Abstract 4930: Comparing the methylation status in cervical squamous intraepithelial lesions using quantitative real-time methylation specific PCR (QMSP) and pyrosequencing
Authors: Riggs Bridget , Delmas Amber , Brown Kevin , Siegel Erin M. 
Published: February 2011

Abstract
Background: Tumor suppressor genes, adenomatous polyposis coli (APC) and retinoic acid receptor b-2 (RARB) have been shown to be methylated early in cervical carcinogenesis; however results vary across studies. There are several approaches that can be taken to assess cervical methylation status, each of which may influence the results. Quantitative real-time methylation specific PCR (QMSP) amplifies methylated DNA and quantifies target methylation level relative to house-keeping genes (e.g. b-actin). Pyrosequencing, on the other hand, amplifies bisulfite converted genomic DNA using primers independent of methylation status and quantifies the percentage of methylated CpG dinucleotides with single base resolution within a targeted loci.
Objectives: The purpose of this study was to determine the prevalence of methylation in two tumor suppressor genes, APC and RARB in liquid-based cytology specimens diagnosed as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL) using QMSP and pyrosequencing.
Methods: We examined 63 LSIL and 32 HSIL residual liquid-based cytology specimens. Methylation of APC and RARB was measured using QMSP and pyrosequencing in the same genomic region. HPV genotyping was conducted using the Roche Linear Array HPV Genotyping Test which enables PCR-based identification of 37 high- and low-risk HPV genotypes.
Results: QMSP detected APC methylation more often than pyrosequencing (36% vs. 6%), with QMSP identifying all pyrosequencing positive samples. RARB was rarely methylated in SILs in both assays (QMSP vs. Pyrosequencing: 4% vs. 3%) and there was no overlap in methylation between the methods. Overall, there were no differences in methylation for APC between HPV positive and HPV negative samples and LSIL and HSIL. RARB was significantly more likely to be methylated in HSIL using QMSP but not when measured by pyrosequencing, however there was no difference among HPV positive and HPV negative specimens.
Conclusions: The use of different methylation detection methods on exfoliated cervical specimens provided different methylation prevalence estimates. Exfoliated cervical specimens (e.g. Pap Smears) contain a high proportion of normal cervical cells and relatively few abnormal cells. This heterogeneity may lead to misclassification of lesion methylation status when using methylation independent assays. Methylation biomarker detection in exfoliated cells collected for clinical use is critical for the translation to clinical testing; however, detection methods need to be sensitive enough to identify biomarkers in a high background of normal cells. While QMSP does not provide methylation frequency at individual CpG sites, this study suggests QMSP may be well suited for the assessment of methylation within heterogeneous samples. Larger studies are needed to confirm these results.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4930.