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Discovery and validation of candidate host DNA methylation markers for detection of cervical precancer and cancer.

ABSTRACT: Human papillomavirus (HPV) testing has been recently introduced as an alternative to cytology for cervical cancer screening. However, since most HPV infections clear without causing clinically relevant lesions, additional triage tests are required to identify women who are at high risk of developing cancer. We performed DNA methylation profiling on formalin-fixed, paraffin-embedded tissue specimens from women with benign HPV16 infection and histologically confirmed cervical intraepithelial neoplasia grade 3, and cancer using a bead-based microarray covering 1,500 CpG sites in over 800 genes. Methylation levels in individual CpG sites were compared using a t-test, and results were summarized by computing p-values. A total of 12 candidate genes (ADCYAP1, ASCL1, ATP10, CADM1, DCC, DBC1, HS3ST2, MOS, MYOD1, SOX1, SOX17 and TMEFF2) identified by DNA methylation profiling, plus an additional three genes identified from the literature (EPB41L3, MAL and miR-124) were chosen for validation in an independent set of 167 liquid-based cytology specimens using pyrosequencing and targeted, next-generation bisulfite sequencing. Of the 15 candidate gene markers, 10 had an area under the curve (AUC) of???0.75 for discrimination of high grade squamous intraepithelial lesions or worse (HSIL+) from

SUBMITTER: Clarke MA

PROVIDER: S-EPMC6774256 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Discovery and validation of candidate host DNA methylation markers for detection of cervical precancer and cancer.

Clarke Megan A MA Luhn Patricia P Gage Julia C JC Bodelon Clara C Dunn S Terence ST Walker Joan J Zuna Rosemary R Hewitt Stephen S Killian J Keith JK Yan Liying L Miller Andrew A Schiffman Mark M Wentzensen Nicolas N

International journal of cancer 20170526 4

Human papillomavirus (HPV) testing has been recently introduced as an alternative to cytology for cervical cancer screening. However, since most HPV infections clear without causing clinically relevant lesions, additional triage tests are required to identify women who are at high risk of developing cancer. We performed DNA methylation profiling on formalin-fixed, paraffin-embedded tissue specimens from women with benign HPV16 infection and histologically confirmed cervical intraepithelial neopl ...[more]

PMID: 28500655

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Project description:BackgroundTo discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in in vitro (cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps.AimTo apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer.ResultsThe application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed in silico. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is CCNA1) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (SST, HTRA3 and NPTX1) are not methylated in normal cervix tissue.ConclusionThe application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown in silico and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.

Project description:BackgroundThe development of accurate urinary biomarkers for non-invasive and cost-effective detection of primary and recurrent bladder tumours is recognized as one of the major clinical needs in bladder cancer diagnostics. The purposes of this study were (1) to validate the results of a previous technical comparison by determining the diagnostic performance of nine methylation markers in urine pellet compared to full void urine, and (2) to validate the diagnostic performance of the optimal marker panel GHSR/MAL from a previous exploratory study in a preclinical setting.MethodsUrine samples of 108 patients with bladder cancer and 100 age- and gender-matched controls were prospectively collected for methylation analysis. Urinary methylation levels of the markers FAM19A4, GHSR, MAL, miR-129, miR-935, PHACTR3, PRDM14, SST and ZIC1 were determined with quantitative methylation-specific PCR in urine pellet. Area under the curves (AUCs) were determined for individual markers and the marker panel GHSR/MAL. The diagnostic performance of the marker panel GHSR/MAL was evaluated in the total study population and in different subgroups of patients with bladder cancer using the Chi-square test. The diagnostic accuracy was assessed by leave-one-out cross-validation.ResultsAll nine urinary methylation markers (FAM19A4, GHSR, MAL, miR-129, miR-935, PHACTR3, PRDM14, SST and ZIC1) showed significantly higher methylation levels in bladder cancer patients than in controls (p < 0.001). Area under the curves (AUCs) of the nine methylation markers tested in urine pellet were similar to AUCs in full void urine of an independent previous cohort. GHSR/MAL reached an AUC of 0.89 (95% confidence interval [CI] 0.84-0.94), at 80% sensitivity and 93% specificity. Sensitivity of GHSR/MAL increased with higher tumour grades, higher tumour stages, in primary vs. recurrent tumours, and in males vs. females.ConclusionsThis technical validation supports the robustness of DNA methylation analysis in urine pellet and full void urine for the non-invasive detection of bladder cancer. Subsequent preclinical validation confirmed the diagnostic potential of GHSR/MAL. These findings underline the diagnostic potential of the marker panel GHSR/MAL for future bladder cancer diagnostics.

Project description:BACKGROUND:The current prognosis of thymic epithelial tumors (TETs) is according to the World Health Organization (WHO) histologic classification and the Masaoka staging system. These methods of prognosis have certain limitations in clinical application and there is a need to seek new method for determining the prognosis of patients with TETs. To date, there have been no studies done on the use of DNA methylation biomarkers for prognosis of TETs. The present study was therefore carried out to identify DNA methylation biomarkers that can determine the overall survival in patients with TETs. METHODS:Bioinformatic analysis of TCGA 450 K methylation array data, transcriptome sequencing data, WHO histologic classification and Masaoka staging system was performed to identify differentially expressed methylation sites between thymoma and thymic carcinoma as well as the different DNA methylation sites associated with the overall survival in patients with TETs. Using pyrosequencing, 4 different methylation sites (cg05784862, cg07154254, cg02543462, and cg06288355) were sequenced from tumor tissues of 100 Chinese patients with TETs. A prognostic model for TETs was constructed using these four methylation sites. RESULTS:The TCGA dataset showed 5155 and 6967 hyper- and hypomethylated CpG sites in type A-B3 group and type C group, respectively, of which 3600 were located within the gene promoter regions. One hundred thirty-four genes were silenced by promoter hypermethylation and 174 mRNAs were upregulated. Analysis of univariate and multivariate Cox regression showed significant association between the methylation levels of 187 sites and the overall survival in patients with TETs. cg05784862(KSR1), cg07154254(ELF3), cg02543462(ILRN), and cg06288355(RAG1) were identified as independent prognostic factors for overall survival in patients with TETs after adjusting for Masaoka staging in 100 Chinese patients. The prognostic model which consists of the four abovementioned genes had higher accuracy for predicting the 5-year overall survival in patients with TETs as compared to the Masaoka clinical staging. (Time-dependent ROC analysis AUC 1.000 vs 0.742, P = 2.7 × 10-6). CONCLUSIONS:The methylation levels of cg05784862(KSR1), cg07154254(ELF3), cg02543462(ILRN), and cg06288355(RAG1) sites are associated with the progression of TETs and may serve as new biomarkers for predicting the overall survival in patients with TETs.

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Discovery and validation of candidate host DNA methylation markers for detection of cervical precancer and cancer.

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Discovery and validation of candidate host DNA methylation markers for detection of cervical precancer and cancer.

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