DNA mutations occur randomly and sporadically in growth-related genes, mostly on cytosines. Demethylation of cytosines may lead to genetic instability through spontaneous deamination. Aims were whole genome methylation and targeted mutation analysis of colorectal cancer (CRC)-related genes and mRNA expression analysis of TP53 pathway genes. Long interspersed nuclear element-1 (LINE-1) BS-PCR followed by pyrosequencing was performed for the estimation of global DNA metlyation levels along the colorectal normal-adenoma-carcinoma sequence. Methyl capture sequencing was done on 6 normal adjacent (NAT), 15 adenomatous (AD) and 9 CRC tissues. Overall quantitative methylation analysis, selection of top hyper/hypomethylated genes, methylation analysis on mutation regions and TP53 pathway gene promoters were performed. Mutations of 12 CRC-related genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53) were evaluated. mRNA expression of TP53 pathway genes was also analyzed. According to the LINE-1 methylation results, overall hypomethylation was observed along the normal-adenoma-carcinoma sequence. Within top50 differential methylated regions (DMRs), in AD-N comparison TP73, NGFR, PDGFRA genes were hypermethylated, FMN1, SLC16A7 genes were hypomethylated. In CRC-N comparison DKK2, SDC2, SOX1 genes showed hypermethylation, while ERBB4, CREB5, CNTN1 genes were hypomethylated. In certain mutation hot spot regions significant DNA methylation alterations were detected. The TP53 gene body was addressed by hypermethylation in adenomas. APC, TP53 and KRAS mutations were found in 30, 15, 21% of adenomas, and in 29, 53, 29% of CRCs, respectively. mRNA expression changes were observed in several TP53 pathway genes showing promoter methylation alterations. DNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.

The early molecular detection of the dysplasia-carcinoma transition may enhance the strength of diagnosis in the case of colonic biopsies. Our aims were to identify characteristic transcript sets in order to develop diagnostic mRNA expression patterns for objective classification of benign and malignant colorectal diseases and to test the classificatory power of these markers on an independent sample set. Colorectal cancer (CRC) and adenoma specific transcript sets were identified using HGU133plus2 microarrays and 53 biopsies (22 CRC, 20 adenoma and 11 normal). Ninety-four independent biopsies (27 CRC, 29 adenoma and 38 normal) were analyzed on microarrays for testing the classificatory power of the discriminatory genes. Array real-time PCR validation was done on 68 independent samples (24 CRC, 24 adenoma and 20 normal). A set of 11 transcripts (including CXCL1, CHI3L1 and GREM1) was determined which could correctly discriminate between high-grade dysplastic adenoma and CRC samples by 100% sensitivity and 88.9% specificity. The discriminatory power of the marker set was proved to be high on independent samples in both microarray and RT-PCR analyses. 95.6% of original and 94.1% of cross-validated samples was correctly classified in discriminant analysis. The identified transcripts could correctly characterize the dysplasia-carcinoma transition in biopsy samples, also on a large independent sample set. These markers can establish the basis of gene expression based diagnostic classification of colorectal cancer. Diagnostic RT-PCR cards can become part of the automated routine procedure.

Colorectal cancer (CRC) development is accompanied by changes in expression for several genes; but the details of the underlying regulatory procesess remain unknown. Our aims were to assess the role of epigenetic processes in tumour formation and to identify characteristic DNA methylation and miRNA alterations in the colorectal adenoma-carcinoma sequence. Whole genome expression profiling was performed on colonic biopsy samples (49 healthy normal, 49 colorectal adenoma (AD), 49 CRC); on laser capture microdissected (LCM) epithelial and stromal cells from 6 CRC-normal adjacent tissue (NAT) samples pairs, and on demethylated human CRC cell lines using HGU133 Plus 2.0 microarrays (Affymetrix). Methylation status of genes with gradually altering expression along the AD-CRC sequence was further analysed on 10–10 macrodissected and 5–5 LCM samples from healthy colon, from adenoma and from CRC biopsy samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes was performed with miRWALK algorithm, miRNA expression was analysed on 3 CRC-NAT sample pairs and 3 adenoma tissue samples using the Human Panel I + II (Exiqon). SFRP1 immunohistochemistry experiments were performed. A set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p < 0.01) in the biopsy samples along the adenoma-carcinoma sequence. Three of those (COL1A2, SFRP2, SOCS3) showed hypermethylation and THBS2 showed hypomethylation both in AD and in CRC samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p < 0.01) upregulated in adenoma and tumour samples compared to the healthy colonic tissue controls and could explain the altered expression of genes for which DNA methylation changes do not appear to play role (e.g. BCL2, MAL, PTGS2). Demethylation treatment could upregulate gene expression of genes that were found to be hypermethylated in human CRC tissue samples. Decreasing protein levels of SFRP1 was also observed along the adenoma-carcinoma sequence. Hypermethylation of the selected markers (MAL, PRIMA1, PTGDR and SFRP1) can result in reduced gene expression and may contribute to the formation of colorectal cancer.

Background: Several classification systems are available to assess pathological response to neoadjuvant chemotherapy in breast cancer, but reliable biomarkers to predict the efficiency of primary systemic therapy (PST) are still missing. Deregulation of gap junction channel forming connexins (Cx) has been implicated in carcinogenesis and tumour progression through loss of cell cycle control. In this study we correlated Cx expression and cell proliferation with disease survival and pathological response to neoadjuvant chemotherapy in breast cancers using existing classification systems.Methods: The expression of Cx26, Cx32, Cx43, Cx46 and Ki67 was evaluated in 96 breast cancer patients prior to and after neoadjuvant chemotherapy using duplicate cores in tissue microarrays (TMA). Cx plaques of <1μm were detected with multilayer, multichannel fluorescence digital microscopy. Current classifications to assess residual tumour burden after primary systemic therapy included the EWGBSP, CPS-EG, Miller-Payne, Sataloff and NSABP systems.Results: In our cohort dominated by hormone receptor (ER/PR) positive and HER2 negative cases, only the CPS-EG classification showed prognostic relevance: cases with scores 1–2 had significantly better overall survival (p=0.015) than cases with scores 3–5. Pre-chemotherapy Cx43 expression correlated positively with hormone receptor status both before and after chemotherapy and had a negative correlation with HER2 expression pre-chemotherapy. There was a positive correlation between Cx32 and HER2 expression pre-chemotherapy and between Cx32 and Ki67 expression post-chemotherapy. A negative correlation was found between post-chemotherapy Cx46 and Ki67 expression. Decreased post-chemotherapy Cx26 expression (20%) pre- and post-chemotherapy correlated with significantly better survival in the intermediate prognostic subgroups of EWGBSP TR2b (p_pre-chemo=0.006; Sataloff TB (p_pre-chemo=0.005; p_post-chemo=0.029) and in Miller-Payne G3 (p_pre-chemo=0.002; p_post-chemo=0.012) classifications. Pre-chemotherapy, Cx46 expression was the only marker that correlated with overall survival within these subgroups.Conclusion: Our results suggest that Cx46 and Cx26 expression in breast cancer may improve the assessment of pathological response and refine intermediate prognostic subgroups of residual tumour classifications used after neoadjuvant chemotherapy.

MiRNA expression markers are well characterized in colorectal cancer (CRC), but less is known about miRNA expression profiles in colorectal adenomas. Genome-wide miRNA and mRNA expression analyses were conducted through the colorectal adenoma dysplasia sequence. Furthermore, analysis of the expression levels of miRNAs in matched plasma samples was performed, focusing on biomarker candidates; miRNA and mRNA expression analyses were performed on colorectal biopsies and plasma samples (20 normals; 11 tubular and 9 tubulovillous adenomas; 20 colorectal carcinomas) by miRNA 3.0 and Human Transcriptome Array (Affymetrix) and validated by RT-qPCR. Microarray data were analyzed using Expression Console and mRNA targets were predicted using miRWALK 2.0. Based on microarray analysis, 447 miRNAs were expressed in tissue and 320 in plasma. Twelve were upregulated (miR-31, 8-fold p < 0.001) and 11 were downregulated (miR-10b 3-fold p < 0.001) in neoplastic lesions compared to normal group. Eleven miRNAs showed altered expression between adenoma subtypes (miR-183 2.8-fold change, p < 0.007). Expression level of 24 miRNAs differed between adenoma and CRC groups (including miR-196a, 3.5-fold). Three miRNAs (miR-31, miR-4506, miR-452*) were differentially expressed in adenoma compared to normal both in tissue and plasma samples. miRNA expression data were confirmed by RT-PCR both in plasma and matched tissue samples. MiRNAs showed characteristic expression changes during CRC development in tissue. miRNAs were also presented in plasma and positively correlated with matched tissue expression levels. The identified miRNA expression changes could be verified RT-PCR methods facilitating routine application.

The incidence of malignant melanoma, one of the deadliest cancers, continues to increase. Here we tested connexin (Cx) expression in primary melanocytes, melanoma cell lines and in a common nevus, dysplastic nevus, and thin, thick, and metastatic melanoma tumor progression series involving the tumor microenvironment by utilizing in silico analysis, qRT-PCR, immunocyto-/histochemistry and dye transfer tests. Primary melanocytes expressed GJA1/Cx43, GJA3/Cx46 and low levels of GJB2/Cx26 and GJC3/Cx30.2 transcripts. In silico data revealed downregulation of GJA1/Cx43 and GJB2/Cx26 mRNA, in addition to upregulated GJB1/Cx32, during melanoma progression. In three melanoma cell lines, we also showed the loss of GJA1/Cx43 and the differential expression of GJB1/Cx32, GJB2/Cx26, GJA3/Cx46 and GJC3/Cx30.2. The dominantly paranuclear localization of connexin proteins explained the ~10–90 times less melanoma cell coupling compared to melanocytes. In melanocytic tumor tissues, we confirmed the loss of Cx43 protein, fall of cell membrane and elevated paranuclear Cx32 with moderately increased cytoplasmic Cx26 and paranuclear Cx30.2 positivity during tumor progression. Furthermore, we found Cx43, Cx26 and Cx30 proteins upregulated in the melanoma adjacent epidermis, and Cx43 in the tumor flanking vessels. Therefore, differential connexin expression is involved in melanocytic tumor progression where varying connexin isotypes and levels reflect tumor heterogeneity-related bidirectional adaptive interactions with the microenvironment.