Background: Techniques for the accurate identification of activating mutations of BRAF in metastatic melanoma are of great clinical importance, due to the availability of targeted therapies for these tumors. There is uncertainty regarding the frequency with which BRAF status differs between primary and metastatic sites. Methods: Between 2011 and 2016, 219 melanoma cases underwent BRAF testing in our institution. In 53 of these cases, paired primary and metastatic specimens were available for polymerase chain reaction (PCR) and immunohistochemical evaluation. Results: Fifty-two out of 53 cases (98%) showed concordant BRAF status between primary and metastatic site by immunohistochemistry (IHC). In one case, a metastasis and its matched primary were positive by IHC, but the metastasis was negative on PCR. On further investigation, PCR was positive in the primary, and repeat PCR in the metastasis was positive, following macrodissection. Conclusions: Our results suggest that discordance of BRAF mutational status between primaries and metastases is a rare occurrence. In one case, IHC provided strong evidence that initial PCR testing had provided a false-negative result due to low tumor volume. Thus, in cases where tissue is difficult to obtain from a metastasis or unavailable, the primary tumor can be used with confidence.

AimsThe assessment of B-raf proto-oncogene, serine/threonine kinase (BRAF) gene status is now standard practice in patients diagnosed with metastatic melanoma with its presence predicting a clinical response to treatment with BRAF inhibitors. The gold standard in determining BRAF status is currently by DNA-based methods. More recently, a BRAF V600E antibody has been developed. We aim to investigate whether immunohistochemical detection of BRAF mutation is a suitable alternative to molecular testing by polymerase chain reaction (PCR).MethodsWe assessed the incidence of BRAF mutation in our cohort of 132 patients, as determined by PCR, as well as examining clinical and histopathological features. We investigated the sensitivity and specificity of the anti-BRAF V600E VE1 clone antibody in detecting the presence of the BRAF V600E mutation in 122 cases deemed suitable for testing.ResultsThe incidence of BRAF mutation in our cohort was 28.8% (38/132). Patients with the BRAF mutation were found to be significantly younger at age of diagnosis. BRAF-mutated melanomas tended to be thinner and more mitotically active. The antibody showed a sensitivity of 86.1% with a specificity of 96.9%. The positive predictive value was 96.9%; the negative predictive value was 94.4%. The concordance rate between PCR and immunohistochemical BRAF status was 95.1% (116/122).ConclusionsThe rate of BRAF mutation in our cohort (28.8%) was lower than international published rates of 40%–60%. This may reflect ethnic or geographic differences within population cohorts. The high concordance rate of PCR and immunohistochemical methods in determining BRAF status suggests that immunohistochemistry is potentially a viable, cost-effective alternative to PCR testing and suitable as a screening test for the BRAF mutation.

e21076 Background: Activating BRAF V600 mutations have been shown to occur in 40%–60% of melanomas, although the mutation rate among the Irish melanoma population has been reported to be lower (30%). The current standard for determining BRAF mutation status is with the use of the Cobas 4800 PCR test. IHC analysis to determine BRAF mutation status has been made possible by the development of monoclonal antibodies directed at mutant BRAF protein expression, which may be of use as an alternative to molecular testing. Methods: We identified 132 patients with metastatic melanoma between 2011 and 2014 at our institution that had tumour tested for BRAF mutation using PCR. Of these, 122 cases were suitable for IHC. Tissue samples were obtained and tested with the BRAF V600E antibody. IHC was assessed as 0 (negative), 1, 2 or 3 based on intensity of cytoplasmic staining. We assessed the incidence of BRAF mutation, investigated the sensitivity and specificity of the BRAF V600E antibody in detecting the presence of the BRAF V600E mutation. Clinical response to BRAF inhibitor therapy as per RECIST was correlated to BRAF mutation status by PCR and to IHC expression. Results: The incidence of BRAF mutation as assessed by PCR was 28.8% (38/132). The antibody showed a sensitivity of 86.1% with a specificity of 96.9%. The positive predictive value was 96.9%; the negative predictive value was 94.4%. The concordance rate between PCR and IHC was 95.1% (116/122). One false positive case and five false negative cases were observed. The results of clinical outcomes for the PCR positive cohort and the IHC level of expression cohorts are shown in the table. Conclusions: The high concordance rate of PCR and IHC methods in determining BRAF status and predicting response to therapy suggests that antibody testing is a viable and cost effective alternative to PCR testing. The BRAF V600E antibody may be suitable as a screening test for the BRAF mutation, with all negative cases being submitted for PCR testing to identify variant BRAF mutations not detected by IHC. Response Rate (%) Median Duration of Response (months) Median Overall Survival (months) PCR (n = 25) 92 7 12 IHC 3+ (n = 10) 83 9.5 12 IHC 2+ (n = 4) 100 7 10 IHC 1+ (n = 1) 100 18 31 IHC 0 (n = 5) 100 6 8