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
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.
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.
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.
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.
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%).
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 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.
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 summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant (V600E)B-Raf signaling cascade to inhibit melanoma metastases.
The BRAF(V600E) mutation was detected in 88 of the tumors examined, with significant differences between groups with and without lymph node (LN) metastases; the mean age of patients with BRAF(V600E) mutation was significantly higher than that of patients without mutations.
BRAFV600E was associated with extrathyroidal invasion (P < 0.0001), multicentricity (P = 0.0026), presence of nodal metastases (P = 0.0009), class III vs. classes I and II (P < 0.00000006), and absence of tumor capsule (P < 0.0001), in particular in follicular- and micro-PTC variants.
We therefore analyzed the correlation of BRAFV600E and ERK-activation in 20 malignant melanomas and 21 subsequently evolved, paired metastases of the same donor by BRAF exon 15 DNA sequencing and phospho-specific immunohistochemistry for ERK.
Treatment of orthotopic thyroid tumors, initiated 1 week after tumor cell implantation with PLX4720, an orally available selective inhibitor of B-Raf(V600E), caused a significant tumor growth delay and decreased distant metastases, without evidence of toxicity.
Because we were interested in diagnostic and therapeutic consequences, we studied the KRAS, NRAS, PIK3CA exon 20, and BRAF genotypes in synchronous and metachronous primary CRCs; in addition, we studied their available metastases.