Development of an Automated Liquid Biopsy Check for updates Assay for Methylated Markers in Advanced Breast Cancer

Abstract

Current molecular liquid biopsy assays to detect recurrence or moni- and the reference assay, cMethDNA, among 66 paired plasma samples tor response to treatment require sophisticated technology, highly trained [MBC N = 40, controls N = 26; Spearman r = 0.891; 95% confidence personnel, and a turnaround time of weeks. We describe the devel- interval (CI) = 0.825–0.933, P < 0.0001]. LBx-BCM achieved a ROC opment and technical validation of an automated Liquid Biopsy for AUC = 0.909 (95% CI = 0.836–0.982), 83% sensitivity and 92% specificity; Breast Cancer Methylation (LBx-BCM) prototype, a DNA methylation cMethDNA achieved a ROC AUC = 0.896 (95% CI = 0.817–0.974), 83% detection cartridge assay that is simple to perform and quantitatively de- sensitivity and 92% specificity in test set samples. The automated LBx-BCM tects nine methylated markers within 4.5 hours. LBx-BCM demonstrated cartridge prototype is fast, with performance levels equivalent to the highly high interassay reproducibility when analyzing exogenous methylated sensitive, manual cMethDNA method. Future prospective clinical stud-DNA (75–300 DNA copies) spiked into plasma (coefficient of variation, ies will evaluate LBx-BCM detection sensitivity and its ability to monitor CV = 7.1%–10.9%) and serum (CV = 19.1%–36.1%). It also demonstrated therapeutic response during treatment for advanced breast cancer. high interuser reproducibility (Spearman r = 0.887, P < 0.0001) when Significance: We technically validated an automated, cartridge-based, samples of metastatic breast cancer (MBC, N= 11) and normal control liquid biopsy prototype assay, to quantitatively measure breast cancer (N = 4) were evaluated independently by two users. Analyses of interplat- methylation in serum or plasma of patients with MBC, that demonstrated form reproducibility indicated very high concordance between LBx-BCM high sensitivity and specificity.