Mutated BRAF (BRAF V600E ) is a potential immunotherapeutic target for melanoma because of its tumor specificity and expression in the majority of these lesions derived from different patients. BRAF V600E is expressed intracellularly and not on the cell surface, therefore providing a target for T cells but not B cells. Demonstration of patients' T cell responses to BRAF V600E would suggest the feasibility of active specific immunotherapy targeting the mutation in these patients. In the present study, BRAF V600E peptides with putative binding sites for human leukocyte antigen (HLA)-A2 were used to stimulate T lymphocytes of HLA-A2-positive melanoma patients. Four of five patients with BRAF V600E -positive lesions showed lymphoproliferative responses to BRAF V600E peptide stimulation. These responses were specific for the mutated epitope and HLA-A2 was restricted in three patients. Lymphocytes from these three patients were cytotoxic against HL A-A2-matched BRAF V600E -positive melanoma cells. None of the four patients with BRAF V600E -negative lesions and none of five healthy donors had lymphoproliferative responses specific for the mutated epitope. The high prevalence (f50%) of HLA-A2 among melanoma patients renders HLA-A2-restricted BRAF V600E peptides attractive candidate vaccines for these patients. (Cancer Res 2006; 66(6): 3287-93)

Deleterious BRCA1 mutations have significant clinical implications for the patients that carry them. Point mutations in critical functional domains and frameshift mutations that lead to early termination of protein translation are associated with a 60-80% risk of breast cancer and a 20-40% risk of ovarian cancer. In contrast, the significance of mutations located in intronic regions of BRCA1, even in the setting of a family history of breast and ovarian cancer, is not always clear. Some of these mutations occur in splice donor/acceptor consensus sites. These mutations can affect heteronuclear RNA (hnRNA) processing, leading to the loss of functional BRCA1 protein and thus may be disease-associated. However, it is important to verify the effect of these mutations, because splicing alterations cannot be predicted from genomic sequence alone. We report here the characterization of two novel BRCA1 mutations identified in families seen in our cancer risk evaluation clinic that alter splice donor sites of BRCA1. We show that both mutations alter transcript splicing and result in truncated BRCA1. IVS17 + 1G --> T leads to inclusion of part of intron 17 after the coding sequence of exon 17, resulting in early termination of BRCA1 protein following codon 1692. 252del5insT abolishes the splice donor site in exon 3, leading to the skipping of exon 5 and BRCA1 protein truncation following codon 45. Thus, both mutations result in loss of BRCA1 function, and carriers of these mutations should be counseled in the same manner as carriers of other truncating BRCA1 mutations.