A Prospective Study Tracking Longitudinal Changes In Genome-Wide Cell-Free Dna (Cfdna) Methylation To Identify Early Nonresponders To Cancer Treatment.

3042 Background: Methylation is an epigenetic modification linked to cancer pathogenesis. The aim was to determine if changes in cfDNA methylation patterns before and after initiation of treatment could predict non-response to treatment prior to routine imaging and clinical follow-up. Methods: We prospectively collected clinical data and blood from 28 patients with metastatic malignancies (13 lung, 11 breast, 4 other). Blood was drawn prior to start of a new treatment, after first cycle (median 30 days), and/or second cycle (median 57 days). We performed whole-genome (WG) bisulfite sequencing (median depth 18X) on plasma cfDNA to determine methylation levels. By tracking how methylation levels deviate from unaffected individuals, from baseline to subsequent timepoints, we classified patients as either progressors (greater deviance) or non-progressors. Treatment response at first follow-up imaging (FUI) was determined by RECIST 1.1. Study endpoints were agreement with first FUI and progression-free survival (PFS) by cfDNA classification. Results: The cohort consisted of 68% females and the median age was 70. Main treatment regimens were chemo- (N = 12), immuno- (6), endocrine (5), or targeted-therapy (5). PFS was significantly shorter (log-rank p = 8 x 10-7) in patients classified as progressors by cfDNA (N = 8; median: 62 days) compared to non-progressors (N = 20, median: 263 days). For patients classified as progressors, the cfDNA assay preceded imaging and clinical evaluation by a median of 34 days. 7 out of 8 patients classified as cfDNA progressors were later confirmed to progress at first follow-up evaluation (88% positive predictive value). The one patient who was classified as progressor based on cfDNA was stable based on FUI (day 93 of treatment) but was later confirmed as progression on day 128 by FUI. For the remaining patients, 18 of 20 did not progress (90% negative predictive value). Thus, sensitivity for the assay for identifying progression was 78% and specificity was 95%. Conclusions: Our results show that WG cfDNA methylation change is a novel signature with potential to identify patients whose treatment regimen is ineffective prior to imaging.