Algorithm Optimization in Methylation Detection with Multiple RT-qPCR.

Lele Song,Jianming Wang,Yuemin Li,Jia Jia,Xiaoliang Han,Sanjun Cai,Jianqiu Sheng,Peng Jin,Guangpeng Zhou,Qian Kang,Guoxiang Cai,Ken Mills

doi:10.1371/journal.pone.0163333

Abstract

Epigenetic markers based on differential methylation of DNA sequences are used in cancer screening and diagnostics. Detection of abnormal methylation at specific loci by real-time quantitative polymerase chain reaction (RT-qPCR) has been developed to enable high-throughput cancer screening. For tests that combine the results of multiple PCR replicates into a single reportable result, both individual PCR cutoff and weighting of the individual PCR result are essential to test outcome. In this opportunistic screening study, we tested samples from 1133 patients using the triplicate Epi proColon assay with various algorithms and compared it with the newly developed single replicate SensiColon assay that measures methylation status of the same SEPT9 gene sequence. The Epi proColon test approved by the US FDA (1/3 algorithm) showed the highest sensitivity (82.4%) at a lower specificity (82.0%) compared with the Epi proColon 2.0 CE version with 2/3 algorithm (75.1% sensitivity, 97.1% specificity) or 1/1 algorithm (71.3% sensitivity, 92.7% specificity). No significant difference in performance was found between the Epi proColon 2.0 CE and the SensiColon assays. The choice of algorithm must depend on specific test usage, including screening and early detection. These considerations allow one to choose the optimal algorithm to maximize the test performance. We hope this study can help to optimize the methylation detection in cancer screening and early detection.

Highlights

Worldwide, colorectal cancer (CRC) is the third most common malignancy in men and the second in women [1]
PCR result are essential to test outcome. In this opportunistic screening study, we tested samples from 1133 patients using the triplicate Epi proColon assay with various algorithms and compared it with the newly developed single replicate SensiColon assay that measures methylation status of the same SEPT9 gene sequence
No significant difference in performance was found between the Epi proColon 2.0 CE and the SensiColon assays

Summary

Introduction

Colorectal cancer (CRC) is the third most common malignancy in men and the second in women [1]. Regular screening and early detection of CRC can achieve effective prevention. 60%-70% of CRC patients are not diagnosed until they are symptomatic at later stages, and only 11.8% of cases are detected at early stages [2]. It is urgent to reduce the CRC morbidity and mortality by improving participation in screening. Participation in screening using current methods, including colonoscopy and fecal blood testing, varies widely, and there are significant barriers posed by the methods that limit their use. PLOS ONE | DOI:10.1371/journal.pone.0163333 November 29, 2016

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