformatic analysis pipeline that enables accurate genome-wide determination of allele-specific copy number, estimation of tumor ploidy and purity, and detection of single nucleotide and indel variants in target genes focused on DDR pathways, all from tumor-only samples. Here we describe the development and accuracy of SNiPDx for detection of LOH and bi-allelic LOF genetic alterations in FFPE samples.
Methods: Genomic DNA (&gt;50 ng) was extracted from FFPE samples of multiple solid tumor types (n = 43). Next-generation sequencing was performed on anchored multiplex PCR libraries, constructed using probes that incorporate unique molecular identifiers and span 26 genes and 5,000 genome-wide common germline single-nucleotide polymorphisms (SNPs). Unmatched non-tumor samples (n = 24) were used to generate a reference baseline dataset. The FACETS algorithm, optimized to account for differential DNA fragmentation across samples, was used to assess copy number imbalance in heterozygous SNPs and to quantify tumor purity. Allele fractions at each heterozygous SNP were used to estimate allelic imbalances across chromosomal regions. A reference dataset was derived from matched FFPE tumor samples by whole genome sequencing (WGS) and analysis of sequence data using 3 complementary algorithms. Allele-specific copy number analysis and tumor purity estimation from SNiPDx and WGS data were compared.
Results: Copy number was evaluable in 605 genes from 24 matched tumor samples that passed quality control filters. Median sequencing depth across samples by SNiPDx and WGS were 1346x and 18.6x, respectively. LOH detection by SNiPDx was reproducible (100%) across 170 genes from 7 samples sequenced and analyzed in duplicate. A strong correlation was observed between sample purity estimates by WGS and SNiPDx (Pearson’s r = 0.81, p &lt; 0.001). Compared with WGS-derived calls, the sensitivity and specificity of LOH detection by SNiPDx were 95% and 90%, respectively, rising to 97% and 91% in regions with LOH agreement by all 3 WGS algorithms, and to 99% and 97% in diploid regions with no subclonal alterations.
b The SNiPDx panel is a novel clinical test for biallelic loss in FFPE tumor-only samples with high accuracy as validated through concordance with a WGS-derived dataset.
Citation Format: Dominik Glodzik, Pier Selencia, Ryan Rogge, Ian M. Silverman, Michael Zinda, Maria Koehler, Robert D. Daber, Verity Johnson, Jorge S. Reis-Filho, Victoria Rimkunas. Detection of biallelic loss of DNA repair genes in formalin-fixed, paraffin embedded (FFPE) tumor samples using a novel tumor-only sequencing panel with error correction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2801.