Background:The detection of V600E BRAF mutation in melanoma is fundamental since here BRAF inhibitors represent an effective treatment. Non-V600E BRAF mutations that may also respond are not detected by certain screening methods. Thus, knowledge about detection of these mutations is needed.Methods:A total of 276 tumour samples from 174 melanoma patients were investigated for BRAF mutations by pyrosequencing. Rare mutations were confirmed by capillary sequencing and compared with findings from COBAS test and immunohistochemistry using a novel BRAF antibody. Melanoma type, localisation, and survival were summarised.Results:BRAF mutations were found in 43% of patients (124 tumours in 75 patients). Among those, 14 patients (18.7%) exhibited rare mutations. The V600EK601del and V600DK601del mutations have never been described before in melanoma. Furthermore, V600K, V600E2, and V600D, V600G, V600R, and L597S mutations were detected. Mutations were not detected by COBAS test in 7 out of these 14 patients and immunohistochemistry only reliably detected patients with the V600E2 and V600EK601del mutation.Conclusion:Accurate diagnosis of rare BRAF mutations is crucial. We show that pyrosequencing is accurate, highly sensitive, reliable, and time saving to detect rare BRAF mutations. Missing these rare variant mutations would exclude a subset of patients from available effective BRAF-targeting therapy.

The prognostic impact of BRAF-V600 tumor mutations in stage I/II melanoma patients has not yet been analyzed in detail. We investigated primary tumors of 437 patients diagnosed between 1989 and 2006 by Sanger sequencing. Mutations were detected in 38.7% of patients and were associated with age, histological subtype as well as mitotic rate. The mutational rate was 36.7% in patients with disease-free course and 51.7% in those with subsequent distant metastasis (p = 0.031). No difference in overall survival (p = 0.119) but a trend for worse distant-metastasis-free survival (p = 0.061) was observed in BRAF mutant compared to BRAF wild-type patients. Independent prognostic factors for overall survival were tumor thickness, mitotic rate and ulceration. An interesting significant prognostic impact was observed in patients with tumor thickness of 1 mm or less, with the mutation present in 6 of 7 patients dying from melanoma. In conclusion, no significant survival differences were found according to BRAF-V600 tumor mutations in patients with primary melanoma but an increasing impact of the mutational status was observed in the subgroup of patients with tumor thickness of 1 mm or less. A potential role of the mutational status as a prognostic factor especially in this subgroup needs to be investigated in larger studies.

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The heterodimeric integrins are important receptors for the attachment of cells to their extracellular matrix. Here, we studied the attachment of human mesenchymal stem cells (MSCs) to type I collagen (col-1), which is part of the extracellular matrix in bone, skin, and connective tissues. Furthermore, we examined how TGF-beta influences the integrin expression and attachment of MSC. Using flow cytometry, immunoblot, and RT-PCR, we report that MSC express several integrin subunits, including the alpha(2)beta(1) integrin (VLA-2, CD49b/CD29). TGF-beta increases the expression of integrin subunits alpha(2), alpha(6), and beta(1) in MSC, thereby enhancing the attachment of MSC to col-1. The TGF-beta-mediated up-regulation of the expression of the integrin subunits alpha(2) and alpha(6) is mainly mediated in MSC by Smad2.

BackgroundThe impact of BRAF tumor mutations on the natural course of disease of melanoma patients is controversial.Patients and MethodsWe analyzed the mutational status and overall survival of 215 patients receiving treatment with dacarbazine or temozolomide. All patients who started first-line treatment at our institution between 2000 and 2010 were included to prevent selection and bias due to thereafter arising therapeutic options.ResultsNo patient received BRAF- or MEK-inhibitors during follow-up. Survival was associated with the pattern of visceral involvement, the presence of brain metastases and the serum lactate dehydrogenase level (all p<0.001). The BRAF-V600 mutational status was not associated with survival and no differences in overall survival were detected according to age, gender or to the cytotoxic agent used for therapy. In Cox regression analysis the presence of brain metastases (hazard ratio 2.3; p<0.001) and an elevated serum LDH (hazard ratio 2.5; p<0.001) were the only factors, which independently predicted survival.ConclusionsNo differences in prognosis were observed according to the BRAF mutational status in patients with distant metastasis treated with monochemotherapy.

C57BL ⁄ 6 CD4 + T cells providing a possible explanation for our findings (17). Thus, in Leishmania-resistant C57BL ⁄ 6 mice -a good surrogate model for human, self limited CL -IL-1 signalling is dispensable for protective immunity.

:  Several viruses are known to cause cancer, such as human herpes virus 8 in Kaposi sarcoma and human papilloma viruses in cervical cancer. Recently, Merkel cell polyoma virus (MCPyV) has been described in 80% of Merkel cell carcinomas (MCC). Similarly to MCC and Kaposi sarcoma, melanoma incidence is increased in immunosuppressed patients. We asked whether infection by known or yet unknown viruses may play a role in melanoma development as well. To detect viral sequences expressed in melanoma cells, we analysed three melanoma metastases by whole‐transcriptome sequencing and digital transcriptome subtraction. None of the samples investigated harboured viral sequences. In contrast, artificial viral sequences and MCPyV transcripts used as a positive control for the bioinformatics analysis were detected. This renders it less likely that viruses are frequently involved in melanoma induction. A larger number of melanoma transcriptome sequencings are required to rule out viruses as a relevant pathogen.

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