Abstract 1976: Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH-1promoter methylation
Authors: WANG DANYI , O'Rourke Dennis , Sanchez-Garcia Jorge F. , Cai Ti , Scheuenpflug Juergen , Feng Zheng 
Published: September 2020

Purpose Studies have shown that MutL Homolog 1 (MLH1) promotor methylation is closely associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. ctDNA methylation analysis has advantages in performance over mutation analysis, such as higher sensitivity, broader dynamic range and increased stability. However, current methods for methylation analysis involving bisulfite treatment possess many challenges which limit broad clinical applications. Here, we report the development of a fit-for-purpose digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC.

Study Procedure Primers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to methylation sensitive restrictive enzyme (MSRE) HpaII digestion. The performance of the MSRE ddPCR assay was evaluated as compared to MSRE qPCR assay. Plasma samples from healthy donors and CRC patients were analyzed with the optimal primer/probe sets using ddPCR. Assay performance was evaluated by determining the limit of detection (LOD), linearity of the primer/probe sets and the correlation coefficient. Receiver operating characteristic (ROC) curves were generated to determine the optimal assay sensitivity and specificity.

Data Summary Using methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Three sets of primer/probes were optimized for robust amplification in ddPCR. Using 0.375ng synthetic DNA input, the LOD was measured as 1 positive droplet for set A, 4 positive droplets for set B, and 3 positive droplets for set C . Based on the results of ddPCR assay using 1ng cfDNA input from healthy donors or CRC samples, ROC curves were generated for each primer/probe set with area under the curve values ranging from 0.85 to 0.95. The optimal assay sensitivity and specificity were displayed when 5 positive droplets was used as acceptance criteria (93% sensitivity and 95% specificity). The optimal cutoff value of MLH-1 promoter methylation in ctDNA associated with patient diagnosis remains to be further defined.

Conclusion The liquid biopsy assay for MLH-1 promoter methylation detection using purely quantitative ddPCR is a simple and ultrasensitive procedure that provides reliable methylation detection in ctDNA. The MRSE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development.

Citation Format: DANYI WANG, Dennis O'Rourke, Jorge F. Sanchez-Garcia, Ti Cai, Juergen Scheuenpflug, Zheng Feng. Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH-1promoter methylation [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 1976.