Abstract A31: Association between TERT promoter mutations and BRAF/NRAS mutations in patients with primary and metastatic melanoma tumors
Authors: Chang Gregory A. , Tadepalli Jyothirmayee S. , Fleming Nathaniel H. , Lui Kevin , Shao Yongzhao , Darvishian Farbod , Pavlick Anna , Berman Russell , Shapiro Richard , Osman Iman , Polsky David 
Published: July 2015

Abstract
Background: BRAF and NRAS are well-established driver oncogenes in melanoma. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERT) increase gene expression and have been identified at high rates in metastatic melanoma, and rarely in familial melanoma. One recent study found an association between TERT mutations and BRAF or NRAS mutations. We developed mutation-specific assays to detect BRAF V600 and NRAS Q61 mutations along with TERT C228T and C250T promoter mutations to identify the prevalence of these mutations in melanoma tumor samples, association with BRAF/NRAS mutations, and potential use of TERT as a biomarker for blood-based monitoring.
Methods: We analyzed 316 primary and metastatic tumors from 114 patients with unresectable stage III/IV melanoma, enrolled and followed in the NYU Melanoma Biorepository program. Each patient had &gt;1 tumor samples available for analysis. We used a combination of allele-specific PCR (Taqman) and SNaPshot assays to identify BRAF V600E/K/R, NRAS Q61L/K/R and TERT promoter C228T and C250T mutations in tumors from 114 patients. We tested the association between TERT promoter mutations and BRAF or NRAS mutations using Fisher's Exact Test. We also analyzed plasma for TERT promoter mutations using samples from a separate group of 20 stage IV patients with stable or active disease at the time of blood draw.
Results: Among the 114 patients, 49, 40 and 22 had 2, 3 or 4 tumors available for analysis respectively. There were 3 patients from whom we analyzed only 1 tumor. We amplified the TERT promoter in 304/316 (96%) samples, and detected mutations in 177/304 (58%) tumors. We found C228T or C250T mutations in 39/77 (51%) primaries and 138/227 (61%) metastases. C228T and C250T mutations were present in 86/304 (28%) and 87/304 (29%) tumors respectively. Among patients with &gt;2 tumor samples we found that 40/111 (36%) had tumors that were discordant for TERT mutations (e.g. &gt;1 TERT mutant and &gt;1 TERT wild-type). Six patients had tumors with both C228T and C250T mutations, present either within different individual tumors (n=2 patients) or within the same tumor (n=4 patients). We amplified BRAF and NRAS in 307/316 (97%) tumor samples. We detected BRAF mutations in 95/307 (31%) tumors. BRAF V600E/K/R mutations were found in 22/77 (29%) primaries and 73/227 (32%) metastases. We detected NRAS mutations in 54/307 (18%) tumors. NRAS Q61K/L/R mutations were found in 7/77 (9%) primaries and 47/227 (21%) metastases. Among 177 tumors with TERT mutations, we found BRAF V600 mutations in 72/177 (41%) and NRAS Q61 mutations in 34/177 (19%). There was a statistically significant association between the occurrence of TERT mutations and BRAF or NRAS mutations for both primary (OR=4.32, p&lt;0.01, 95% CI 1.57-11.91) and metastatic tumors (OR=3.16, p&lt;0.01, 95% CI 1.79-5.56). Overall, 21/74 (28%) primaries and 85/222 (38%) metastases had mutations in both TERT and BRAF or NRAS. On a per-patient basis, 64/114 (56%) patients had at least 1 NRAS-mutant tumor or 1 BRAF-mutant tumor; 86/114 (75%) had at least 1 TERT mutant tumor. Overall, 98/114 (86%) patients had either a TERT mutation or a BRAF/NRAS mutation. We detected a TERT mutation in the plasma of 1/20 (5%) patients with an available sample.
Conclusions: C228T/C250T TERT promoter mutations occur more frequently than BRAF mutations in primary and metastatic melanomas. TERT mutations preferentially associated with BRAF or NRAS mutations suggesting a possible cooperative role with these oncogenes. The high frequency of TERT tumor mutations on a per-patient basis combined with the presence of inter-tumor heterogeneity and the detection of mutated TERT DNA in the plasma suggest that TERT may be a useful disease biomarker in a large proportion of patients.
Citation Format: Gregory A. Chang, Jyothirmayee S. Tadepalli, Nathaniel H. Fleming, Kevin Lui, Yongzhao Shao, Farbod Darvishian, Anna Pavlick, Russell Berman, Richard Shapiro, Iman Osman, David Polsky. Association between TERT promoter mutations and BRAF/NRAS mutations in patients with primary and metastatic melanoma tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A31.