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Purpose: The mutational spectra of melanoma has been well characterized; however, the presence of distinct subclones among multiple tumors from a given patient has been less well described. As mutational heterogeneity has been associated with decreased responses to treatments in other cancers, we sought to estimate the occurrence of distinct subclones within individual melanoma patients by analyzing commonly mutated melanoma genes using a multi-platform mutation-detection approach. Methods: We analyzed 271 formalin-fixed, paraffin embedded tumors from 99 patients with stage III or IV melanoma enrolled in the NYU Melanoma Biorepository. All patients had two or more available tumor specimens, and complete clinical data. All samples were reviewed for adequate tumor content, and extracted DNA was assessed for mutations at BRAFV600, NRASQ61, and TERT-124C>T and TERT-146C>T using a combination of multiplex SNaPshot assays, Sanger Sequencing, Allele-specific real-time PCR, or droplet digital PCR (ddPCR). Samples undergoing ddPCR analysis for TERT mutations were treated with uracil-DNA glycolase prior to amplification to remove C>T artifacts from formalin-fixation. Results: Sixty patients had a primary plus one or more metastatic tumors available; 39 patients had multiple metastatic tumors, but no primary tumor available. Overall, 88% of patients had tumors with at least one BRAF, NRAS and/or TERT mutation. We identified inter-tumor mutational heterogeneity in 20/99 (20%) patients, with TERT mutational heterogeneity present in 15 of these patients. Among 14 patients with heterogeneity between their primary and metastatic tumors, 6/14 (43%) had additional mutations in their metastases compared to their primaries. Most interestingly, 8/14 (57%) patients had mutations in their primary that were undetectable in at least one of their metastases; 5 of these patients had TERT mutational heterogeneity. Three patients had a BRAF mutation in their primary that was undetectable in at least 1 of their metastases. One patient had a BRAFV600E/NRASWILD-TYPE/TERTWILD-TYPE primary on the leg and 3 regional metastases lacking BRAF mutations; but carrying NRASQ61K and TERT-124C>T mutations. Another patient had 3 different metastatic tumors, with 3 different mutational spectra. We did not detect any tumors with simultaneous BRAF and NRAS mutations; however, we did detect both TERT-124C>T and TERT-146C>T mutations in 7 tumors from 7 individual patients. Four of these were primary tumors, and metastases from these patients lacked 1 of the 2 TERT mutations identified in the primary. Conclusion: Clonal heterogeneity in melanoma is fairly common as evidenced by divergent detection of TERT, BRAF and NRAS mutations using high sensitivity multi-platform mutation detection analyses of multiple melanoma tumors from individual patients. Heterogeneity appears to occur in primary tumors. Citation Format: Gregory Chang, Broderick Corless, Nathaniel Fleming, Cindy Spittle, Farbod Darvishian, Anna Pavlick, Russell Berman, Richard Shapiro, George Karlin-Neumann, Iman Osman, David Polsky. Identification of melanoma mutational tumor heterogeneity using BRAF, NRAS and TERT-promoter mutation-detection assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4704.

Purpose Melanoma patients with BRAFmutant tumors often develop resistance to BRAF-inhibitor therapies. Co-occurring mutations, present at the time of treatment initiation, have been identified in patients with primary treatment resistance, and NRAS mutations have been associated with secondary resistance. We tested the ability of multiplex mutation detection assays to identify possible co-occurring and resistance mutations in cell-free, circulating tumor DNA (ctDNA) from patients undergoing treatment with BRAF-targeted therapies. Methods Purified ctDNA samples remaining from a previous longitudinal study of metastatic melanoma patients, which measured BRAFmutant and NRASmutant ctDNA using droplet digital PCR (ddPCR) duplex assays, were used for this study. Twelve samples from 6 patients with BRAF V600E mutant (V600E) tumors, who were receiving BRAF-inhibitor therapy, were separately analyzed in different laboratories using the two different multiplex assays (Oncomine and OnTarget). Oncomine used approximately 20ng DNA; OnTarget used approximately 30ng DNA. Patient samples were chosen based on clinical response or disease progression at the time of blood draw. Results Ten of 12 samples had V600E ctDNA detected by ddPCR. The On-Target detected V600E in 9/10 samples; the Oncomine detected V600E in 8/9 samples (1 sample was not analyzed due to limited sample availability). The 1 sample with a ddPCR detected mutation that was not detected by either OnTarget or Oncomine had a 0.01% fractional abundance, which is below the detection limit of these multiplex assays. Neither assay detected V600E DNA when it was not detected by ddPCR. One patient had 2 samples with a co-occurring p53 (R273H) mutation detected by OnTarget and Oncomine. This mutation was present at the time of partial response, and at disease progression with an associated increased fractional abundance as determined by both assays. The OnTarget detected NRASmtant ctDNA (Q61K x 2, Q61H x1) in 3 of the V600E samples; the Oncomine detected the Q61K in 1 of these 2 samples (the third sample was not analyzed as noted above). The NRAS mutations arose at times of disease progression. Oncomine also detected additional p53, GNAS, and FBXW7 mutations at generally low fractional abundances that were not detected by OnTarget. Conclusion Co-occurring and potential resistance mutations are detectable in the plasma of metastatic melanoma patients using OnTarget or Oncomine assays. The assays demonstrate high sensitivity, as evidenced by their ability to identify V600E ctDNA. These findings suggest that studies analyzing patient ctDNA for resistance mutations while they are undergoing treatment are feasible. Results of such studies may eventually help inform treatment choices, such as switching therapies when resistance mutations emerge. Citation Format: Broderick Corless, Greg Chang, Weihua Liu, Jin Li, Andre Marziali, Laura Mai, Matthew Wiggin, Melissa Wilson, Anna Pavlick, Iman Osman, George Karlin-Neumann, Cindy Spittle, David Polsky. Detection of co-occurring and potential resistance mutations in cell-free, circulating tumor DNA from patients with BRAFmutant metastatic melanoma undergoing treatment with BRAF-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5531.

Purpose: Our goal is to develop response predictive biomarkers for melanoma patients that are more sensitive than standard measures of disease activity. TERT promoter mutations occur frequently in many cancers, especially melanoma. We developed droplet digital PCR assays that detect the 2 most frequently occurring TERT promoter mutations, C228T and C250T. We previously demonstrated that these assays could detect TERTmutant cell-free, circulating tumor DNA (ctDNA) in the plasma of metastatic melanoma patients. In the current study we sought to compare the clinical sensitivity and specificity of these blood-based assays to the current blood biomarker, lactate dehydrogenase (LDH), in detecting the presence of metastatic melanoma and disease progression among patients with unresectable Stage III/IV disease. Methods: We analyzed 71 patients' tumors with unresectable stage III/IV metastatic melanoma undergoing treatment with immuno-oncology therapies, prospectively acquired by the NYU Interdisciplinary Melanoma Cooperative Group. TERT mutations were identified in tumors using droplet digital PCR (ddPCR) or SNaPShot assays (prior to the development of the ddPCR assays). Patients with TERT mutant tumors were selected for further study based on the availability of serial blood collections and radiographic scan data. ddPCR assays used to analyze ctDNA from these patients were chosen based on the tumor mutation detected. Plasmas used for ctDNA analysis ranged from 1-5 mL (73% having at least 4 mL). The ctDNA results were compared with LDH levels and disease status based on radiographic scans and clinical notes. Results: We identified TERT mutations in 31/71 (44%) tumors (18/31 C228T and 13/31 C250T). Our final data set consisted of 26 patients with 85 plasmas. Among patients with available pretreatment samples, ctDNA and LDH were elevated in 14/24 (58%) and 5/24 (21%) patients, respectively (p-value=0.012, McNemar exact test). Among patients whose disease progressed, ctDNA and LDH were elevated at the time of progression in 11/16 (69%) and 4/16 (25%) patients, respectively (p=0.039). Among the patients who had either a complete response (n=2), partial response (n=3), or stable disease (n=3), 1/8 had a slightly elevated ctDNA (3.8 copies/mL with normal level Conclusion: These data demonstrate that ddPCR assays detecting TERT C228T or C250T mutations in ctDNA of metastatic melanoma patients have a greater sensitivity to detect the presence of metastatic disease and disease progression than the currently used standard, LDH. Citation Format: Mahrukh M. Syeda, Broderick Corless, Melissa Wilson, Yesung Lee, Jeremy Tchack, Todd Wechter, Una Moran, George Karlin-Neumann, Anna C. Pavlick, Iman Osman, Yongzhao Shao, David Polsky. Analysis of TERTmutant circulating tumor DNA as a potential biomarker of disease activity in patients with unresectable stage III/IV melanoma receiving immuno-oncology therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5534.

Purpose: Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. Mutations in 1 of 2 hot spots in the TERT promoter sequence are found in several cancers, including up to 85% of melanomas and the majority of cases that lack BRAF or NRAS mutations (about one-third of melanomas). Due to the high G-C content of the TERT promoter sequence these mutations can be difficult to detect using NGS approaches. We developed novel droplet digital PCR (ddPCR) assays to detect these 2 mutations with high sensitivity and specificity, and demonstrate the application of these assays in melanoma clinical samples. Methods: Assays were optimized using cell lines with Sanger sequencing-confirmed mutations: glioblastoma A172 (C228T), and melanoma NYU12-126 (C250T). We varied assay designs and amplification conditions to optimize probe-based detection using the Bio-Rad QX-200 ddPCR system. Assay sensitivities and specificities at various DNA input levels were determined using serial dilutions with 3 replicate wells for each condition. Sensitivity is defined as the lowest mutant allele dilution for which the confidence interval did not overlap with that of the 0% mutant wells. We used normal and cancer-derived DNA sources of different quality (e.g. normal human DNA (Promega), cancer cell lines, plasma and FFPE-derived DNAs) with and without the mutations, and compared the efficiency of detection of amplicons of 88, 113 and 163 base-pairs. We compared efficiencies to assays of similar size for RPP30, a housekeeping gene. Patient-matched metastatic melanoma tumors and plasma samples were analyzed to explore the clinical utility of these assays. Results: The assays showed greater sensitivity when higher amounts of DNA were analyzed. For C228T the limit of detection (LOD) of the mutant allele was 1%, 0.25% and 0.1% for 6.6ng/well, 33ng/well and 66ng/well respectively; for C250T the LODs were 0.25%, 0.05% and 0.05% respectively. Using normal human DNA, the efficiency of the TERT assays averaged approximately 90% of that for RPP30 across assays of similar size, and no decrease in assay efficiency was observed as amplicon length increased. In contrast, whereas amplicon size had only a modest effect on assay efficiency in plasma cfDNA, it gave a more pronounced effect on FFPE DNA’s, decreasing to 38% for the 163bp amplicon. We observed 100% concordance between TERT mutation detection by SNaPshot and ddPCR in 10 FFPE tumor samples, and in plasma samples from 4 metastatic melanoma patients with matching tumor samples. Conclusion: We developed robust ddPCR assays to detect TERT promoter mutations with high sensitivity and specificity. Mutated TERT DNA can be detected and quantitated in the plasma of patients with metastatic melanoma, and is likely to be present in the plasma of other cancer patients in whom TERT mutations occur. Citation Format: Broderick Corless, Gregory Chang, Samantha Cooper, Mahrukh Syeda, Iman Osman, George Karlin-Neumann, David Polsky. Detection of TERT C228T and C250T promoter mutations in melanoma tumor and plasma samples using novel mutation-specific droplet digital PCR assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2017-743