Algorithm Optimization in Methylation Detection with Multiple RT-qPCR

PLoS One. 2016 Nov 29;11(11):e0163333. doi: 10.1371/journal.pone.0163333. eCollection 2016.

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.

MeSH terms

  • Aged
  • Algorithms*
  • Colorectal Neoplasms / genetics
  • DNA Methylation*
  • Female
  • Gastrointestinal Diseases / genetics
  • Genomics
  • Humans
  • Male
  • Middle Aged
  • Real-Time Polymerase Chain Reaction / methods*
  • Septins / genetics

Substances

  • SEPTIN9 protein, human
  • Septins

Grants and funding

This study was supported by BioChain (Beijing) Science and Technology, Inc. The funder provided support in the form of salaries for authors [LS, GZ, JW, XH] and had an additional role in the study design, data analysis, preparation of the manuscript and publication of the study. The specific roles of these authors are articulated in the ‘author contributions’ section. This work was also supported by the Beijing Municipal Science and Technology Project (capital public health project) No. Z151100003915092, sponsored by the Beijing Municipal Science and Technology Commission.