Epigenetic differences are a common feature of many diseases, including cancer, and disease-associated changes have even been detected in bodily fluids. DNA modification studies in circulating DNA (cirDNA) may lead to the development of specific non-invasive biomarkers. To test this hypothesis, we investigated cirDNA modifications in prostate cancer patients with locally confined disease (n = 19), in patients with benign prostate hyperplasias (n = 20) and in men without any known prostate disease (n = 20). This initial discovery screen identified 39 disease-associated changes in cirDNA modification, and seven of these were validated using the sodium bisulfite-based mapping of modified cytosines in both the discovery cohort and an independent 38-patient validation cohort. In particular, we showed that the DNA modification of regions adjacent to the gene encoding ring finger protein 219 distinguished prostate cancer from benign hyperplasias with good sensitivity (61%) and specificity (71%). We also showed that repetitive sequences detected in this study were meaningful, as they indicated a highly statistically significant loss of DNA at the pericentromeric region of chromosome 10 in prostate cancer patients (p = 1.8 × 10(-6)). Based on these strong univariate results, we applied machine-learning techniques to develop a multi-locus biomarker that correctly distinguished prostate cancer samples from unaffected controls with 72% accuracy. Lastly, we used systems biology techniques to integrate our data with publicly available DNA modification and transcriptomic data from primary prostate tumors, thereby prioritizing genes for further studies. These data suggest that cirDNA epigenomics are promising source for non-invasive biomarkers.

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Background. Glioma is the most common and deadly malignancy of the central ner vous system. Capillary hemangioblastoma (CHB) is a rare and usually benign tumour of brain, occurring as part von Hippel-Lindau (VHL) disease or as a sporadic entity. For an improved understanding of the molecular mechanisms of the pathogenesis of VHL-related tumours and gliomas, we analysed the expression of the tumour suppressor proteins p53 and p16, and promoter methylation of the tumour suppressor genes (TSGs) MGMT and RASSF1A.Materials and methods. Expression of p53 and p16 was analysed in 27 cases of glioma, 5 cases of breast cancer, and 17 cases with VHL-related tumours, including CHBs and pheochromocytomas (PCCs), by means of immunohistochemistry. For the gene promoter methylation and deletion assessment, methylation-specific PCR and differential polymerase chain reaction combined with on-chip electrophoresis were used, respectively.Results. Aberrant expression of the p53 protein was frequent (p < 0.01) in malignant tumours, including gliomas and breast carcinomas, but not in benign tumours (PCC and CHB). Protein p53 alterations were most frequent (65%) in glioblastomas, and a low number (20%) of patients with p53-positive gliomas survived more than one year after diagnosing the disease. Other biomarkers, including loss of p16 expression, promoter hypermethylation of the MGMT gene and promoter hypermethylation or deletion of the RASSF1A gene, were frequent in all groups of tumours.Conclusions. Alterations of the tumour suppressors p16, MGMT and RASSF1A are frequent in VHL-related tumours, while aberrant expression of p53 is only detectable in malignant tumours -gliomas and breast carcinomas.

Background. Both capillary hemangioblastoma (CHB) and pheochromocytoma (PCC) are rare, usually benign tumours occurring sporadically or as part of familial cancer syndromes. The genetic background of most of the inherited cases is well established, but the molecular causes of sporadic cases remains poorly characterized. To better understand the molecular mechanisms of CHB and PCC pathogenesis, we analysed the genetic and epigenetic alterations of the p16 and p14 genes at the CDKN2A locus. Materials and methods. Aberrant methylation of the p16 and p14 genes was analysed in 16 cases with CHB or PCC by means of methylation-specific PCR. The differential polymerase chain reaction was used to prove the occurrence of the genetic deletion of p16. For comparison, 28 cases of glioma-a highly malignant tumour of the brain-was included into the study. Results. Data of our study show that gene p16 is hypermethylated in 25% of CHBs and in 25% of PCCs, while in gliomas this alteration is more frequent (35%) and predominantly occurs in low-grade tumours (67%). Frequent hypermethylation of the p14 gene was observed in PCCs (50%) and CHBs (37.5%), but was less prevalent in gliomas (26%). When all alterations in the CDKN2A locus were considered, including hypermethylation of p16 and p14, and genetic deletion of p16, 75% of PCC, 62.5% of CHB, and 64% of gliomas had at least one alteration of this locus. Conclusions. Our study adds new data to understanding the involvement of the CDKN2A locus in the pathogenesis of CHB and PCC-two of the most common VHLrelated tumours. Furthermore, aberrant methylation in the CDKN2A locus is also frequent in gliomas.