Abstract 5925: Circulating cell-free methylated tumor DNA measurements correlate with plasma VAF-based tumor fraction estimates

Abstract Introduction: Cell-free DNA (cfDNA) consists of circulating tumor DNA (ctDNA) and DNA shed by non-tumor cells such as immune and endothelial cells. Tumor fraction (TF) is the proportion of ctDNA present in cfDNA. TF is often estimated by summary statistics on somatic variants such as the mean or maximum variant allele fraction (VAF). The Northstar Response (NSR) assay quantifies ctDNA by accurately and precisely counting methylated cfDNA molecules across over 500 loci uniquely methylated in tumor tissue but not in any non-cancerous cells. We hypothesize that the NSR quantification of plasma methylated DNA, via the Tumor Methylation Score (TMS), correlates with tumor fraction as estimated by VAF. Methods: A pan-cancer cohort of 271 longitudinal blood samples across 74 patients was gathered and run on the Northstar Select (NSS), an 84-gene liquid biopsy panel for somatic variant detection, and NSR assays. Samples were required to have positive detection in both assays to ensure precise TF measurements (max(VAF)>0.01%, TMS>25 methylated molecules). Tumor fractions were estimated by removing germline variants and evaluating the max(VAF) and sum(VAF), with the latter measurement paralleling the summation of methylated molecules used by the TMS. Due to the high variability of tumor mutational burden (TMB) and tumor methylation levels between tumors, correlations between VAF and TMS were calculated on a per-patient basis for patients with at least 3 longitudinal samples. Results: TMS ranged from 25 to 253k molecules with a mean of 16k; sum(VAF) ranged from 0.06 to 782 with a mean of 67; max(VAF) ranged from 0.06% to 100% with a mean of 34%. TMS correlated most strongly with sum(VAF) with mean(R2)=0.77, median(R2)=0.96, whereas for max(VAF), mean(R2)=0.67, median(R2)=0.91. A linear regression model between max(VAF) and TMS demonstrated a mean increase of 1.3k methylated molecules per unit percent increase in max(VAF). Conclusions: Previous studies have demonstrated that VAF decreases are prognostic to targeted therapy response including immune checkpoint blockade and tyrosine kinase inhibitors. However, VAF-based tumor burden measurements are limited by the paucity of somatic variants. In contrast, aberrant DNA methylation is a well-known hallmark of cancer that is an abundant source of ctDNA. The strong, linear correlation between TMS and VAF shown here suggests that TMS is an effective measure of ctDNA burden and can similarly be used to monitor response to therapy. Furthermore, the large amount of methylated DNA in patient samples (mean=16k molecules) suggests that epigenetic approaches can be highly sensitive even at very low tumor fraction (<1%). Citation Format: Lee Davis McDaniel, David Tsao. Circulating cell-free methylated tumor DNA measurements correlate with plasma VAF-based tumor fraction estimates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5925.