Project description:The aim of this study was to identify new candidate genes that are differentially methylated in squamous cell carcinoma compared to the DNA samples from cervical intraepithelial neoplasia grade 3 (CIN3) and normal cervical scrapes. The Illumina Infinium Human Methylation 450 K BeadChip method identifies genome-wide DNA methylation changes in CpG islands, CpG shores and shelves. In this study 20 normal cervical samples (HPV negative), 18 samples with CIN3 lesions (HPV positive) and 6 cervical cancer tissues (HPV positive) were included.
Project description:Cervical cancer is characterized by a well-defined pre-malignant phase, cervical intraepithelial neoplasia (CIN). Identification of high grade CIN lesions by population-based screening programs and their subsequent treatment has led to a significant reduction of the incidence and mortality of cervical cancer. Cytology-based testing of cervical smears is the most widely used cervical cancer screening method, but is not ideal, as the sensitivity for detection of CIN2 and higher (CIN2+) is only ~55%. Therefore, more sensitive and specific biomarkers for cervical cancer and its precancerous stages are needed.
Project description:Epigenetic modifications, such as aberrant DNA promoter methylation is frequently observed in cervical cancer. Identification of hypermethylated regions maybe useful for discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3) or worse may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions were characterised using genome-wide methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methyl-DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium resulting in the identification of hypermethylated differentially methylated regions (DMRs). Validation of 9 selected DMRs by MSP or BSP in cervical tissue revealed methylation in 63.2-94.7% high-grade CIN and in 59.3-100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was applied exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples (p<0.001). Clinical validation of both markers in cervical scrapings from patients referred with an abnormal cervical smear, confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion (p<0.001) and the ROC analysis was discriminative (p<0.005). These possible methylation markers represent COL25A1 and KATNAL2 promoters and their observed increased methylation upon progression is in agreement with their biological function (cytoskeleton regulation). In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and could be potential biomarkers for early detection. Epigenetic modifications, such as aberrant DNA promoter methylation is frequently observed in cervical cancer. Identification of hypermethylated regions maybe useful for discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3) or worse may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions were characterised using genome-wide methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methyl-DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium resulting in the identification of hypermethylated differentially methylated regions (DMRs). Validation of 9 selected DMRs by MSP or BSP in cervical tissue revealed methylation in 63.2-94.7% high-grade CIN and in 59.3-100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was applied exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples (p<0.001). Clinical validation of both markers in cervical scrapings from patients referred with an abnormal cervical smear, confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion (p<0.001) and the ROC analysis was discriminative (p<0.005). These possible methylation markers represent COL25A1 and KATNAL2 promoters and their observed increased methylation upon progression is in agreement with their biological function (cytoskeleton regulation). In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and could be potential biomarkers for early detection. MeDIP with subsequent microarray analysis was performed on DNA isolated from frozen macrodissected epithelial tissue of CIN3 lesions (n=15) and normal cervices (n=10).
Project description:Comparative analysis of gene expression prodfiles of cervical biopsy from two (2) patients with pre-invasive neoplastic lesions (carcinonma in citu, or high-grade cervical intraepithelial neoplasia) and 2 patients with earliest stage of invasive cancer
Project description:The aim of this study was to identify new candidate genes that are differentially methylated in squamous cell carcinoma compared to the DNA samples from cervical intraepithelial neoplasia grade 3 (CIN3) and normal cervical scrapes. The Illumina Infinium Human Methylation 450 K BeadChip method identifies genome-wide DNA methylation changes in CpG islands, CpG shores and shelves.
Project description:Genome-wide DNA methylation profiles in liquid based cytology (LBC) cervical scrapes samples was assessed using the Illumina Infinium Methylation850 BeadChip V1.0B4. The purpose of this study was to identify new candidate genes that are differentially methylated in squamous cell carcinoma compared to the DNA samples from cervical intraepithelial neoplasia grade (CIN) and normal cervical scrapes.
Project description:Genome wide DNA methylation profiling of four control and cancer cell lines where apoptosis was induced using BH3 mimetics. The Illumina Human MethylationEPIC methylation profiling array was used to obtain DNA methylation profiles across approximately 850,000 CpGs. 8 samples were from treated cells harvested at two different time points representing early and late apoptotic stages, and 4 samples were controls combined from the two timepoints. 4 additional samples was left over DNA from the Nalm6 cell line ran on the array to fill up the bead chips. Data analysis will be presented in the manuscript "HPV-induced host epigenetic reprogramming is lost upon progression to high-grade cervical intraepithelial neoplasia" accepted for publication in the International Journal for Cancer.
Project description:Using the pools of DNA from squamous cervical carcinomas (SCC) and DNA from normal scraping cells discovered the abnormal changes of genomic wide methylation by methylation BeadChip. The 61 genes were selected to validate by quantitative methylation specific PCR (qMSP) and bisulfite pyrosequencing in further processes. A CpG islands methylator phenotype (CIMP) was confirmed at 14 candidates in an independent set contained a spectrum of scraping cells form abnormal cervical lesions. In addition, we found among of 7 genes were announced to imply to potentially biological functions of β-catenin signal. A pool DNA collected from equal amount of DNA of 38 SCC, and the other pool collected from equal amount of DNA of 19 normal cervical scraping cells. Based on 40 percent of different methylation in two pools of samples, we selected 91 genes showed abnormal methylation in SCC. After the confirmation of gene restored expression in cervical cell lines, there 61 genes showed upregulation in the treatment with demethylating drug. In addition, gel based of MSP were contributed to confirm in a small size of samples, and quantitative MSP was contributed to validated in an independent set. The methylation status of qMSP was also confirmed by bisulfite pyrosequence. Finally, we selected 14 genes demonstrated clinical relevance in the high methylation of CIMP associated to a risk factor in cervical intraepithelial neoplasia grade 3 (CIN3) and worse lesions. isk factor in cervical intraepithelial neoplasia grade 3 (CIN3) and worse lesions.