ERK/pERK expression and B-raf mutations in colon adenocarcinomas: correlation with clinicopathological characteristics.

BACKGROUND: Colorectal (CRC) carcinogenesis through various morphological stages has been linked to several genetic and epigenetic changes. The Raf/MEK/ERK (MAPK) signal transduction cascade is an important mediator of a number of cellular fates. METHODS: In this study, we investigated the presence of B-raf and K-ras mutations in 94 consecutive cases of primary colon adenocarcinoma in correlation with the immunohistochemical expression of total and activated ERK and the expression of mismatch repair proteins (MMR) hMLH1 and hMSH2 as well as their correlations with standard clinicopathological parameters. RESULTS: The immunostaining pattern for total and activated ERK was nuclear and cytoplasmic. hMLH1 and hMSH2 proteins were preserved in 45/63 (71.43%) cases and 35/53 (66.04%) cases respectively. Total ERK nuclear expression, was positively correlated with tumor stage (p = 0.049), whereas nuclear pERK expression was positively correlated with histological grade (p = 0.0113) and tumor stage (p = 0.0952), although the latter relationship was of marginal significance. DNA sequencing showed that 12 samples (12.7%) had a mutation in B-RAF Exon 15 and none in Exon 11, whereas 22 (23.4%) had a K-ras mutation. Disruption of the MAP kinase pathway-either through K-ras or B-raf mutation-was detected in 37% of all the examined cases, although the overexpression of total and activated ERK1/2 was not correlated with the mutational status of K-ras or B-raf genes. Finally, the preservation of hMLH1 or hMSH2 immunoexpression was not correlated with the presence of B-raf and/or K-ras mutations. CONCLUSIONS: In this study, we present evidence that ERK activation occurs in a K-ras or B-raf -independent manner in the majority of primary colon cancer cases. Moreover, B-raf mutations are not associated with mismatch-repair deficiency through loss of hMLH1 or hMSH2 expression. Activated ERK could possibly be implicated in tumor invasiveness as well as in the acquisition of a more aggressive phenotype.

TGF-betaRII, BAX, IGFIIR, caspase-5, hMSH3 and hMSH6 alterations are not associated with microsatellite instability or p53 mutations in invasive urothelial carcinoma of the urinary bladder.

AIM: The aim of this study was to determine the potential synchronous contribution of alterations in TGF-betaRII, BAX, IGFIIR, caspase-5, hMSH3 and hMSH6 genes to the development and clinical outcome of bladder cancer, in relation to p53 mutations, microsatellite status and hMLH1/hMSH2 expression. METHODS: Molecular biology techniques as well as immunohistochemistry were applied in 69 samples from patients with urothelial carcinoma. RESULTS: Microsatellite alterations were observed in TGF-betaRII(A)(10 )(16%) and BAX(G)(8 )(3%), irrespective of the presence of p53 mutations, but not in IGFIIR(G)(8), caspase-5(A)(10, ) hMSH3(A)(8) and hMSH6(C)(8). A statistically significant correlation could be found only between hMLH1 expression and the presence of microsatellite instability (Fisher's exact test, p = 0,013). Survival analysis indicated that apart from grade and T-category, hMLH1 expression was the only parameter significantly affecting overall survival (p = 0.021 in univariate and p = 0.015 in multivariate analysis) and recurrence-free survival (p = 0.0463 in univariate and p = 0.022 in multivariate analysis). CONCLUSIONS: We conclude that alterations of the examined target genes of MSI are rare in urinary bladder carcinoma and they are not associated with microsatellite instability or the presence of p53 mutations.

Mutations of microsatellite instability target genes in sporadic basal cell carcinomas.

Microsatellite instability (MSI) caused by a defective DNA mismatch repair (MMR) system is one of the phenotypes of genomic instability, accounting for the tumorigenesis of certain types of cancers conveying clinical and prognostic significance. Genes such as TGF-betaRII, IGFIIR, hMSH3, and hMSH6 include coding mononucleotide repeats that are known targets for mutations in MSI-high tumors. The aim of our study was to investigate the prevalence of mutations in the above 4 MSI target genes in correlation with the MSI status of 75 basal cell carcinomas (BCCs), including aggressive-growth BCCs and cases with perineural invasion. TGF-betaRII or hMSH3 frameshift mutations were identified in 5% of the BCCs, including two cases of aggressive-growth subtype, whereas there were no microsatellite alterations in the IGFIIR and hMSH6 genes. Mutations at the mononucleotide repeats within the hMSH3 and TGF-betaRII genes occurred in certain BCCs, not always in association with MSI. It seems likely that microsatellite alterations may be important in the development of individual cases of BCCs despite the low frequency of MSI in our cohort.

Expression of mismatch repair enzymes, hMLH1 and hMSH2 is not associated with microsatellite instability and P53 protein accumulation in basal cell carcinoma.

Microsatellite instability (MSI) constitutes an alternative-to the chromosomal instability-pathway of carcinogenesis for certain tumour types with prognostic and therapeutic significance for the respective patients. MSI is caused by mutations in mismatch repair (MMR) genes, mainly hMLH1, hMSH2, leading to a defective MMR system. The role of MSI in basal cell carcinoma (BCC) has not been clearly delineated yet. p53 gene as a target for ultraviolet radiation-induced mutations may enhance genomic instability in BCC, with loss of its function. Our aim was to investigate the involvement of MSI and expression of hMLH1 and hMSH2 in parallel with P53 protein accumulation in the pathogenesis of BCC and its possible correlation to the clinicopathological features of the patients. The presence of MSI was investigated in 76 BCCs using mononucleotide microsatellite markers, BAT-25, BAT-26 and TGF-beta receptor type II (TGF-beta-RII). Additionally, 3 dinucleotide markers were analysed in 20 cases in which matched normal tissue was available. The expression of hMLH1, hMSH2 and P53 proteins was evaluated by immunohistochemical analysis. Alterations of the BAT-26 marker were observed in one fibroepithelioma of Pincus, one nodular and one multifocal superficial BCC. A keratotic BCC showed an altered BAT-25 locus. Two samples, a multifocal superficial and a nodular BCC, displayed MSI at two markers (BAT-25 and BAT-26; and BAT-25 and TGF-beta-RII, respectively). Three more cases, a metatypical, a multifocal superficial and a signet ring BCC exhibited frameshift mutations in the TGF-beta-RII. No sample showed length alterations at the dinucleotide markers examined. hMLH1 and hMSH2 protein immunohistochemical expression was scored positive in 46 and 49 out of 52 cases respectively. P53 accumulation was observed in 27 out of 56 samples. Correlation of the molecular and immunohistochemical findings with the clinicopathological parameters produced no statistically significant results. No correlation between MSI and hMLH1, hMSH2 or P53 protein expression was determined. MSI appears to play a minor role in the pathogenesis of BCCs being present only in a small subset of such tumours.

Epigenetic alterations in sporadic basal cell carcinomas.

Basal cell carcinoma (BCC) is the most common malignant human neoplasm characterized by slow growth and virtual absence of metastases. Recently, it has become evident that along with genetic mutations epigenetic alterations play a key role in the pathogenesis of human cancer. We searched for promoter methylation of hMLH1, RASSF1A, DAPK, APC, DCR1 and DCR2 genes and BRAF mutations in BCCs in association with the clinicopathological parameters and the histological subtypes of the tumours. Fifty-two BCCs, 17 FFPE along with 35 fresh tissue samples with matching normal tissues for 26 cases were analyzed by methylation-specific PCR to assess the methylation status of hMLH1, RASSF1A, DAPK, APC, DCR1 and DCR2 genes after sodium bisulfite treatment of the tumour and normal DNA. hMLH1 and DCR1 gene expression was investigated by immunohistochemistry. BRAF mutations were studied by high resolution melting analysis. Methylation was detected at a variable frequency of 44, 33, 32.5, 32 and 14 % of DCR2, APC, DCR1, RASSF1 and DAPK promoters, respectively, whereas methylation of hMLH1 promoter was absent. No BRAF mutations were found. There was no correlation between the frequency of the promoter methylation of the above-mentioned genes and the clinicopathological features or the histological subtypes of the tumours. The relatively high frequency of RASSF1A, DCR1, DCR2 and APC promoter methylation may imply that methylation constitutes an important pathway in the tumourigenesis of BCC that could provide new opportunities in developing epigenetic therapies for BCC patients. Nevertheless, further studies are needed to establish the above-mentioned hypothesis.

B-Raf mutations, microsatellite instability and p53 protein expression in sporadic basal cell carcinomas.

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.

Is there any potential link among caspase-8, p-p38 MAPK and bcl-2 in clear cell renal cell carcinomas? A comparative immunohistochemical analysis with clinical connotations.

BACKGROUND: Clear cell renal cell carcinomas (ccRCCs) constitute the most common renal carcinomas, characterized by a relatively aggressive clinical course. Thus, scientific research is targeting towards the identification of immunohistochemical and molecular markers that could be useful regarding diagnosis, appropriate therapy and prediction of prognosis. In the present study we assessed and correlated the expression of caspase-8, phosphorylated p38 mitogen-activated protein kinase (p-p38) and bcl-2 protein with histopathological features and clinical outcome of 27 patients with ccRCCs. METHOD: Immunohistochemistry in formalin-fixed and paraffin-embedded tissue sections was performed. The associations among various features were assessed utilizing statistical analysis. RESULTS: We found that increased expression of cytoplasmic caspase-8 and bcl-2 protein was strongly associated with low Fuhrman's grade of carcinomas (p = 0.019 and p = 0.041, respectively). On the other hand, increased p-p38 expression was significantly related to high Fuhrman's grade (p = 0.006). Moreover, high bcl-2 expression was correlated with low pathological stage of ccRCCs (p = 0.026). Increased expression of cytoplasmic caspase-8 as well as low-grade tumors (grade 1 and 2) implied a greater probability of patients' survival, in univariate statistical analysis (p = 0.037 and p = 0.019, respectively). Neither p-p38 nor bcl-2 expression was significantly linked to patients' survival. There were not emerged statistically significant associations among caspase-8, p-p38 kinase and bcl-2 protein. CONCLUSION: For the first time the prognostic impact of caspase-8 and p-p38 was studied in a series of ccRCCs, using immunohistochemistry in formalin-fixed and paraffin-embedded tissue sections. The suggestive relationship of caspase-8 with patients' clinical outcome, as well as the role of p-p38 within different grade categories, mandates further studies in larger cohorts of RCCs.

Comparative immunohistochemical analysis of aurora-A and aurora-B expression in human glioblastomas. Associations with proliferative activity and clinicopathological features.

In the present study, we carried out a comparative immunohistochemical analysis of aurora-A and aurora-B expression in 40 patients with primary glioblastomas, and attempted to identify any associations with Ki-67 index and the patients' clinical features. The impact of various treatment modalities and proliferative activity on patient outcome was also assessed. Immunohistochemistry was carried out using formalin-fixed and paraffin-embedded tissue sections. Aurora-A expression was higher in tumors with high Ki-67 expression (p=0.01) and was positively, though marginally, related to aurora-B expression (p=0.085). Aurora-B expression was not linked to Ki-67 expression (p=0.182). Lower aurora-A immunohistochemical expression, chemotherapy administration, and tumor localization in one lobe of the brain implied a greater probability of patient survival in univariate analysis (p=0.044, p=0.008, p=0.041, respectively). Ki-67 and aurora-B immunoreactivities were not associated with patient survival (p=0.918 and p=0.539, respectively). To our knowledge, for the first time, the association between aurora-A and aurora-B expression, the correlation of aurora-A with Ki-67 index, and the prognostic impact of aurora-A expression were assessed in glioblastomas. Although we addressed a prognostic connotation of aurora-A, we presume that aurora-A and aurora-B play a complicated role within glioblastomas. Further examinations of larger series are required, so that definite conclusions can be drawn.