Genomic analysis of free circulating DNA (fcDNA) has a number of potential clinical applications, including cancer studies. Tumor-derived fcDNA harbors the same molecular aberrations, including mutations and methylation, as the derivative tumor. As it can be collected by non-invasive means, fcDNA is particularly promising as a cancer detection tool. In addition, molecular characterization of fcDNA in cancer patients is holds promise for determining tumor sub-types and monitoring response to treatment. However, because fcDNA concentrations are relatively low in the circulation, whole-genome analysis for molecular characterization is quite challenging. Standardization of fcDNA collection and processing for downstream analysis is imperative to help address these challenges.
Our studies focus on the development of an early detection test for high-grade serous ovarian cancer (HGSOC), using whole-genome methylation analysis in fcDNA from HGSOC patients. DNA methylation plays a key role in the development of many cancer types, and thus carries great potential as a cancer diagnostic biomarker. We propose that DNA methylation changes in HGSOC tumors can be detected in the patient's fcDNA and can be used as a blood-based test to detect HGSOC. We have carried out Next Generation Sequencing (NGS) of methylation-enriched fcDNA from 3 HGSOC patients and 5 healthy controls with the aim of identifying a list of differentially methylated loci in fcDNA that can distinguish between HGSOC and control plasma.
We also sought to generate standard protocols for collection and processing of fcDNA for whole-genome studies. First we analyzed effects of blood storage time on fcDNA recovery and quality and determined that storage for ≥8 hrs prior to plasma separation leads to increased DNA concentrations, as well as an appearance of a band that co-migrates with high-molecular weight genomic DNA. This increased DNA concentration likely represents genomic DNA contamination as a result of leukocytes lysis during storage.
We next analyzed the effects of processing and scaling on enriched fcDNA recovery and quality. We used a methyl-binding protein-based (MBD2) protocol to enrich for methylated sequences in fcDNA isolated from plasma samples. Following application of a modified protocol due to low concentrations of fcDNA in plasma, we obtained a 10.2-14.9% enrichment of methylated fragments. In preparing the sample libraries for NGS, the predominant fcDNA size of ~180 base pairs allows minimal sample loss during the size restriction step. NGS analysis provided 37-86x10^6 unique mappable reads per fcDNA sample, representing >50% of the total mappable reads. These read counts indicate a satisfactory level of library complexity was achieved from minimal fcDNA input.
Our standardization protocols provide the ability to comprehensively sequence patient fcDNA using half of the recommended sample input. This optimized approach allows in-depth whole genome characterization and comparison of fcDNA isolated from HGSOC and healthy controls to develop diagnostic tools and investigate biological mechanisms driving tumor progression.
Citation Format: Kristina Warton, Lin Vita, Nicola J. Armstrong, Warren Kaplan, Kevin Ying, Helena Mangs, Neville F. Hacker, Robert L. Sutherland, Susan J. Clark, Goli Samimi. Whole genome sequencing of free circulating DNA from plasma of HGSOC patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A4.