title: Constitutive activation of the Ras-Raf signaling pathway in metastatic melanoma is associated with poor prognosis.
abstract: BACKGROUND
  Genes of the Raf family encode kinases that are regulated by Ras and mediate cellular responses to growth signals. Recently, it was shown that activating mutations of BRaf are found with high frequency in human melanomas. The Ras family member most often mutated in melanoma is NRas.
  
  
  METHODS
  The constitutive activation of the Ras/Raf signaling pathway suggests an impact on the clinical course of the tumor. To address this notion, we analyzed tumor DNA from 114 primary cutaneous melanomas and of 86 metastatic lesions obtained from 174 patients for mutations in BRaf (exons 15 and 11) and NRas (exons 1 and 2) by direct sequencing of PCR products and correlated these results with the clinical course.
  
  
  RESULTS
  In 57.5% of the tumors either BRaf or NRas were mutated with a higher incidence in metastatic (66.3%) than in primary lesions (50.9%). Although the majority of BRaf mutations affected codon 599, almost 15% of mutations at this position were different from the well-described exchange from valine to glutamic acid. These mutations (V599R and V599K) also displayed increased kinase and transforming activity. Surprisingly, the additional BRaf variants D593V, G465R and G465E showed a complete loss of activity in thein vitrokinase assay; however, cells overexpressing these mutants displayed increased Erk phosphorylation. The correlation of mutational status and clinical course revealed that the presence of BRaf/NRas mutations in primary tumors did not negatively impact progression free or overall survival. In contrast, however, for metastatic lesions the presence ofBRAF/NRASmutations was associated with a significantly poorer prognosis, i.e. a shortened survival.
  
  
  CONCLUSIONS
  We demonstrate a high - albeit lower than initially anticipated - frequency of activating BRaf mutations in melanoma in the largest series of directly analyzed tumors reported to date. Notably, the clinical course of patients harboring activating BRaf mutations in metastatic melanoma was significantly affected by the presence of a constitutive BRaf activation in these.
authors.affiliation: Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Universität Würzburg, Versbacher Str. 5, D-97078 Würzburg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Houben
authors.affiliation: Klinik und Poliklinik für Haut- und Geschlechtskrankheiten, Universität Würzburg, Josef Schneider Str. 2, D-97078 Würzburg, Germany.
authors.forename: Jürgen C
authors.initials: JC
authors.lastname: Becker
authors.affiliation: Nanogen Recognomics GmbH, Industrial Park Höchst, Building G 830, D-65926 Frankfurt am Main, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kappel
authors.affiliation: Klinik und Poliklinik für Haut- und Geschlechtskrankheiten, Universität Würzburg, Josef Schneider Str. 2, D-97078 Würzburg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Terheyden
authors.affiliation: Klinik und Poliklinik für Haut- und Geschlechtskrankheiten, Universität Würzburg, Josef Schneider Str. 2, D-97078 Würzburg, Germany.
authors.forename: Eva-B
authors.initials: EB
authors.lastname: Bröcker
authors.affiliation: Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Universität Würzburg, Versbacher Str. 5, D-97078 Würzburg, Germany.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Goetz
authors.affiliation: Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Universität Würzburg, Versbacher Str. 5, D-97078 Würzburg, Germany.
authors.forename: Ulf R
authors.initials: UR
authors.lastname: Rapp