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

Simple Summary Tumor cells shed pieces of DNA that circulate in biofluids like blood and urine. The amount of circulating tumor DNA is related to the amount of cancer in the body. Knowing the amount of cancer helps doctors predict outcomes and decide treatments for patients. Tumor DNA has methylation patterns that makes it distinct from non-cancer DNA. In this study, we describe a new method called tumor methylated fraction (TMeF) that quantifies the cancer-indicative methylation patterns within circulating tumor DNA from blood samples. This method is non-invasive because it does not need tumor tissue to estimate the amount of cancer in the body. In the future, doctors could use TMeF to supplement current cancer screening methods.Abstract 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.