Abstract
BackgroundDownstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of mutations within the kinase domain including the activating V599E and V599K transitions.MethodsWe here investigated a representative series of 60 resection specimens of cutaneous and subcutaneous melanoma metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) gel electrophoresis.ResultsSequencing of cloned PCR-SSCP amplicons resulted in 24 (40%) samples harbouring somatic mutations which is not exceeding the mutation frequency in recently investigated primary melanomas. The activating mutation T1796A was present in 24/60 (40%) resection specimens, followed in frequency by the oncogenic g1795A mutation in 8/60 (13%) cases. As to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 19/60 (32%) and 6/60 (10%) cases, resepectively, but were not associated with enhanced risk for subsequent metastasis in patients' follow up. In comparison to the primary melanomas that we recently investigated, the spectrum of predicted B-raf protein mutations narrowed significantly in the cutaneous/subcutaneous metastases. Unexpectedly, V599 and V599E mutations were absent in cutaneous/subcutaneous metastases derived from acrolentiginous melanomas as preceding primary tumours.ConclusionDuring transition from primary melanomas towards cutaneous/subcutaneous metastases, the spectrum of predicted B-raf mutations narrows significantly. Focusing on the V599E and V599K, these oncogenic mutations are likely to affect melanocyte-specific pathways controlling proliferation and differentiation.
Highlights
Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of mutations within the kinase domain including the activating V599E and V599K transitions
Pattern of B-raf exon 15 mutations Assessing the presence of amplifyable DNA by polymerase chain reaction (PCR) amplification of a 184 bp human A-myb gene fragment, 60 resection specimens of melanoma cutaneous/subcutaneous metastases were positive for both cancerous and matched noncancerous tissues
These melanoma resections specimens were analysed by PCR and single-strand conformation polymorphism (SSCP) gel electrophoresis for mutations in exon 15 of the B-raf gene. 24/60 (40%) cases exhibited SSCP patterns distinctly different obtained from corresponding adjacent normal tissues (Figure 1)
Summary
Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of mutations within the kinase domain including the activating V599E and V599K transitions. The B-raf oncogene has been reported to be mutated, among other carcinomas, in a majority of melanoma cell lines [3,4]. All mutations were within the kinase domain, with a single amino acid substitution (V599E) accounting for 95% of B-raf mutations in melanoma cell lines [3,4] and leading to constitutive kinase activity [3]. In preliminary series of primary melanomas, 5/9 [3] and 4/5 [5] of the resection specimens harboured this V599E mutation, and only a single different mutation occurred [3]. Investigating a representative series of 50 resection specimens of primary cutaneous melanomas, we detected 12/50 cases (24%) to harbour this V599E transition [6]
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