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Introduction: Primary human cancers are thought to arise from genetic and epigenetic alterations of tumor suppressor genes and oncogenes. Transcriptional and DNA copy-number studies have improved our understanding and classification of solid tumors. Recent studies have established that like mutation, methylation-mediated gene silencing often leads to tumorigenesis. The aim of this study was to comprehensively explore the “ovarian cancer methylome” and to validate the novel methylation marker AGTR1 in a large independent series of ovarian cancer samples. Patients and methods: Fifteen ovarian cancer samples and 10 normal ovarian surface epithelium brushings were hybridized to Affymetrix U133 plus 2.0 arrays. Furthermore, 3 normal ovarian and 3 isogenic (resistant and sensitive to chemotherapy) ovarian cancer cell lines, with or without treatment with a demethylating agent were hybridized to the same array. Selection of cancer specific methylated genes was based on differential expression between normal and cancer samples, with cancer samples having lower expression than normal samples, and upregulation in cancer cell lines after treatment with a demethylating agent. A number of selected genes that showed low or no expression in primary cancer tissues and re-expressed after treatment with a demethylating agent were then tested for promoter methylation by bisulfite sequencing. Quantitative methylation specific PCR (QMSP) was developed for the AGRT1 gene and tested in a total of 353 ovarian cancer, 17 ovarian cystadenoma and 16 ovarian borderline tumor samples. Results: Fourteen primary ovarian tumor DNA samples were available from the samples that were hybridized for expression array analysis. Twelve out of 14 samples were methylated for AGRT1 which is inversely correlated with expression from our array analysis. We then tested 13 normal ovarian epithelium samples and no methylation was detected in any of the samples. Validation in a large independent set of samples confirmed that ovarian cancer samples (207/353, 53%) were more frequently methylated than ovarian cystadenoma (3/17, 18%; P=0.004). Conclusion: We have identified a set of potential tumor suppressor genes by a comprehensive discovery approach to uncover the cancer methylome. Among these genes, AGTR1 methylation was identified as a potential cancer specific methylated gene that may hold promise for further studies to establish it as a biomarker. Functional studies are warranted to evaluate the biologic role of AGTR1 methylation in the carcinogenic process of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4035. doi:1538-7445.AM2012-4035

Objective: To improve current screening modalities, additional molecular markers that allow detection of ovarian cancer at an early stage are needed. The aim of the current study was to identify novel methylation-based biomarkers for ovarian cancer. Methods: Genes frequently expressed at low levels were identified by comparing global expression levels from 232 primary ovarian cancers to Universal Reference RNA. Ten genes possessing a CpG island in the promoter region that showed frequent low expression in serous cancers and at least one other histological subtype were selected. The methylation status of candidate genes was verified in 50 sporadic ovarian cancers, 11 hereditary cancers, 13 borderline cancers and 12 cystadenomas using methylation specific PCR (MSP). Results: Three candidate genes (IGFBP1, LIN28 and ZNF582) showed frequent methylation in cancers and were unmethylated in normal leukocyte DNA and human ovarian surface epithelial cells. Promoter methylation of any of the three candidate genes was observed in 88% of stage III/IV cancers and 72% of stage I/II cancers. In contrast, only 9% of hereditary cancers showed evidence of methylation for any of the three genes (p Conclusions: Using oligonucleotide microarray data, three novel methylation-based biomarkers for sporadic epithelial ovarian cancer were discovered. Further studies should elucidate the methylation status of these genes in larger cohorts of ovarian cancers and investigate their methylation status in serum. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 161.

Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. GULP1, a molecule not previously related to cancer, is a cytoplasmic adaptor protein with a phosphotyrosine binding domain that plays a role in one of two partially redundant pathways that lead to the engulfment and clearance of apoptotic cells, according to several genetic studies performed in Caenorhabditis elegans. Performing a pharmacologic unmasking technique we observed that this molecule is downregulated in ovarian tumor tissues when compared to normal ovary. Ovarian cancer is the leading cause of death among gynaecological cancers worldwide, this due to the fact that women are diagnosed with advanced stage disease and because of the lack of truly sensitive/specific screening techniques and unavailability of individualized therapy. We performed an expression microarray on 15 ovarian tumor samples, 10 ovarian normal surface epithelium, 3 ovarian cancer cell lines and 3 normal cell lines. All cells have been treated with a demethylating agent which comprehensively uncovers genes silenced by promoter hypermethylation. The selection of the best candidates was based upon the differential expression between normal and tumor samples (or cell lines), them being downregulated in cancer when compared to normal, and finally the re-activation after the treatment with the demethylating agent. One of these promising genes was GULP1, which expression pattern was then assessed by Reverse Transcriptase PCR and Western Blot and compared to its methylation status in the same six ovarian cell lines and showed correlation between absence of expression and presence of methylation (or the opposite situation). We developed quantitative fluorogenic methylation specific PCR (QMSP) for GULP1 and profiled 437 ovarian tumor samples, 17 borderline tumors, 19 cystadenoma samples and 13 normal ovarian samples, finding 34.7% (151/437), 11.7% (2/17), 10.5% (2/19) and 0% (0/13) of methylation frequency, respectively, (establishing an empiric cutoff). Using fisher's exact test, we observe a significant increase in methylation when comparing tumors with cystadenomas and normals (p=0.0439 and p=0.0131, respectively). Late stage tumors also showed a higher frequency of methylation versus early stage (p=0.004). So far, we have demonstrated reduced log growth rates after transiently overexpressing GULP1 in one ovarian cell line (IGROVCP). To assess its potential as a TSG, we have ongoing experiments to assess its biological function as ectopic overexpression of this gene in ovarian cancer cell lines as well as creating knock downs using shRNAs. These preliminary data indicate that GULP1 may be an ovarian cancer biomarker. Further studies on the biology of this gene are needed to evaluate its potential as a TSG and its role in ovarian cancer carcinogenesis. 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 4891.