Matthias Dürst

Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression.

BackgroundCervical Cancer (CC) exhibits highly complex genomic alterations. These include hemizygous deletions at 4p15.3, 10q24, 5q35, 3p12.3, and 11q24, the chromosomal sites of Slit-Robo pathway genes. However, no candidate tumor suppressor genes at these regions have been identified so far. Slit family of secreted proteins modulates chemokine-induced cell migration of distinct somatic cell types. Slit genes mediate their effect by binding to its receptor Roundabout (Robo). These genes have shown to be inactivated by promoter hypermethylation in a number of human cancers.ResultsTo test whether Slit-Robo pathway genes are targets of inactivation at these sites of deletion, we examined promoter hypermethylation of SLIT1, SLIT2, SLIT3, ROBO1, and ROBO3 genes in invasive CC and its precursor lesions. We identified a high frequency of promoter hypermethylation in all the Slit-Robo genes resulting in down regulated gene expression in invasive CC, but the inhibitors of DNA methylation and histone deacetylases (HDACs) in CC cell lines failed to effectively reactivate the down-regulated expression. These results suggest a complex mechanism of inactivation in the Slit-Robo pathway in CC. By analysis of cervical precancerous lesions, we further show that promoter hypermethylation of Slit-Robo pathway occurs early in tumor progression.ConclusionTaken together, these findings suggest that epigenetic alterations of Slit-Robo pathway genes (i) play a role in CC development, (ii) further delineation of molecular basis of promoter methylation-mediated gene regulation provides a potential basis for epigenetic-based therapy in advanced stage CC, and (iii) form epigenetic signatures to identify precancerous lesions at risk to progression.

Candidate locus analysis of the TERT-CLPTM1L cancer risk region on chromosome 5p15 identifies multiple independent variants associated with endometrial cancer risk

Several studies have reported associations between multiple cancer types and single-nucleotide polymorphisms (SNPs) on chromosome 5p15, which harbours TERT and CLPTM1L, but no such association has been reported with endometrial cancer. To evaluate the role of genetic variants at the TERT–CLPTM1L region in endometrial cancer risk, we carried out comprehensive fine-mapping analyses of genotyped and imputed SNPs using a custom Illumina iSelect array which includes dense SNP coverage of this region. We examined 396 SNPs (113 genotyped, 283 imputed) in 4,401 endometrial cancer cases and 28,758 controls. Single-SNP and forward/backward logistic regression models suggested evidence for three variants independently associated with endometrial cancer risk (Pxa0=xa04.9xa0×xa010−6 to Pxa0=xa07.7xa0×xa010−5). Only one falls into a haplotype previously associated with other cancer types (rs7705526, in TERT intron 1), and this SNP has been shown to alter TERT promoter activity. One of the novel associations (rs13174814) maps to a second region in the TERT promoter and the other (rs62329728) is in the promoter region of CLPTM1L; neither are correlated with previously reported cancer-associated SNPs. Using TCGA RNASeq data, we found significantly increased expression of both TERT and CLPTM1L in endometrial cancer tissue compared with normal tissue (TERTPxa0=xa01.5xa0×xa010−18, CLPTM1LPxa0=xa01.5xa0×xa010−19). Our study thus reports a novel endometrial cancer risk locus and expands the spectrum of cancer types associated with genetic variation at 5p15, further highlighting the importance of this region for cancer susceptibility.

RUNX3 and CAMK2N1 hypermethylation as prognostic marker for epithelial ovarian cancer

Treatment of epithelial ovarian cancer consists of surgery plus platinum‐taxane based chemotherapy. Neither prognostic nor predictive serum or tissue markers except BRCA1/2 mutations are available thus precluding individualized treatment. Aim of this study is the identification and validation of DNA‐methylation markers with prognostic value. Genome‐wide array analyses were used to determine methylation patterns in groups of serous EOC with different outcome (PFSu2009<u2009vs.u2009>u20093 years, each nu2009=u20096) but comparable clinical parameters. Two hundred and twenty differentially methylated regions in tumor tissue of patients with short vs. long PFS (106 hypo‐ and 114 hypermethylated regions) were identified. Thirty‐five of 37 selected CpG islands were validated by MSP using the same samples as for microarray analyses. Six of these regions were analyzed by targeted next‐generation bisulfite‐sequencing confirming array and MSP results. Validation experiments with an enlarged patient group of Type II EOC samples (PFS <3 years nu2009=u200930; >3 years nu2009=u200918) revealed the CpG island of RUNX3 as significantly more often methylated in patients with short PFS (10/30 vs. 0/18; pu2009<u20090.01). Marker combinations with significantly different methylation frequencies in patient groups reached an increased sensitivity with equal specificity (RUNX3+CAMK2N1; sens 40%; spec 100%; pu2009<u20090.01). RUNX3/CAMK2N1 methylation‐positive patients of the array‐independent subset (nu2009=u200936) showed a significantly lower PFS (pu2009<u20090.01) but no other difference in clinical parameters compared to methylation‐negative patients. Genome‐wide methylation analyses reliably identified markers of potentially prognostic value. Hypermethylation of RUNX3/CAMK2N1 is associated with poor clinical outcome in Type II EOC, also after macroscopic complete resection.

Genome-Wide Association Study for Ovarian Cancer Susceptibility Using Pooled DNA

Recent Genome-Wide Association Studies (GWAS) have identified four low-penetrance ovarian cancer susceptibility loci. We hypothesized that further moderate- or low-penetrance variants exist among the subset of single-nucleotide polymorphisms (SNPs) not well tagged by the genotyping arrays used in the previous studies, which would account for some of the remaining risk. We therefore conducted a time- and cost-effective stage 1 GWAS on 342 invasive serous cases and 643 controls genotyped on pooled DNA using the high-density Illumina 1M-Duo array. We followed up 20 of the most significantly associated SNPs, which are not well tagged by the lower density arrays used by the published GWAS, and genotyping them on individual DNA. Most of the top 20 SNPs were clearly validated by individually genotyping the samples used in the pools. However, none of the 20 SNPs replicated when tested for association in a much larger stage 2 set of 4,651 cases and 6,966 controls from the Ovarian Cancer Association Consortium. Given that most of the top 20 SNPs from pooling were validated in the same samples by individual genotyping, the lack of replication is likely to be due to the relatively small sample size in our stage 1 GWAS rather than due to problems with the pooling approach. We conclude that there are unlikely to be any moderate or large effects on ovarian cancer risk untagged by less dense arrays. However, our study lacked power to make clear statements on the existence of hitherto untagged small-effect variants.

Cervical cancer, HPV 16 E6, variant genotypes, and serology

1 Hill MD, Hachinski V. Stroke treatment: time is brain. Lancet 1998; 352 (suppl III): 10–14. 2 Chinese Acute Stroke Trial collaborative group. CAST: a randomised placebocontrolled trial of early aspirin use in 20 000 patients with acute ischaemic stroke. Lancet 1997; 349: 1641–49. 3 International Stroke Trial Collaborative Group. The International Stroke Trial (IST): a randomised trial of aspirin, subcutaneous heparin, both, or neither among 19 435 patients with acute ischaemic stroke. Lancet 1997; 349: 1569–81. 4 Antiplatelet Trialists’ Collaboration. Collaborative overview of randomised trials of antiplatelet therapy. I: Prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. BMJ 1994; 308: 81–106. multivariants V-131/350 (9%), and V345/350 (3%). Seroreactivity with the prototype antigen was observed in 79%. Of the seven E6 seronegative patients, the prevalence rate of the prototype was 29%, of the variant V-350 43%, and of the variant V-131/350 29%. All serum samples were reanalysed with five different variant antigens.* All the 27 seropositive serum samples remained seropositive independent of the antigen. No HPV 16 E6 antibodies were detectable in the seven seronegative cases, irrespective of the antigen used, and all seronegative sera were positive for IgG antibodies against Epstein-Barr virus, cytomegalovirus antigens, or both. Age of the seronegative patients (mean 46 years) and stages of disease (71% FIGO stage I, 29% stage II) did not differ from the HPV-seropositive group. Do different HPV 16 serotypes exist? By contrast to extensive genotype analyses, only serological studies using virus-like particles of L1 from different HPV 16 variants have been done and no different serotypes were found. In our study, the same seroreactivity was seen for six different 16 E6 variant antigens, suggesting that different HPV 16 E6 serotypes do not exist. Moreover, HPV 16 E6 negative serum samples cannot be explained on the basis of variant proteins. The presence of HPV 16 E6 variants will not, therefore, alter the outcome of past or current screening tests for cervical cancer that are all based on the prototype E6 antigen.

Tribbles 2 mediates cisplatin sensitivity and DNA damage response in epithelial ovarian cancer

Aim was to identify methylated genes with functional involvement in cisplatin‐resistance development of epithelial ovarian cancer (EOC). Genome‐wide analyses of hypermethylated CpG‐islands in resistant cell lines in combination with qRT‐PCR analyses were used to identify epigenetically silenced genes. EOC‐Type‐II tumors were analyzed for gene methylation and expression and TCGA data were interrogated in‐silico. Experiments revealed 37 commonly hypermethylated genes in resistant cells of which Tribbles 2 (TRIB2) showed the most pronounced downregulation on mRNA level and was characterized further. TRIB2 showed a reactivation after 5′‐Aza‐Cytidine treatment in resistant cells but a cisplatin‐dependent, prominent upregulation on mRNA level in sensitive cells, only. Re‐expression in resistant A2780 cells increased the sensitivity to cisplatin and other DNA‐damaging agents, but not taxanes. Contrary, knockdown of TRIB2 increased resistance to cisplatin in sensitive cells. TRIB2 was involved in the induction of a cisplatin‐dependent cell cycle arrest and apoptosis by influencing p21 and survivin expression. An increased Pt‐DNA‐adduct formation in TRIB2 re‐expressing cells did not translate in higher levels of dsDNA damage (yH2AX‐foci). Thus, TRIB2 is potentially involved in the signal transduction from nucleotide excision repair of intrastrand cross links. Importantly, patient stratification of two homogenous cohorts of EOC‐Type‐II patients from Jena (nu2009=u200938) and the TCGA (nu2009=u2009149) by TRIB2 mRNA expression consistently revealed a significantly decreased PFS for patients with low TRIB2 levels (log‐rank pu2009<u20090.05). Tumors from resistant patients expressed the lowest levels of TRIB2. Downregulation of TRIB2 contributes to platin‐resistance and TRIB2 expression should be validated as prognostic and predictive marker for EOC.

Evaluating the ovarian cancer gonadotropin hypothesis: A candidate gene study - eScholarship

OBJECTIVEnOvarian cancer is a hormone-related disease with a strong genetic basis. However, none of its high-penetrance susceptibility genes and GWAS-identified variants to date are known to be involved in hormonal pathways. Given the hypothesized etiologic role of gonadotropins, an assessment of how variability in genes involved in the gonadotropin signaling pathway impacts disease risk is warranted.nnnMETHODSnGenetic data from 41 ovarian cancer study sites were pooled and unconditional logistic regression was used to evaluate whether any of the 2185 SNPs from 11 gonadotropin signaling pathway genes was associated with ovarian cancer risk. A burden test using the admixture likelihood (AML) method was also used to evaluate gene-level associations.nnnRESULTSnWe did not find any genome-wide significant associations between individual SNPs and ovarian cancer risk. However, there was some suggestion of gene-level associations for four gonadotropin signaling pathway genes: INHBB (p=0.045, mucinous), LHCGR (p=0.046, high-grade serous), GNRH (p=0.041, high-grade serous), and FSHB (p=0.036, overall invasive). There was also suggestive evidence for INHA (p=0.060, overall invasive).nnnCONCLUSIONSnOvarian cancer studies have limited sample numbers, thus fewer genome-wide susceptibility alleles, with only modest associations, have been identified relative to breast and prostate cancers. We have evaluated the majority of ovarian cancer studies with biological samples, to our knowledge, leaving no opportunity for replication. Using both our understanding of biology and powerful gene-level tests, we have identified four putative ovarian cancer loci near INHBB, LHCGR, GNRH, and FSHB that warrant a second look if larger sample sizes and denser genotype chips become available.