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