Analytical validation of a circulating tumor DNA assay using PhasED-Seq technology for detecting residual disease in B-cell malignancies.

BACKGROUND: Circulating tumor DNA (ctDNA) can be used as a tool to detect minimal residual disease (MRD) which can provide important prognostic information in diffuse large B-cell lymphomas (DLBCL). Here, we present an ultra-sensitive MRD assay reliant on Phased Variant Enrichment and Detection Sequencing (PhasED-Seq), which leverages phased variants to detect ctDNA. METHODS: Blank plasma samples were used to assess assay specificity and a limiting dilution series with a DLBCL clinical-contrived sample was performed to assess assay sensitivity and precision. DLBCL plasma patient samples with MRD comparator assay results were tested with PhasED-Seq technology to assess assay accuracy. RESULTS: The assay's false positive rate was 0.24% and the background error rate was 1.95E-08. The limit of detection at 95% detection rate with 120 ng of input DNA was 0.7 parts in 1,000,000 and precision was >96%. Positive percent agreement for the MRD assay was 90.62% (95% CI 74.98%, 98.02%) and negative percent agreement was 77.78% (95% CI 52.73, 93.59) using a single nucleotide variant-based method as reference. CONCLUSIONS: The PhasED-Seq-based MRD assay has strong analytical and clinical performance in B-cell malignancies. Improved ctDNA detection methods such as this may improve patient outcomes through detection of residual disease or early relapse.