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9019 Background: Management of melanoma suffers from a lack of robust biomarkers of disease activity. In this study, we tested the ability of droplet digital PCR (ddPCR) to quantitatively measure levels of circulating BRAF and NRAS mutant DNA in the plasma of metastatic melanoma patients undergoing treatment with BRAF-targeted therapy or immunotherapy. Methods: We analyzed plasma samples from 45 patients with stage IV melanoma prospectively enrolled in the NYU Melanoma Biorepository program. All patients were commercially genotyped for BRAF V600E. SNaPshot assays were used to identify NRAS Q61 mutations in BRAF-WT tumors. Each patient had at least 3 serially collected plasma samples including one drawn prior to treatment, one or more after treatment began and one upon signs of disease progression. ddPCR was used to measure mutant copies/ml of BRAF V600E and NRAS Q61K/L/R DNA in plasma samples. Results: Among 45 patients, 28 patients had BRAF and 8 patients had NRAS tumor mutations. We extracted DNA from 1...

9023 Background: Patients with metastatic melanoma are eligible for BRAF inhibitor therapy if the BRAF V600E mutation can be identified in their tumor specimen. Patients lacking an available specimen for genotyping are unable to receive inhibitor therapy. We developed two mutation-specific genotyping platforms and tested their ability to detect BRAF and NRAS mutations in archived plasma and tumor samples to determine the potential utility of blood-based tumor genotyping in melanoma. Methods: We analyzed a group of 96 patients with stage III or IV melanoma, prospectively enrolled and followed in the NYU Melanoma Biorepository program. Each patient had a plasma sample and one or more tumor samples available for analysis. We used a combination of allele-specific PCR (Taqman) and SNaPshot assays to identify BRAF V600 and NRAS Q61 mutations in the tumor and plasma samples. Results: Among the 96 patients, 51 had stage III disease at the time of analysis; 45 had stage IV disease. Seventy-two patients had 2 or more tumor samples available for analysis, for a total of 204 tumors analyzed. In total, 52/96 (54%) patients had one or more BRAF or NRAS mutant tumors, including one patient with separate BRAF and NRAS mutant tumors (BRAF, n=35 (36%); NRAS, n=18 (19%)). We successfully amplified plasma DNA from 39/52 (75%) patients with tumor-associated mutations. Among those patients with amplifiable plasma DNA we detected mutations in 7 (18%) patients including 3 BRAF V600E, one V600K, 2 NRAS Q61K and one Q61L. Plasma-based mutations matched tumor-associated mutations in all 7 patients. All 7 patients had active disease at the time of blood draw. There were 32 patients with tumor-associated mutations in which a mutation could not be detected in the plasma. Only 15 of those 32 (47%) had active disease at the time of blood draw. There were no mutations detected in the plasma of the 44 patients whose tumors lacked BRAF or NRAS mutations. Conclusions: These data suggest that plasma-based detection of BRAF and NRAS mutations has a high specificity for tumor-associated mutations. It may prove to be a useful adjunct to tumor-based genotyping in patients with active disease, especially those lacking an available tumor specimen.