Impact of panel size on minimum residual disease (MRD) assay performance.

Abstract

e15022 Background: Sequencing circulating tumor DNA is a promising method for monitoring cancer treatment response and detecting recurrence, but sensitivity at the low tumor fractions typical of early-stage, post-treatment, and early recurrent tumors is limited by the small number of variants targeted in commercially available assays. Methods: We developed a tumor-informed minimum residual disease (MRD) assay using tumor-normal whole-genome sequencing (WGS) followed by interrogation of select somatic variants in cell-free DNA (cfDNA). We describe theoretical limits on panel designability as a function of tumor purity and sequencing depth using simulations and a discovery cohort of 31 patient samples. cfDNA assay performance was tested by creating serial dilutions of patient plasma before somatic enrichment and sequencing. Simulations and in-silico downsampling of target sites were used to test sensitivity as a function of panel size. A bioinformatics pipeline incorporating somatic calling, copy number inference, error assessment, and tumor-fraction estimation was developed to analyze results. Results: Across a range of tissues, tumor-normal WGS identified from hundreds to tens of thousands of somatic variants to inform an MRD capture panel. 97% of samples tested returned at least 300 high-confidence somatic variants, and targeted sequencing demonstrated over 95% positive predictive value in somatic variant calls. In cfDNA dilution experiments, we observed > 99% sensitivity at a tumor fraction of 0.01%. Expanding the panel size from 16 to 300 target variants increased sensitivity at 0.005% tumor fraction from approximately 20% to > 95%. Our tumor fraction model returned accurate quantification down to the parts per hundred thousand (0.001%) range, even when only a single tumor molecule could be observed at each target site. Conclusions: Increasing the number of targeted variants in an MRD assay increases sensitivity at low tumor fractions, and WGS-driven panel design ensures a sufficient number of interrogated variants while minimizing the failure rate due to panel designability. High sensitivity MRD has the potential to enable earlier recurrence detection while also giving researchers and clinicians new tools for monitoring patient treatment responses.