Abstract 788: Novel multiplex methylation-specific qPCR strategy for early CRC detection

Abstract

Abstract Colorectal cancer (CRC) can be cured if detected early. Analysis of abnormalities in cfDNA methylation is an emerging and powerful strategy for early cancer detection. Recently, targeted methylation NGS methods have shown encouraging performance in CRC detection, but their high cost and complicated workflow can be challenging for many clinical settings. At the same time, the alternative, single methylation biomarker PCR assays, demonstrate limited sensitivity and specificity. An accurate, accessible and affordable assay ideal for clinical applications requires an easily scalable set-up and a set of high quality biomarkers providing sufficient predictive power. We aim to develop an assay fulfilling the CMS definitive performance guideline of LBx CRC screening – at least 74% sensitivity and 90% specificity. Here we report the development of a novel multiplex methylation-specific qPCR (MMS-qPCR) strategy for early CRC detection. Previously, we established detailed methylation profiles of our proprietary CRC targeted methylation sequencing panel with 560 biomarkers, based on more than 500 plasma samples of both healthy controls and CRC patients of various stages. We selected 9 biomarkers that are both highly sensitive and specific for CRC. These 9 markers show methylation signatures prevalent in patients and have very low noise in healthy controls. Along with one reference gene, the 9 CRC hypermethylated biomarkers can be multiplexed and analyzed in a single methylation-specific qPCR reaction. Analytical sensitivity and specificity were determined using a model system where hypermethylated DNA molecules were spiked into normal cell free DNA background at various percentages. With 10 ng of input, we can detect a single copy of the hypermethylated genome, achieving 0.006% limit of detection. Plasma samples from 10 stage I and 11 stage II CRC patients as well as 104 age-matched control subjects were subjected to the PCR analysis to yield a specificity of 96% and detection rate of 50% for stage I and 81% for stage II. The total turnaround time for analysis was <4 hours including DNA isolation, bisulfite conversion and qPCR analysis. The MMS-qPCR described here provides an accurate, accessible, and affordable liquid biopsy assay for the early detection of CRC. The same development strategy can also be applied to many other cancer types. Citation Format: Yun Bao, Heng Wang, Peter Vu, Grace Zhao, Shengrong Lin. Novel multiplex methylation-specific qPCR strategy for early CRC detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 788.