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

Abstract

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 BRAF^V600, NRAS^Q61, TERT^-124C>T, and TERT^-146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERT^mutant 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 BRAF^mutant 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.

Figure 1:. Multiplatform analysis for quality control.

Mutational profiles were determined using a combination of multiplex SNaPshot, Sanger sequencing, MS-PCR, or ddPCR. (a) Summary of BRAF and NRAS mutation detection methods and flow chart depicting the sequence of sample analysis. All 271 samples were tested by SNaPshot. One hundred and seven samples underwent MS-PCR validation. Samples with sufficient DNA were sent for Sanger Sequencing, including 138 analyzed by SNaPshot and 71 analyzed by both SNaPshot and MS-PCR. Thirty samples with sufficient DNA remaining were tested by ddPCR. (b) Summary of TERT promoter mutation detection methods and results by method. Samples with sufficient DNA were tested by SNaPshot (268/271). Samples which presented with TERT promoter heterogeneity by SNaPshot (n=89) were tested using ddPCR. One sample was tested by ddPCR only.

Figure 2:. Representative patients exhibiting inter-tumor heterogeneity.

(a) Patient 06–001 was diagnosed with a BRAF^V600E/NRAS^wild-type/TERT^wild-type** primary tumor (P) and developed a BRAF^wild-type/NRAS^Q61K/TERT^−124[C>T] satellite metastasis (M1), 310 days after primary resection. The second metastasis was an in-transit lesion diagnosed 3667 days after initial diagnosis and was BRAF^wild-type/NRAS^Q61K/TERT^−124[C>T]. The patient subsequently developed a regional lymphatic metastasis of the same genotype. Sample marked as ^** was wild-type by SNaPshot but failed ddPCR analysis (this patient was not included in the TERT heterogeneity group). Scale bars = 800 μm in (P) and (M1), 4 mm in (M2) and 3 mm in (M3). (b) Patient 06–075 had three metastatic tumors which presented with two unique genotypic profiles, M1: BRAF^V600E/NRAS^Q61K/TERT^wild-type, M2: BRAF^wild-type/NRAS^Q61K/TERT^wild-type and M3: BRAF^wild-type/ NRAS^Q61K/TERT^wild-type. The patient’s primary tumor was located on the trunk, in the mid back region, but tissue was not available for mutational analysis. Scale bars = 2 mm in (M1), 5 mm in (M2) and 4 mm in (M3).

Figure 3:. Representative droplet digital PCR two dimensional plots of TERT promoter mutations from primary tumor DNA (patient 04–050).

(a) Two dimensional plot of TERT mutation −124[C>T], fractional abundance of 21.9%. (b) Two dimensional plot of TERT mutation −146 [C>T], fractional abundance of 0.97%. Y-axis: mutant allele. X-axis: WT allele. Droplets containing different fragments of DNA are displayed as follows: Single positive mutant allele: upper left quadrant (blue); single positive wild-type allele: lower right quadrant (green); double positive mutant/wild-type alleles: upper right quadrant (orange) and droplets not containing either allele: lower left quadrant (grey).