A Novel Tissue-Free Method to Estimate Tumor-Derived Cell-Free DNA Quantity Using Tumor Methylation Patterns.

Estimating the abundance of cell-free DNA (cfDNA) fragments shed from a tumor (i.e., circulating tumor DNA (ctDNA)) can approximate tumor burden, which has numerous clinical applications. We derived a novel, broadly applicable statistical method to quantify cancer-indicative methylation patterns within cfDNA to estimate ctDNA abundance, even at low levels. Our algorithm identified differentially methylated regions (DMRs) between a reference database of cancer tissue biopsy samples and cfDNA from individuals without cancer. Then, without utilizing matched tissue biopsy, counts of fragments matching the cancer-indicative hyper/hypo-methylated patterns within DMRs were used to determine a tumor methylated fraction (TMeF; a methylation-based quantification of the circulating tumor allele fraction and estimate of ctDNA abundance) for plasma samples. TMeF and small variant allele fraction (SVAF) estimates of the same cancer plasma samples were correlated (Spearman's correlation coefficient: 0.73), and synthetic dilutions to expected TMeF of 10-3 and 10-4 had estimated TMeF within two-fold for 95% and 77% of samples, respectively. TMeF increased with cancer stage and tumor size and inversely correlated with survival probability. Therefore, tumor-derived fragments in the cfDNA of patients with cancer can be leveraged to estimate ctDNA abundance without the need for a tumor biopsy, which may provide non-invasive clinical approximations of tumor burden.

Evaluation of cell-free DNA approaches for multi-cancer early detection.

In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.