Molecular residual disease assessment in colorectal and bladder cancer by somatic structural variant analysis of cell-free DNA whole-genome sequencing data

Whole-genome sequencing (WGS)-based methods for circulating tumor DNA (ctDNA) detection typically rely on tumor-informed identification of somatic single nucleotide variants (SNVs). Somatic structural variants (SVs) are another type of cancer-specific genomic alteration, which owing to their larger genomic footprint and unique breakpoint junctions, are easier to distinguish from sequencing noise than SNVs. They are, however, rarely used for ctDNA detection because of (1) artifacts from WGS procedures that SV callers may falsely interpret as genuine SVs. This makes it difficult to establish high-confidence SV catalogos from short-read tumor WGS and can cause false-positive ctDNA detections. (2) Lack of robust strategies to quantify SV-supporting reads in plasma WGS. To address these barriers and enable integration of SV biomarkers into WGS-based ctDNA detection, we present a bioinformatic framework for algorithmic curation of somatic SV calls from fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tumors, coupled with a novel approach for sensitive, accurate mapping and quantification of SV breakpoint-supporting reads in plasma WGS. Tumor, normal and plasma WGS data from 144 patients with stage III colorectal cancer was used to establish the bioinformatic framework. This included ~30x WGS data from 1564 serially collected plasma samples. The framework was validated using tumor/normal/plasma WGS data from 32 patients with muscle-invasive bladder cancer. SV-based ctDNA detection was benchmarked against previously published SNV-based ctDNA results for the same samples. After curation of SV calls and quantification in plasma WGS, our SV-based approach enabled robust ctDNA detection with overall specificity exceeding 99% in plasma samples. Furthermore, we observed strong concordance (Pearson’s r > 0.93, p < 2.2 × 10 − 16 ) between ctDNA-positive samples identified by our SV-based method and previous SNV-based analyses, validating the reliability of our approach. Finally, we demonstrated application of the method in an independent bladder cancer cohort, highlighting its generalizability and potential clinical use. We provide a bioinformatic framework that establishes somatic SVs as ultra-specific biomarkers for WGS-based, tumor-informed ctDNA detection. The approach delivers specific detection even when the SV catalogos are established from FFPE samples. The SV framework can stand alone or enhance SNV-based analysis pipelines.