Purpose: We previously demonstrated that high levels of circulating methylated DNA are associated with subsequent disease progression in women with metastatic breast cancer (MBC). In this study, we evaluated the clinical utility of a novel Liquid Biopsy-Breast Cancer Methylation (LBx-BCM) prototype assay using the GeneXpert® cartridge system for early assessment of disease progression in MBC. Experimental Design: The 9-marker, LBx-BCM prototype assay was evaluated in TBCRC-005, a prospective biomarker study, using plasma collected at baseline, week 4 and week 8 from 144 MBC patients. Results: At week 4 MBC patients with high cumulative methylation (CM) had a significantly shorter median PFS (2.88 months v 6.60 months, p = 0.001) and OS (14.52 months v 22.44 months, p=0.005) compared to those with low CM. In a multivariable model, high versus low CM was also associated with shorter PFS (HR = 1.90, 95%CI 1.20-3.01; p =0.006). Change in CM from baseline to week 4 (OR = 4.60, 95%CI 1.77, 11.93; p = 0.002) and high levels of CM at week 4 (OR = 2.78, 95%CI 1.29, 5.99; p = 0.009) were associated with progressive disease at the time of first restaging. A robust risk model based on week 4 circulating CM levels was developed to predict disease progression as early as 3 months after initiating a new treatment. Conclusions: The automated LBx-BCM prototype assay is a promising clinical tool for detecting disease progress a month after initiating treatment in women with MBC undergoing routine care. The next step is validating its clinical utility for specific treatments.

<div>AbstractPurpose:<p>We previously demonstrated that high levels of circulating methylated DNA are associated with subsequent disease progression in women with metastatic breast cancer (MBC). In this study, we evaluated the clinical utility of a novel liquid biopsy-breast cancer methylation (LBx-BCM) prototype assay using the GeneXpert cartridge system for early assessment of disease progression in MBC.</p>Experimental Design:<p>The 9-marker LBx-BCM prototype assay was evaluated in TBCRC 005, a prospective biomarker study, using plasma collected at baseline, week 4, and week 8 from 144 patients with MBC.</p>Results:<p>At week 4, patients with MBC with high cumulative methylation (CM) had a significantly shorter median PFS (2.88 months vs. 6.60 months, <i>P</i> = 0.001) and OS (14.52 months vs. 22.44 months, <i>P</i> = 0.005) compared with those with low CM. In a multivariable model, high versus low CM was also associated with shorter PFS (HR, 1.90; 95% CI, 1.20–3.01; <i>P</i> = 0.006). Change in CM from baseline to week 4 (OR, 4.60; 95% CI, 1.77–11.93; <i>P</i> = 0.002) and high levels of CM at week 4 (OR, 2.78; 95% CI, 1.29–5.99; <i>P</i> = 0.009) were associated with progressive disease at the time of first restaging. A robust risk model based on week 4 circulating CM levels was developed to predict disease progression as early as 3 months after initiating a new treatment.</p>Conclusions:<p>The automated LBx-BCM prototype assay is a promising clinical tool for detecting disease progression a month after initiating treatment in women with MBC undergoing routine care. The next step is to validate its clinical utility for specific treatments.</p></div>

<p>Supplementary figure 5 displays time updated ROC curves for (A) CM level at week 8, (B) Change in CM level (Week8 – Week4) and (C) the Combination of week 8 CM level and Change in CM level (Week 8-Week 4) using linear discriminant analysis.</p>

<p>Study Schema</p>

<p>Supplementary Figure 3 displays baseline CM level by disease status at first restaging.</p>