Abstract
BackgroundStudies of methylation biomarkers for cervical cancer often involved only few randomly selected CpGs per candidate gene analyzed by methylation-specific PCR-based methods, with often inconsistent results from different laboratories. We evaluated the role of different CpGs from multiple genes as methylation biomarkers for high-grade cervical intraepithelial neoplasia (CIN).ResultsWe applied a mass spectrometry-based platform to survey the quantitative methylation levels of 34 CpG units from SOX1, PAX1, NKX6-1, LMX1A, and ONECUT1 genes in 100 cervical formalin-fixed paraffin-embedded (FFPE) tissues. We then used nonparametric statistics and Random Forest algorithm to rank significant CpG methylations and support vector machine with 10-fold cross validation and 200 times bootstrap resampling to build a predictive model separating CIN II/III from CIN I/normal subjects. We found only select CpG units showed significant differences in methylation between CIN II/III and CIN I/normal groups, while mean methylation levels per gene were similar between the two groups for each gene except PAX1. An optimal classification model involving five CpG units from SOX1, PAX1, NKX6-1, and LMX1A achieved 81.2% specificity, 80.4% sensitivity, and 80.8% accuracy.ConclusionsOur study suggested that during CIN development, the methylation of CpGs within CpG islands is not uniform, with varying degrees of significance as biomarkers. Our study emphasizes the importance of not only methylated marker genes but also specific CpGs for identifying high-grade CINs. The 5-CpG classification model provides a promising biomarker panel for the early detection of cervical cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0037-1) contains supplementary material, which is available to authorized users.
Highlights
Studies of methylation biomarkers for cervical cancer often involved only few randomly selected CpGs per candidate gene analyzed by methylation-specific PCR-based methods, with often inconsistent results from different laboratories
Our results showed that choosing the right CpG unit to assay is critical, and a panel of multiple specific CpG methylation constructed by computerized algorithm allowed us to separate high-grade Cervical intraepithelial neoplasia (CIN) from low-grade or healthy subjects with high accuracy, providing a candidate biomarker panel for early detection of cervical cancer development
Survey of CpG methylation of five genes by MALDI-TOF-based EpiTYPER assay A total of 100 formalin-fixed paraffin-embedded (FFPE) cervical samples with histopathological classifications of normal (N = 16), CIN I (N = 31), and CIN II or CIN III (N = 53 including 4 CIN II and 49 CIN III) were obtained retrospectively from a cohort of ethnic Han Chinese women
Summary
Studies of methylation biomarkers for cervical cancer often involved only few randomly selected CpGs per candidate gene analyzed by methylation-specific PCR-based methods, with often inconsistent results from different laboratories. We evaluated the role of different CpGs from multiple genes as methylation biomarkers for high-grade cervical intraepithelial neoplasia (CIN). Cervical cancer screening aims to identify high-grade intraepithelial lesions (corresponding to histological grades CIN II and CIN III) which require surgical procedures to prevent further progression. Low-grade intraepithelial lesions (corresponding to histological grade CIN I), on the other hand, should not be over-treated for such procedures as they have high potential to spontaneously regress to normal [2]. A third strategy is direct colposcopy [10], which requires interpretational expertise, is not amenable to high throughput processing, and has low positive predictive values for lowgrade squamous intraepithelial lesions [4,11]. An objective, high-throughput approach with high sensitivity and specificity is urgently needed for early diagnosis of cervical cancer
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