Preponderance of the oncogenic V599E and V599K mutations in B-raf kinase domain is enhanced in melanoma cutaneous/subcutaneous metastases

Abstract

Background: 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.

Methods: We 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.

Results: Sequencing 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, respectively, 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.

Conclusion: During 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.

Figure 1

PCR-SSCP analysis of B-raf exon 15 in resection specimens from cutaneous/subcutaneous melanoma metastases. Cases 7, 23, and 54 exhibit different DNA fragment mobilities when compared to the respective normal tissues from patients. Suspecting these tumour specimens to harbour mutations, these samples were subjected to DNA sequencing.

Figure 2

Part of the DNA sequence of the activation segment of the B-raf kinase domain. Representative melanoma resection specimens are shown which harbour missense mutations. In comparison to wild type DNA sequence (upper electropherogram, forward sequence on the left, reverse sequence on the right), melanoma cases 7, 23 and 54 exhibit nucleotide transitions, among them the activating T1796A mutation.