Abstract 1973: A novel method for highly sensitive, facile and inexpensive quantitative assessment of aberrant DNA methylation in liquid biopsies
Authors: Nomura Sachio , Kobayashi Toshiyuki , Sugimoto Kiichi , Arai Masami , Pisanic Thomas R. , Momose Hirotaka , Sakamoto Kazuhiro , Hino Okio 
Published: September 2020

Abstract
Introduction:
There remains a considerable need for the development of new, simple methods for assessing methylation of cell-free DNA obtained from liquid biopsies. In particular, methods that can achieve highly-sensitive quantitative analysis are needed to enable estimation of tumor burden, invasiveness and recurrence. While standard methylation-sensitive high resolution melt (MS-HRM) analysis methods allow for semi-quantitative assessment of DNA methylation, achieving sufficient sensitivity and accuracy to enable reliable detection of rare circulating tumor DNA (ctDNA) in a large background of predominantly healthy cfDNA remains challenging. We developed a novel method that drastically improves the analytical sensitivity of HRM analysis in order to quantify epiallelic fractions as low as 0.001%. This method was named HiQASP (Highly Quantitative Allele Specific PCR). We demonstrate use of HiQASP for ultra-high-sensitive and quantitative methylation assessment of the promoter region of SEPT9, which is currently used in colorectal cancer screening kits. Here, we utilize this method to assess SEPT9 methylation in cfDNA derived from with liquid biopsies of colorectal cancer patients.
Methods:
All human blood samples were obtained with patient's consent and relevant IRB approval. The plasma specimens were separated with twice centrifugation and cfDNA purified with Qiagen's QIAamp Circulating Nucleic Acid Kit. Bisulfite conversion was performed using Zymo EZ DNA Methylation-Lightning kit according to the manufacturer's instructions. Plasma DNA concentrations were calculated using DX-11Fx (DeNovix) with QuantiFluor (Promega). Analytic specificities and sample epiallelic quantification were determined using synthetic oligonucleotide standards for the bisulfite-converted sequence of methylated and unmethylated SEPT9 were obtained from IDT and GenScript, respectively. Validation of the SEPT9 HiQASP assay was performed using serial dilutions of synthetic oligonucleotides. EpiTaq HS (TaKaRa) and EvaGreen (BioTium) were used to amplify and detect the PCR products, respectively. The equipment QuantStudio 3 (ThermoFisher) was used to amplify and execute HRM analysis.
Results:
Custom HiQASP primers were designed to maximize assay analytical sensitivity and specificity for methylation analysis at the SEPT9 locus. Overall, the SEPT9 HiQASP assay achieved an analytical sensitivity of 0.0005% for both bisulfite-converted unmethylated and methylated synthetic oligonucleotide targets. The entire assay could be performed in only 45 minutes. The potential clinical utility of the assay was also demonstrated using cfDNA derived from of a plasma sample obtained from a colorectal cancer patient. HiQASP analysis indicated the percentage of methylated SEPT9 in this specimen to be 3.8% yielding a total copy concentration of 35 copies of methylated SEPT9 per ml of plasma.
Conclusion:
HiQASP is a highly-sensitive locus-specific assay for facile, rapid and low-cost quantitative assessment of methylated DNA in challenging specimens such as liquid biopsies.
Citation Format: Sachio Nomura, Toshiyuki Kobayashi, Kiichi Sugimoto, Masami Arai, Thomas R. Pisanic, Hirotaka Momose, Kazuhiro Sakamoto, Okio Hino. A novel method for highly sensitive, facile and inexpensive quantitative assessment of aberrant DNA methylation in liquid biopsies [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 1973.