Associations between TERT Promoter Mutations and Survival in Superficial Spreading and Nodular Melanomas in a Large Prospective Patient Cohort.

Survival outcomes in melanoma and their association with mutations in the telomerase reverse transcriptase gene TERT promoter remain uncertain. In addition, few studies have examined whether these associations are affected by a nearby common germline polymorphism or vary on the basis of melanoma histopathological subtype. We analyzed 408 primary tumors from a prospective melanoma cohort for somatic TERT-124[C>T] and TERT-146[C>T] mutations, the germline polymorphism rs2853669, and BRAFV600 and NRASQ61 mutations. We tested the associations between these variants and clinicopathologic factors and survival outcomes. TERT-124[C>T] was associated with thicker tumors, ulceration, mitoses (>0/mm2), nodular histotype, and CNS involvement. In a multivariable model controlling for the American Joint Committee on Cancer stage, TERT-124[C>T] was an independent predictor of shorter recurrence-free survival (hazard ratio = 2.58, P = 0.001) and overall survival (hazard ratio = 2.47, P = 0.029). Patients with the germline variant and TERT-124[C>T]-mutant melanomas had significantly shorter recurrence-free survival than those lacking either or both sequence variants (P < 0.04). The impact of the germline variant appeared to be more pronounced in superficial spreading than in nodular melanoma. No associations were found between survival and TERT-146[C>T], BRAF, or NRAS mutations. These findings strongly suggest that TERT-124[C>T] mutation is a biomarker of aggressive primary melanomas, an effect that may be modulated by rs2853669.

TERT, BRAF, and NRAS Mutational Heterogeneity between Paired Primary and Metastatic Melanoma Tumors.

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n = 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAFV600, NRASQ61, TERT-124C>T, and TERT-146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERTmutant discordant tumors; seven of these had a single tumor with both TERT-124C>T and TERT-146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAFmutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples.

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERTmutant DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERTmutant tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERTmutant DNA in tumors and plasma of cancer patients.

Sensitivity of plasma BRAFmutant and NRASmutant cell-free DNA assays to detect metastatic melanoma in patients with low RECIST scores and non-RECIST disease progression.

Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p = 0.006) patients respectively. In patients with RECIST scores <5 cm prior to treatment initiation, ctDNA levels were elevated in 5/7 (71%) patients compared to LDH which was elevated in 1/13 (8%) patients (p = 0.007). Among all disease progression events the modified bootstrapped sensitivities for ctDNA and LDH were 82% and 40% respectively, with a median difference in sensitivity of 42% (95% confidence interval, 27%-58%; P < 0.001). In addition, ctDNA levels were elevated in 13/16 (81%) instances of non-RECIST disease progression, including 10/12 (83%) instances of new brain metastases. In comparison LDH was elevated 8/16 (50%) instances of non-RECIST disease progression, including 6/12 (50%) instances of new brain metastases. Overall, ctDNA had a higher sensitivity than LDH to detect disease progression, including non-RECIST progression events. ctDNA has the potential to be a useful biomarker for monitoring melanoma disease activity.

Mutational Heterogeneity in Melanoma: An Inconvenient Truth.

Identification of oncogenic BRAF mutations in primary and metastatic melanomas supports a linear model of clonal evolution in cancer. Some mutational studies, however, have failed to identify BRAF mutations in metastatic tumors from patients with BRAFmutant primary melanomas. Using a combination of methods, Riveiro-Falkenbach et al. (2015) assert that technical issues, and not clonal heterogeneity, may explain prior discordant mutational results.