Abstract 3154: Cell-free circulating tumor DNA methylation in high-grade serous ovarian cancer
Authors: Davis Kayleigh R. , Flower Kirsty J. , Borley Jane V. , Wilhelm-Benartzi Charlotte SM , Brown Robert 
Published: July 2016

Abstract
Background Ovarian cancer is often diagnosed late leading to poor patient survival. Over two thirds of women with ovarian cancer are diagnosed with Stages III/IV where the relative 5year survival is &lt;20%, compared to &gt;90% for Stage I. New strategies for early detection and patient stratification are urgently required, as well as non-invasive ways of monitoring tumour evolution. Circulating tumour DNA (ctDNA) can be detected in plasma from ovarian cancer patients. Genome-wide methylation analysis of tissue has identified tumour specific changes in DNA methylation which could be present in ctDNA. However, lysis of peripheral blood mononuclear cells (PBMCs) can release DNA into the plasma confounding detection of tumour DNA. If specific methylation changes that differentiate tumour DNA from PBMC DNA are identified, this would allow DNA methylation in ctDNA to be more accurately quantified.
Methods In this study, publically available genome-wide array-based DNA methylation datasets (Cancer Genome Atlas Research, 2011) (Fridley et al., 2014) were interrogated to identify regions that are highly methylated in high-grade serous ovarian carcinoma, (HGSOC), and hypomethylated in PBMCs and vice versa. The methylation status of these loci was then confirmed in an independent dataset of HGSOC and benign
tissue. Sodium heparin blood samples were collected from healthy volunteers and patients suspected of having HGSOC at Hammersmith Hospital, London. Blood samples were separated by low speed followed by high speed centrifugation, DNA extracted and analysed using bisulfite pyrosequencing.
Results Ten loci were identified from the publically available data that had &gt;80% methylation in HGSOC (n = 342) as well as &lt;10% methylation in PBMCs (n = 276). Conversely, 2 loci identified had &lt;10% methylation in HGSOC and &gt;80% methylation in PBMCs. Seven of these have been independently validated as differentially methylated by bisulphite pyrosequencing in HGSOC versus PBMCs from the Hammersmith patient cohort. Methylation can be detected using pyrosequencing using DNA amounts less than 10ng. For three loci analysed so far, we have shown significant differences in methylation between DNA isolated from plasma of ovarian cancer patients compared to healthy volunteers. One locus was capable of distinguishing ovarian cancer patients from healthy volunteers with 100% sensitivity and specificity.
Conclusions We have identified differentially methylated loci between HGSOC and PBMCs with high sensitivity and specificity. We have shown that these epigenetic changes can be detected in plasma ctDNA from patients with ovarian cancer.
References Cancer Genome Atlas Research Network., Integrated genomic analyses of ovarian carcinoma. Nature 2011, 474:7353 Fridley et al., Methylation of leukocyte DNA and ovarian cancer: relationships with disease status and outcome. BMC Med Genomics 2014, 7:21
Citation Format: Kayleigh R. Davis, Kirsty J. Flower, Jane V. Borley, Charlotte SM Wilhelm-Benartzi, Robert Brown. Cell-free circulating tumor DNA methylation in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3154.