Abstract 731: Deciphering the origin of circulating cell-free DNA and its alterations during menstruation
Authors: Yuwono Nicole Laurencia , Warton Kristina , Henry Claire , Ford Caroline 
Published: September 2020

Abstract
Background and aim
Tumor derived cell-free DNA (cfDNA) in blood is a promising biomarker for cancer. Understanding factors that influence cfDNA concentrations and tissues of origins will assist with cancer biomarker development by excluding cfDNA changes driven by normal physiological processes. Endothelial cells make up 10% of the plasma cfDNA pool and undergo angiogenesis in the endometrium during the recurrent cycles of menstruation. Therefore, endothelial cfDNA changes are a potential source of false positive signals in cancer screening of pre-menopausal women. The aim of this study is to determine if cfDNA derived from the endothelium is increased during menstruation.
Methods
80 matched blood samples were collected from 40 healthy female donors at non-menstruating (NM) and menstruating (M) phases. CfDNA was extracted from 5 mL of plasma and quantified by qPCR by targeting a short (ALU115) and long region (ALU247) of ALU sequences. Methylation specific primers were designed to interrogate VE-cadherin DNA region at sequences downstream of the transcriptional start site (from +122 bp to +218 bp) to generate a 96 bp amplicon. The primer was tested against human primary aortic and blood-brain barrier endothelial cells as positive controls. To detect and measure endothelium derived sequences, cfDNA equivalent of 4 mL of plasma was bisulfite converted for quantification of endothelial specific VE-cadherin region by qPCR.
Results
VE-cadherin was identified from the literature and selected as a gene with methylation unique to endothelial cells. The VE-cadherin primer, designed specifically to amplify unmethylated version of the region, was tested against bisulfite-converted human aortic and blood-brain barrier genomic DNA with detection levels down to 111 pg and 125 pg input, respectively.
There was no statistically significant change in total cfDNA amount during menstruation and, based on the ratio of ALU115 and ALU247, no change in the integrity and size distribution of cfDNA was observed. We also found no correlation in total cfDNA amount between the NM and M phases. However, there was a positive correlation in the size ratio (r = 0.53), meaning that while individual cfDNA levels fluctuate over time, the size distribution stays relatively similar. Additionally, no significant changes were observed in the endothelium-derived cfDNA concentration between NM and M phases in the 40 matched plasma, including when the proportion is adjusted against the total cfDNA concentration. Positive correlation was also found between NM and M samples in the endothelium-derived cfDNA levels (r = 0.4).
Conclusion
This absence of change suggests a specific release pathway for cfDNA to enter the bloodstream rather than simply inefficiency in apoptotic debris clean-up and provides evidence that women can be screened for early detection of ovarian cancer using tests targeting cfDNA methylation regardless of their menstrual status.
Citation Format: Nicole Laurencia Yuwono, Kristina Warton, Claire Henry, Caroline Ford. Deciphering the origin of circulating cell-free DNA and its alterations during menstruation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 731.