an Assay to Detect Hypomethylation in Circulating Cell Free DNA and Monitor Cancer Burden

Abstract Circulating, cell free DNA (cfDNA) is a non-invasive sample source that contains tumor associated DNA. Next generation sequencing (NGS) of cfDNA has shown that tumor specific mutations can be detected, providing an effective means to monitor disease, treatment efficacy, and characterize the genome wide methylation state of cfDNA. While hypermethylation is observed for specific gene promoters, on a genome wide perspective, hypomethylation of cfDNA is observed in cancer, making cfDNA an attractive target to assess cancer burden. The challenges in deep sequencing of cfDNA include: mandatory fast turnaround times due to sample degradation, limited sample material, short DNA fragments, and limit of detection issues caused by the presence of both normal and tumor DNA. This study describes a novel library preparation for the Illumina platforms that is cost-effective, sensitive, and specific to assess the methylation status of cfDNA. To characterize the methylation status of cfDNA, NGS libraries were generated utilizing a chemistry that sequentially ligates the adapters to each end of the DNA molecules. Since the library is generated after bisulfite treatment, a high recovery of DNA library molecules is observed, thus enabling high complexity library preparation from 5 ng of cfDNA. Upon establishment of this technique, we obtained cfDNA from healthy subjects as well as from subjects with a spectrum of cancers. Following bisulfite-conversion and library preparation of these samples, we sequenced each sample on the Illumina MiSeq to a depth of 10 million reads. A minimum threshold of 1.1% was used to determine significant hypomethylation. Upon analysis, preliminary analysis of the hypomethylation status of the cfDNA from 4 of the 5 cancer subjects ranged from 3% to 9% when compared to the healthy controls. The cfDNA sample which was negative for hypomethylation originated from the plasma of a subject with a high grade serous adenocarconima in the fallopian tube. The most hypomethylated cfDNA came from the plasma of subject with metastatic adenocarcinoma of the colon which had metastasized to the liver. Other tumor types which resulted in cfDNA hypomethylation between these two extremes included invasive breast carcinoma as well as pancreatic ductal carcinoma. This method provides the basis for a technique to reliably prepare and characterize cfDNA via NGS deep sequencing. We obtained results 5 days after resection and reliably made high complexity library using only 5 ng of cfDNA. We have demonstrated reproducibility and sensitivity for genome wide methylation status, while also observing that the utility of this assay may vary with cancer type. To further define biologically significant thresholds for methylation status, we will present a longitudinal study examining the cfDNA methylation status of individuals before, during, and after treatment, thereby generating a methylation gradient as a function of time and treatment. Citation Format: Cassie A. Schumacher, Sukhinder Sandhu, Vladimir Makarov. An assay to detect hypomethylation in circulating cell free DNA and monitor cancer burden. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1051. doi:10.1158/1538-7445.AM2015-1051