Epigenetic analysis of sporadic and Lynch-associated ovarian cancers reveals histology-specific patterns of DNA methylation

<div><p>Diagnosis and treatment of epithelial ovarian cancer is challenging due to the poor understanding of the pathogenesis of the disease. Our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis and, especially, whether tumors with different histological subtypes or hereditary background (Lynch syndrome) exhibit differential susceptibility to epigenetic inactivation of growth regulatory genes. Gene candidates for epigenetic regulation were identified from the literature and by expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents. Thirteen genes were chosen for methylation-specific multiplex ligation-dependent probe amplification assays on 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (i.e., hypermethylation) of variable degree was characteristic of ovarian carcinomas relative to the corresponding normal tissues, and hypermethylation was consistently more prominent in non-serous than serous tumors for individual genes and gene sets investigated. Lynch syndrome-associated clear cell carcinomas showed the highest frequencies of hypermethylation. Among endometrioid ovarian carcinomas, lower levels of promoter methylation of <i>RSK4</i>, <i>SPARC</i>, and <i>HOXA9</i> were significantly associated with higher tumor grade; thus, the methylation patterns showed a shift to the direction of high-grade serous tumors. In conclusion, we provide evidence of a frequent epigenetic inactivation of <i>RSK4</i>, <i>SPARC</i>, <i>PROM1</i>, <i>HOXA10</i>, <i>HOXA9</i>, <i>WT1-AS</i>, <i>SFRP2</i>, <i>SFRP5</i>, <i>OPCML</i>, and MIR34B in the development of non-serous ovarian carcinomas of Lynch and sporadic origin, as compared to serous tumors. Our findings shed light on the role of epigenetic mechanisms in ovarian tumorigenesis and identify potential targets for translational applications.</p></div

Candidate colorectal cancer predisposing genes with Sanger validated truncating variants in familial CRC cases.

<p>Gene, transcript and chromosomal positions taken from Ensembl build 37 (<a href="http://www.ensembl.org" target="_blank">http://www.ensembl.org</a>).</p>a<p>fs = frameshift insertion and deletion variant, sp = splice site variant.</p>b<p>Counts include both exome data controls and Sanger sequenced controls.</p

Clinical characteristics of the 96 cases with familial colorectal cancer.

<p>NOTE: some of the numbers do not match due to missing data.</p><p>Abbreviations: MSI, microsatellite instability; MSS, microsatellite stable.</p>*<p>Distal, from splenic flexure to rectum; proximal, from cecum to transverse colon.</p

Schematic representation of the overall study design.

<p>We performed exome sequencing analysis of germline DNA from 96 independent familial CRC cases. Initially, quality, frequency and control filtering were applied to the exome data. Next, genes with putative truncating loss-of-function variants in at least 2/96 cases were validated by Sanger sequencing. Confirmed truncating variants were then screened in Finnish population matched controls. Loss of heterozygosity was analyzed in the respective tumor tissues. Variants in genes showing loss of the wild-type allele in tumor tissue were genotyped in a set of validation phase samples. Overall, 11 novel candidate CRC predisposing genes were identified. CRC, colorectal cancer; MAF, minor allele frequency; LOH, loss of heterozygosity.</p

Examples of DNA sequence chromatograms.

<p>Chromatograms on the top demonstrate c.3346C>T, p.Q1116X in <i>UACA</i>. Chromatograms on the bottom demonstrate c.121C>T, p.Q41X in <i>TWSG1</i>. DNA extracted from tumor tissue shows LOH with retention of the mutated alleles (right). The wild-type alleles can still be seen in the tumor chromatograms, due to normal tissue contamination in the tumor samples.</p

Pedigrees of families found to carry <i>UACA</i> and <i>TWSG1</i> truncating variants.

<p>Carrier status is depicted for all the cases for whom readily extracted DNA was available. The individuals that underwent exome sequencing are marked with an arrow. Numbers represent the age at diagnosis of the affected individuals. The following abbreviations are used: CRC, colorectal cancer; BCC, basal cell carcinoma, MG, meningioma; HD, Hodgkin lymphoma; PC, prostate cancer; MM, melanoma and LC, lung cancer.</p

Additional file 1 of Colorectal cancer incidences in Lynch syndrome: a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium

Additional file 1