Abstract 3763: Detection of tumor-associated gene inactivation in clinical blood draws via cell-free DNA methylation profiling
Authors: Chen Sai , Zhang Shile , Jiang Tingting , Yen Jennifer , He Yupeng , Jaimovich Ariel , Kim Yvonne , Ma Dustin , Tran Giao , Gaile Daniel P. , Nagy Rebecca J. , Helman Elena , Chuang Han-Yu 
Published: June 2022

Abstract
Introduction: The ability to detect genome-wide epigenetic changes, such as DNA methylation, has expanded translational applications in oncology settings. Because these changes occur early in carcinogenesis, they can be used for early cancer detection when genomic technologies fall short due to lower sensitivity, and in the early and late-stage cancer setting for minimal residual disease detection, disease monitoring and therapy selection. In this analysis, we demonstrated our detection of differential methylations that classify cancer from healthy normals, as well as the quantification of promoter methylation, using a highly sensitive targeted assay that simultaneously captures both genomic alterations and methylation signatures in cell-free DNA (cfDNA).
Methods: Methylation signals were profiled with a broad genomic panel (15.2 Mb) targeting regions that are unmethylated in plasma cfDNA from cancer-free donors. The panel covers the promoter regions of 925 out of 1,217 known tumor suppressor genes (TSGs) (e.g. TP53, APC, RB1, PTEN), homologous recombination and repair (HRR) genes (e.g. ATM, BRCA1/2, CDK12, RAD51C/D). We applied our genomic and epigenomic assay on cfDNA from 1,968 colorectal cancer (CRC) patients, 480 patients with other 6 common cancers, and 2,037 cancer-free donors. To test the sensitivity of our epigenomic assay, we generated an in-silico dataset by computationally mixing reads from the cancer patients with those from cancer-free donors at a low tumor fraction (TF) of 0.1%.
Results: Among 62 clinically relevant TSG and HRR genes, 56 (90%) were differentially methylated (Wilcoxon p&lt;0.05) between the 2,448 cancer vs. 2,037 cancer-free donor samples. In the in-silico dataset, 51 of the 56 differentially methylated genes remained statistically significant (Wilcoxon p&lt;0.05) at 0.1% TF. In the 1,968 CRC patients, we observed a significant association (Fisher’s p&lt;1e-05) between MLH1 promoter methylation and microsatellite instability (MSI-H): 70% of MSI-H samples has MLH1 promoter methylation above a predefined “high methylation” threshold, while only 7% of microsatellite-stable samples has MLH1 promoter methylation above this threshold. Our result is consistent with previous studies that 54-100% of CRC patients with MSI-H tumors harbor MLH1 promoter methylation.
Conclusion: We demonstrate that our assay can accurately detect cancer-driven DNA methylation across the genome in clinical plasma samples. Detection of differential methylation in cancerous versus non-cancerous tissues could allow for early cancer detection, leading to better survival and prediction of recurrence prior to imaging. The highly sensitive detection of promoter methylation shown with TSG, HRR, and MLH1 genes may provide orthogonal information to oncologists in therapeutic selection with high confidence.
Citation Format: Sai Chen, Shile Zhang, Tingting Jiang, Jennifer Yen, Yupeng He, Ariel Jaimovich, Yvonne Kim, Dustin Ma, Giao Tran, Daniel P. Gaile, Rebecca J. Nagy, Elena Helman, Han-Yu Chuang. Detection of tumor-associated gene inactivation in clinical blood draws via cell-free DNA methylation profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3763.