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ABSTRACT

BackgroundThe approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.MethodsSamples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).ResultsThere was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.ConclusionTherefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.

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Adult human mesenchymal stem cells (hMSC) are involved in wound healing and regeneration of mesodermal tissue, but the underlying homing mechanisms are not well understood. Fibrin clot formation is associated with most wound healing processes and potentially guides the recruitment of hMSC. The objective of this study is the investigation of a fibrinolytic capacity, which is required for hMSC to migrate into a wounded tissue and thus to contribute to tissue regeneration. Using RT-PCR, semiquantitative real-time PCR and ELISA, we detected key components of the fibrinolytic cascade, including the urokinase plasminogen activator (uPA) and its receptor (uPAR), the tissue plasminogen activator (tPA) and the plasminogen activator inhibitor (PAI), suggesting a strong fibrinolytic activity of hMSC. To test this activity in a functional assay, we cultured fibrin-embedded hMSC in vitro for 7 days. The cells efficiently dissolved the surrounding fibrin mesh into the fibrin degradation products, the fibrinopeptides. The fibrinolytic activity of hMSC and human dermal fibroblasts, known to be critically involved in skin wound extracellular matrix remodeling, was similar. Our results suggest that a high intrinsic fibrinolytic capacity of hMSC mediates the invasion into a fibrin clot of a wounded tissue.