These data suggest that melanoma growth, invasion, and metastasis are attributable to constitutively activated ERK apparently mediated by excessive growth factors through autocrine mechanisms and BRAF kinase activation.
Our data show that BRAF mutation is common in melanoma metastases, regardless of their site, that mutations include both exons 11 and 15, and suggest that anti-RAS/RAF strategies may be effective in metastatic melanoma patients.
Examination of 126 patients with papillary thyroid cancer showed that BRAF mutation correlated significantly with distant metastasis (P = 0.033) and clinical stage (P = 0.049).
Thirty-three samples (7 of 25 primaries, 15 of 25 regional metastases, 5 of 25 nodal metastases, and 6 of 10 distant metastases) harbored the V599EB-RAF mutation (39%), 12 contained a Q61R N-RAS mutation and 5 a Q61K N-RAS mutation.
Primary melanomas (n = 71) and corresponding metastases (n = 88) from 71 patients were screened for BRAF exon 11 and exon 15 mutations using single-strand conformational polymorphism and nucleotide sequence analysis
NRAS was exclusively mutated in nine of 47 (19%) cell lines and two of 16 (13%) metastases, whereas BRAF was solely mutated in 28 of 47 (60%) cell lines and nine of 16 (56%) metastases.
BRAF mutations have been found in a high percentage of melanoma cell lines and metastases; however, only a few studies with a limited number of specimens have focused on primary melanomas.
Recently, BRAF has been identified as a common site of activating mutations, and, although many reports focus on its growth-promoting effects, this pathway has also been implicated in progression toward metastatic disease.
BRAF(V599E) tended to be associated, although not significantly, with a greater volume and extension of the tumour and with lymph-nodal metastases at surgery.
Sixty-eight samples (20 of 36 primaries, 18 of 27 regional metastases, 16 of 40 nodal metastases, and 9 of 12 distant metastases) harbored the V599EB-RAF mutation (59%), 17 contained a Q61R N-RAS mutation, and 4 contained a Q61K N-RAS mutation.
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.
In our study of 51 primary nodular melanomas and 18 paired metastases, we found mutations in BRAF (codon 600, previously denoted 599) in 15 primary tumors (29%) and eight metastases (44%).
In line with the previous reports, NRAS/BRAF mutations were rare; only one metastatic tumor had an NRAS E61R mutation, and one primary tumor and two metastases harbored BRAFV599E mutations.
With regard to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 15 (62%) and five (21%) of the 24 positive metastases, respectively.
Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases.
K-Ras, B-Raf and p53 mutations were detected in 27, 3 and 32% of the cases, with K-Ras mutations significantly associated with metastatic tumour (P=0.019).
Multiple metastases were analysed in 57 of the cases and mutations were identical in all except three, indicating that BRAF and NRAS mutations occur before metastasis.
Together, these results suggest that targeting mutant (V600E)B-Raf reduces melanoma cell extravasation by decreasing IL-8 production and interrupting ICAM-1-beta2 integrin binding of melanoma cells to the endothelium mediated by PMNs in the microcirculation, which provides a rationale and mechanistic basis for targeting mutant (V600E)B-Raf to inhibit melanoma extravasation and subsequent metastasis development.
In 42 cases with LN metastases, 23 harbored the BRAF(V600E) mutation in the metastatic tumor and presented a wider diameter of the largest metastatic area, a higher number of involved LNs, and a higher percentage of metastatic lesions with extracapsular extension of LN (ECE-LN).