Abstract. Aberrant DNA methylation is responsible for the epigenetic silencing of genes associated with tumourigenesis and progression of cancer. In this study, we assessed the methylation status of eight genes in 49 snap-frozen primary breast tumours. Epigenetic alterations of 8 genes were analysed with methylation-specific polymerase chain reaction (MS-PCR) (DCR1, DAPK1, RASSF1A and DCR2) or methylationsensitive high-resolution melting analysis (MS-HRM) (APC, MGMT, GSTP1 and PTEN). MS-HRM performance was validated by bisulfite pyrosequencing regarding the methylation levels of MGMT. Promoter methylation was observed in APC 54.34%, 40.4% DCR1, 37.5% DAPK1, 33.3% RASSF1A, 22.44% MGMT, 16.6% GSTP1, 6% PTEN and 0% DCR2 promoters, respectively. Interestingly, 37 out of 49 cases (75.5%) displayed aberrant promoter methylation in at least one gene. An association of MGMT promoter methylation with age and tumour grade was recorded. Moreover, a correlation with advanced T-category was elicited for GSTP1, RASSF1 and DAPK1 promoter methylation. Finally, concurrent methylation of several genes showed a marginal statistical relationship with N-category. We conclude that APC, DCR1, DAPK1 and RASSF1A promoter methylation represents a common event in breast cancer tumourigenesis. Our results suggest that GSTP1, RASSF1, DAPK1 and MGMT may be implicated in the acquisition of a more aggressive phenotype in breast cancer.

Deregulation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is closely associated with cancer development and cancer progression. PIK3CA, AKT1, and PTEN are the fundamental molecules of the PI3K/AKT pathway with increased mutation rates in cancer cases leading to aberrant regulation of the pathway. Even though molecular alterations of the PI3K/AKT pathway have been studied in breast cancer, correlations between specific molecular alterations and clinicopathological features remain contradictory. In this study, we examined mutations of the PI3K/AKT pathway in 75 breast carcinomas using high-resolution melting analysis and pyrosequencing, in parallel with analysis of relative expression of PIK3CA and AKT2 genes. Mutations of PIK3CA were found in our cohort in 21 cases (28 %), 10 (13 %) in exon 9 and 11(15 %) in exon 20. Mutation frequency of AKT1 and PTEN genes was 4 and 3 %, respectively. Overall, alterations in the PI3K/AKT signaling cascade were detected in 35 % of the cases. Furthermore, comparison of 50 breast carcinomas with adjacent normal tissues showed elevated PIK3CA messenger RNA (mRNA) levels in 18 % of tumor cases and elevated AKT2 mRNA levels in 14 %. Our findings, along with those of previous studies, underline the importance of the PI3K/AKT pathway components as potential biomarkers for breast carcinogenesis.

Basal Cell Carcinoma (BCC) is the most common skin malignancy. Genes related to the Ras/Raf signalling pathway have been implicated in the pathogenesis of skin cancer. The objective of this study was to investigate the presence of B-Raf mutations in sporadic BCCs as well as its correlation with the phenotype of microsatellite instability (MSI), the clinicopathological parameters of the tumours and p53 protein expression. 83 BCC specimens were screened for B-Raf mutations, applying polymerase chain reaction, single-stranded conformation polymorphism (PCR-SSCP) and DNA sequencing. MSI status was examined using mononucleotide microsatellite markers and p53 protein expression was demonstrated by immunohistochemical staining. A C to T transition at 1790 nucleotide leading to a silent mutation L597L; and a T to A transversion causing an amino acid change (F610I) have been found. MSI was detected in 5% of the cases and p53 accumulation was present in 37/83 samples studied. Although rare B-Raf alterations have been observed in BCC, none of them harboured the hot-spot mutation T1799A commonly present in melanomas and colon carcinomas. Consequently, no correlation could be determined between B-Raf alterations, MSI status, the clinicopathological features and p53 protein expression. Our results are in favour of a secondary importance for Ras signalling cascade genes in BCC pathogenesis.