Absence of Mutations of the BRAF Gene and Constitutive Activation of Extracellular-Regulated Kinase in Malignant Melanomas of the Uvea

Abstract

SUMMARY:The v-raf murine sarcoma viral homolog B1 (BRAF) gene, one of the human isoforms of RAF, is activated by Ras, leading to cooperative effects in cells responsive to growth factor signals. Recently, somatic missense mutations of the BRAF gene have been detected in more than 66% of malignant melanomas of the skin. We analyzed 42 malignant melanomas of the uvea, 3 corresponding liver metastases, and 10 cutaneous melanomas for possible BRAF mutations: after microdissection, mutation analysis of BRAF and KRAS was performed. The expression of extracellular-regulated kinase 1 and 2 (ERK1/2), an important downstream point of convergence in the Ras-RAF-MEK-Erk pathway, was analyzed immunohistochemically. Interestingly, we failed to detect activating BRAF mutations in uvea melanomas and their corresponding liver metastases. There were no mutations of BRAF in corresponding non-neoplastic uvea specimens, although we detected three BRAF mutations in sporadic cutaneous melanoma that led to a substitution of valine by glutamic acid at position 599 (V599E). KRAS mutations were detected in 1 of 10 cutaneous melanoma but not in uveal or metastatic melanoma. Despite the lack of activating mutations in the BRAF gene, we identified constitutively activated ERK in almost all (86%) uveal melanoma tissues tested but not in corresponding normal retina or uveal cells. Our data indicate that BRAF gene mutations are rare to absent events in uveal melanoma. The finding of activated Erk suggests a causative role for MAPK activation in uveal melanoma independent of activating BRAF or RAS mutations. (Lab Invest 2003, 83:1771-1776.

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS

“…Similarly, our observation of a complete lack of BRAF mutations in primary uveal tumours mirrors the findings of several recent reports (Cohen et al, 2003;Cruz et al, 2003;Edmunds et al, 2003;Rimoldi et al, 2003;Weber et al, 2003;Kilic et al, 2004). Table 3 contains a summary of published reports on RAS and BRAF mutations, as well as studies on other members of the MAPK pathway, in uveal melanomas.…”

“…The signal transducer MEK1/2 phosphorylates extracellular signal-regulated kinase (ERK1/2, p44/42), leading to the activation of these kinases, which in turn activate a variety of transcription factors, including ELK1, again through phosphorylation. It has emerged that BRAF (v-raf murine sarcoma viral oncogene homologue B1) is very frequently activated by mutation in cutaneous melanomas (Brose et al, 2002;Davies et al, 2002;Alsina et al, 2003;Dong et al, 2003;Gorden et al, 2003;Kumar et al, 2003a, b;Maldonado et al, 2003;Omholt et al, 2003;Pollock et al, 2003;Rimoldi et al, 2003;Satyamoorthy et al, 2003;Weber et al, 2003;Cohen et al, 2004;Reifenberger et al, 2004;Shinozaki et al, 2004;Tsao et al, 2004). The frequency of BRAF mutations varies from 8 to 83% depending on the anatomic site of the lesion and its histogenic subtype.…”

The T1799A point mutation is present in posterior uveal melanoma

“…However, there have been several studies, in uveal melanoma including primary and metastatic choroidal and ciliary body melanomas and the BRAF mutation has only been identified in one case (Malaponte et al, 2006). Despite this apparent lack of the characteristic BRAF mutation, inappropriate downstream MAPK component activation has been reported by Weber et al (2003) who failed to identify the BRAF mutation in 42 primary uveal melanoma but showed immunohistochemical staining for ERK in 86% of these cases. Similarly, Zuidervaart et al (2005) found constitutive activation of the MAPK pathway in 11 uveal melanoma cell lines and 19 primary tumours by performing western blot or immunohistochemistry for the various pathway components.…”

“…The majority of other studies have used some form of direct sequencing to identify this mutation (Cruz et al, 2003;Edmunds et al, 2003;Weber et al, 2003). Sequencing would not be able to detect mutant alleles present at low frequency because of somatic mosaicism.…”

“…In particular a single point mutation in exon 15 (T1799A), which results in constitutive kinase activity and unregulated signal transduction, is involved in up to 80% of cases (Brose et al, 2002;Davies et al, 2002). Despite the high incidence of this mutation in cutaneous melanoma, several studies have shown that it is not present in posterior uveal melanoma (Cohen et al, 2003;Weber et al, 2003). The BRAF mutation has however been identified in several primary uveal melanoma cell lines and recently Malaponte et al (2006) identified the mutation in one primary uveal melanoma.…”