The T1799A point mutation is present in posterior uveal melanoma

Abstract

An activating mutation in exon 15 of the BRAF gene is present in a high proportion of cutaneous pigmented lesions. Until recently this mutation had however only been identified in one case of posterior uveal melanoma. Despite this apparent lack of the BRAF mutation, inappropriate downstream activation of the Ras/Raf/MAPK pathway has been described in posterior uveal melanoma. Based on the already recognised morphological and cytogenetic heterogeneity in uveal melanoma, we hypothesised that the BRAF mutation may be present in uveal melanoma but only in some of the tumour cells. In this study, we analysed 20 ciliary body and 30 choroidal melanomas using a nested PCR-based technique resulting in the amplification of a nested product only if the mutation was present. This sensitive technique can identify mutated DNA in the presence of wild-type DNA. The mutation was identified in 4 of 20 (20%) ciliary body and 11 of 30 (40%) choroidal melanomas. Further analysis of separate areas within the same choroidal melanoma demonstrated that the mutation was not present in the entire tumour. In conclusion, the T1799A BRAF mutation is present in a proportion of posterior uveal melanomas but within these tumours the distribution of the mutation is heterogeneous.

Figure 1

Silver-stained polyacrylamide gel showing a single band at 200 nt for human foreskin fibroblast (HFF) DNA (A) and an additional band at 100 nt for SKmel-28 cell DNA (A). Mixed DNA from HFF and SKmel-28 (B) also shows molecular markers at 200 and 100 bp respectively.

Figure 2

(A) Tissue section of enucleation specimen after dissection of four tumour areas (1–4) and one normal tissue area (N). (H&E, magnification × 1.25). (B) Silver-stained polyacrylamide gel showing a single band at 200 bp in the four corresponding tumour samples and in the normal tissue sample indicating lack of the T1799A point mutation in this case. (C) Tissue section of local resection specimen after dissection of four tumour areas (1–4). (H&E, magnification × 2). (D) Silver-stained polyacrylamide gel showing an additional band at 100 nt in samples 2 and 4 indicating the presence of the mutation in these samples. There is an additional band between 200 and 100 nt representing a PCR artefact. Samples 1 and 3 show only one band at 200 nt indicating lack of the mutation.

Figure 3

Kaplan–Meier survival curve showing the relationship between time as surgery and survival rate for patients with tumours with and without the BRAF mutation.