Abstract
In melanoma, the RAS/RAF/MEK/ERK signalling pathway is an area of great interest, because it regulates tumor cell proliferation and survival. A varying mutation rate has been reported for B-RAF and N-RAS, which has been largely attributed to the differential source of tumor DNA analyzed, e.g., fixed tumor tissues or in vitro propagated melanoma cells. Notably, this variation also interfered with interpreting the impact of these mutations on the clinical course of the disease. Consequently, we investigated the mutational profile of B-RAF and N-RAS in biopsies and corresponding cell lines from metastatic tumor lesions of 109 melanoma patients (AJCC stage III/IV), and its respective impact on survival. 97 tissue biopsies and 105 biopsy-derived cell lines were screened for B-RAF and N-RAS mutations by PCR single strand conformation polymorphism and DNA sequencing. Mutations were correlated with patient survival data obtained within a median follow-up time of 31 months. B-RAF mutations were detected in 55% tissues and 51% cell lines, N-RAS mutations in 23% tissues and 25% cell lines, respectively. There was strong concordance between the mutational status of tissues and corresponding cell lines, showing a differing status for B-RAF in only 5% and N-RAS in only 6%, respectively. Patients with tumors carrying mutated B-RAF showed an impaired median survival (8.0 versus 11.8 months, p = 0.055, tissues; 7.1 versus 9.3 months, p = 0.068, cell lines), whereas patients with N-RAS-mutated tumors presented with a favorable prognosis (median survival 12.5 versus 7.9 months, p = 0.084, tissues; 15.4 versus 6.8 months, p = 0.0008, cell lines), each in comparison with wildtype gene status. Multivariate analysis qualified N-RAS (p = 0.006) but not B-RAF mutation status as an independent prognostic factor of overall survival. Our findings demonstrate that B-RAF and N-RAS mutations are well preserved during short term in vitro propagation and, most importantly, differentially impact the outcome of melanoma patients.
Highlights
Malignant melanoma is associated with genetic heterogeneity and a complex etiology
Biopsies from solid lesions were subsequently divided into three parts: one was immediately frozen down in liquid nitrogen until further analysis, the second was used for histopathological confirmation of melanoma, and the third was used for establishing a permanently growing melanoma cell line
Additional analyses were performed dividing the patients into three groups, (i) patients harbouring B-RAF mutations, (ii) patients harbouring N-RAS mutations, and (iii) patients without mutations in both genes. These analyses revealed, that with regard to the entire patient population (n = 109), patients harbouring B-RAF mutations show a similar survival as patients without a mutation in B-RAF or NRAS, whereas patients carrying an N-RAS mutation present with a favorable survival (p = 0.11, tissues, Figure 3C; p = 0.004, cell lines, Figure 3F)
Summary
Malignant melanoma is associated with genetic heterogeneity and a complex etiology. In the majority of melanomas, the RAS/RAF/MEK/ERK signalling pathway is constitutively activated either due to oncogenic mutations in B-RAF and N-RAS genes or through autocrine growth factor stimulation [1]. RAS proteins are membrane-bound small G proteins, whereas RAF, MEK, and ERK are cytosolic protein kinases that form a tiered protein kinase cascade downstream of RAS. Signalling is initiated, when active RAS recruits RAF to the plasma membrane for activation through a complex process requiring lipid and protein binding, conformational changes, and regulatory phosphorylation and dephosphorylation events. There are three RAF proteins in mammals, ARAF, B-RAF, and C-RAF, which can all activate MEK, but clearly perform distinct functions in vivo as shown by the phenotypic differences between A-RAF, B-RAF, and C-RAF knock-out mice
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