Analytical Validation of a Circulating Tumor DNA Assay using PhasED-Seq Technology for Detecting Residual Disease in B-Cell Malignancies

BackgroundCirculating tumor DNA (ctDNA) is a non-invasive biomarker that can be used as a tool to detect minimal residual disease (MRD). MRD can provide important prognostic information in diffuse large B-cell lymphomas (DLBCL). Here, we present an MRD assay with an improved detection method for ctDNA, Phased Variant Enrichment and Detection Sequencing (PhasED-Seq) which leverages phased variants (PVs) to detect ctDNA. MethodsPlasma samples from non-cancer controls were used to assess assay specificity. A limiting dilution series using DLBCL clinical-contrived samples was performed to assess assay sensitivity and precision. The accuracy of the PhasED-Seq-based assay was assessed using plasma samples from individuals with DLBCL and for whom MRD comparator assay results were also available. All samples were sourced from commercial vendors or academic studies. ResultsThe analytical and clinical performance of the MRD assay was evaluated using clinical and clinical-contrived DLBCL samples. The assays false positive rate was 0.24% and the background error rate was 1.95E-08. The limit of detection at 95% detection rate (LoD95) at 120 ng was 0.7 parts in 1,000,000 and precision was >96%. Clinical accuracy was 90.62% PPA and 77.78% NPA. ConclusionsThe PhasED-Seq-based MRD assay has strong analytical and clinical performance in B-cell dyscrasias. Through the development of improved ctDNA detection methods such as that presented here, patient outcomes may be improved through the detection of residual disease or early relapse which may be used to guide treatment decisions. Brief SummaryHere we present the analytical validation of a non-invasive minimal residual disease (MRD) assay which uses Phased Variant Enrichment and Detection Sequencing (PhasED-Seq) to improve the error profile and sensitivity of circulating tumor DNA (ctDNA) detection. The assays performance included a false positive rate of 0.24% and a background error rate of 1.95E-08. The limit of detection at 120 ng was 0.7 parts in 1,000,000 (6.61E-07 PVAF) with precision >96%. Positive and negative agreement were 90.62% and 77.78%, respectively. This suggests that the PhasED-Seq-based MRD assay is accurate and reproducible, thus appropriate for clinical use for individuals with B-cell malignancies.