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.
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:Identification of promoter methylation profile associated with cervical cancer progression. The Ilumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs from 19 cervical samples (including normal, CIN I-II, in situ and invasive cervical cancer tissues).
Project description:We developed a discovery-validation mass-spectrometry based pipeline to identify a set of proteins that are regulated in serum of patients with cervical intraepithelial neoplasia (CIN) and squamous cell cervical cancer using isobaric Tags for Relative and Absolute Quantitation (iTRAQ®), label-free shotgun and targeted mass-spectrometric quantification. At the discovery stage we used a “pooling” strategy for the comparative analysis of immunodepleted serum from patients with early-(CES) and late-stage (CLS) cervical cancer versus healthy controls that revealed 15 up- and 26 down-regulated proteins. The analysis of non-depleted serum samples from patients with CIN, CES, CLS, and healthy controls showed significant changes in abundance of alpha-1-acid glycoprotein 1, alpha-1-antitrypsin, serotransferrin, haptoglobin, alpha-2-HS-glycoprotein and vitamin D-binding protein. To further validate our findings we developed a fast UPLC/MRM method for the simultaneous targeted quantification of these proteins in an independent set of serum from patients with cervical cancer or CIN and healthy controls as well as serum samples from patients with ovarian cancer. The panel of six proteins showed 72 % sensitivity and 82 % specificity for discrimination of patients with CIN from healthy controls, a stage of the disease where current protein-based biomarkers, e.g. squamous cell carcinoma antigen fail to show any discrimination.
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:Using a genome-wide DNA methylation profiling of 186 cervical samples from women with different CIN grades and well-characterized HPV genotyping, we identified novel methylation markers of epigenetic changes that discriminate accurately between clinically significant and transient cervical disease. In particular, a 2-gene DNA methylation classifier (ATP10A and HAS1) showed a promising ability to discriminate among pre-invasive cervical lesion grades. The identified markers are excellent candidates for future diagnostic or prognostic assays in cervical cancer screening.
Project description:Identification of promoter methylation profile associated with cervical cancer progression. The Ilumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs from 19 cervical samples (including normal, CIN I-II, in situ and invasive cervical cancer tissues). Bisulphite converted DNA from the 19 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2
Project description:We developed a discovery-validation mass-spectrometry based pipeline to identify a set of proteins that are regulated in serum of patients with cervical intraepithelial neoplasia (CIN) and squamous cell cervical cancer using isobaric Tags for Relative and Absolute Quantitation (iTRAQ®), label-free shotgun and targeted mass-spectrometric quantification. At the discovery stage we used a “pooling” strategy for the comparative analysis of immunodepleted serum from patients with early-(CES) and late-stage (CLS) cervical cancer versus healthy controls that revealed 15 up- and 26 down-regulated proteins. The analysis of a new of non-depleted serum samples from patients with CIN, CES, CLS, and healthy controls showed significant changes in abundance of alpha-1-acid glycoprotein 1, alpha-1-antitrypsin, serotransferrin, haptoglobin, alpha-2-HS-glycoprotein and vitamin D-binding protein. To further validate our findings we developed a fast UPLC/MRM method for the simultaneous targeted quantification of these proteins in a new set of 48 CIN, 50 CES, 34 CLS and 48 healthy controls as well as 49 serum samples from patients with ovarian cancer. The panel of six proteins showed 62% sensitivity and 82 % specificity for discrimination of patients with CIN grade 2 or worse from healthy controls, a stage of the disease where current protein-based biomarkers fail to show any discrimination.