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. Author manuscript; available in PMC: 2023 Oct 1.
Published in final edited form as: J Invest Dermatol. 2022 Apr 22;142(10):2733–2743.e9. doi: 10.1016/j.jid.2022.03.031

Associations between TERT promoter mutations and survival in superficial spreading and nodular melanomas in a large prospective patient cohort.

Gregory A Chang 1,2,*, Eric Robinson 1,2,*, Jennifer M Wiggins 1,2,*, Yilong Zhang 2,3,4, Jyothirmayee S Tadepalli 1,2, Christine N Schafer 1,2, Farbod Darvishian 2,5, Russell S Berman 2,6, Richard Shapiro 2,6, Yongzhao Shao 2,3, Iman Osman 1,2, David Polsky 1,2,5
PMCID: PMC9509439  NIHMSID: NIHMS1816625  PMID: 35469904

Abstract

Survival outcomes in melanoma, and their association with mutations in the telomerase reverse transcriptase (TERT) promoter, remain uncertain. In addition, few studies have examined whether these associations are affected by a nearby common germline polymorphism, or vary based on 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 AJCC stage, TERT−124[C>T] was an independent predictor of shorter recurrence-free survival (RFS) (HR=2.58, p=0.001), and overall survival (HR=2.47, p=0.029). Patients with the germline variant and TERT−124[C>T] mutant melanomas had significantly shorter RFS than those patients lacking either or both sequence variants (p<0.04). The impact of the germline variant appeared to be more pronounced in superficial spreading compared to nodular melanoma. No associations were found between survival and TER−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.

INTRODUCTION

Nearly all cancers require telomerase expression to maintain telomeres and achieve cellular immortalization (Daniel et al., 2012, Heidenreich and Kumar, 2017). Mutations in the telomerase reverse transcriptase (TERT) promoter, particularly the mutually exclusive hotspot mutations located in positions −124[C>T] (C228T) and −146[C>T] (C250T), occur frequently in multiple cancers and can result in increased TERT expression (Bell et al., 2016, Cancer Genome Atlas, 2015, Heidenreich and Kumar, 2017, Heidenreich et al., 2014, Huang et al., 2013, Liu T. et al., 2016, Remke et al., 2013, Yu et al., 2021). These hotspot mutations have been associated with more aggressive tumor features and poor survival in several cancer types (Bollam et al., 2018, Chen et al., 2021, Griewank et al., 2013b, Griewank et al., 2013c, Griewank et al., 2014b, Heidenreich et al., 2014, Liu C. et al., 2016, Liu T. et al., 2016, Pirker et al., 2020, Spiegl-Kreinecker et al., 2018, Zhang et al., 2021). In many of these cancers the rs2853669 germline variant, a common T>C polymorphism at position −245 bp has been shown to alter the prognostic impact of the promoter mutations (Dratwa et al., 2020, Hsu et al., 2006, Ko et al., 2016, Mosrati et al., 2015, Rachakonda et al., 2013, Shaughnessy et al., 2020, Shen et al., 2017, Spiegl-Kreinecker et al., 2015, Yuan et al., 2019).

Among skin malignancies, TERT promoter mutation frequency varies between the type and subtype of skin cancer (Gandini et al., 2021, Griewank et al., 2013a, Griewank et al., 2013c, Griewank et al., 2014b, Heidenreich et al., 2014). In cutaneous melanoma, these mutations occur at a higher frequency (up to 86%) compared to mutations in either BRAF (~50%) or NRAS (~25%) (Cancer Genome Atlas, 2015, Chang et al., 2020, Hayward et al., 2017). Despite being important oncogenic drivers, mutated BRAF or NRAS alleles have not been consistently associated with poor survival in primary melanoma (Scatena et al., 2021, Thomas et al., 2015, Yang et al., 2020). In contrast, TERT promoter mutation often associates with increased tumor thickness and poor survival outcomes (Gandini et al., 2021). Since no tissue-based prognostic biomarker has met the minimum thresholds of evidence for clinical use according to the NCCN melanoma guidelines (Swetter et al., 2021), we conducted the largest analysis to date of primary tumors from a prospectively accrued cohort of superficial spreading and nodular melanoma patients for the two most common TERT promoter mutations and their association with survival outcomes. As few studies have examined the potential impact of the rs2853669 variant on these associations (Nagore et al., 2016), we also describe the potential impact of this germline variant and histopathologic subtype on tumor phenotypic markers and survival outcomes.

RESULTS

Patient and sample characteristics

We studied 408 patients: 229 male and 179 female. The median age at diagnosis was 61 years (63 for males and 55 for females). The anatomic distribution of the melanomas was 44.1% extremities (180/408); 41.4% trunk (169/408); and 14.5% head/neck (59/408) (Table 1). The median tumor thickness was 0.9 mm (range 0.2 mm - 16 mm); 18.1% (74/408) of tumors were ulcerated. The stage distribution was stage I, 64.2% (262/408); stage II, 26.2% (107/408); and stage III 9.6%, (39/408) (Supplementary Table S1). Superficial Spreading Melanoma (SSM) comprised 72.5% (296/408) and Nodular Melanoma (NM) 27.5% (112/408) of histological subtypes.

Table 1:

Summary of patient demographics and tumor mutational characteristics

Characteristics n (%) (n=408)
Patients
Age (mean – median) 58.6 – 61.0
Gender
 Male 229 (56.1%)
 Female 179 (44.9%)
Primary tumor location
 Extremities 180 (44.1%)
 Trunk 169 (41.4%)
 Head/Neck 59 (14.5%)
Primary tumor thickness (mm)
 Median (range) 0.9 (0.16–16)
Primary tumor ulceration status
 Absent 334 (81.9%)
 Present 74 (18.1%)
Histotype
 Nodular 112 (27.5%)
 Superficial Spreading 296 (72.5%)
Associated Nevus
 Absent 271 (66.4%)
 Present 128 (31.4%)
 Unclassified 9 (2.2%)
AJCC stage at diagnosis
 Stage I 262 (64.2%)
 Stage II 107 (26.2%)
 Stage III 39 (9.6%)
Recurrence 55 (13.5%)
Oncogenic Driver Genotype
 BRAFmutant 137 (33.6%)
 NRASmutant 80 (19.6%)
 BRAFWT/NRASWT 189 (46.3%)
 NRASWT/Failed BRAF 2 (0.5%)
TERT Promoter Genotypes
 −124 [C>T] 97 (23.8%)
 −146 [C>T] 136 (33.3%)
 TERT wild-type 169 (41.4%)
 Failed 6 (1.5%)
BRAF/NRAS status & TERT promoter genotypes
 BRAFmutant or NRASmutant (n=217)
  + TERTmutant 131/217 (60.4%)
  + TERTWT 84/217 (38.7%)
  Failed TERT mutational analysis 2/217 (0.9%)
 BRAFWT/NRASWT (n=189)
  + TERTmutant 100/189 (52.9%)
  + TERTWT 85/189 (45.0%)
  Failed TERT mutational analysis 4/189 (2.1%)
 NRASWT/Failed BRAF (n=2)
  + TERTmutant 2/2 (100.0%)
TERT rs2853669 germline variant and promoter genotypes
 Germline Variant −245 [T>C] 194/408 (47.5%)
  + TERTmutant 107/194 (55.2%)
  + TERTWT 86/194 (44.3%)
  Failed TERT mutational analysis 1/194 (0.5%)
 Consensus Sequence −245 [T] 207/408 (50.7%)
  + TERTmutant 126/207 (60.9%)
  + TERTWT 80/207 (38.6%)
  Failed TERT mutational analysis 1/207 (0.5%)
 Failed/Unavailable for germline analysis 7/408 (1.7%)
  + TERTmutant 0/7 (0.0%)
  + TERTWT 3/7 (42.9%)
  Failed TERT mutational analysis 4/7 (57.1%)
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Detection of Somatic and Germline Sequence Variants

TERT promoter mutations were found in 57.1% (233/408) of tumors; 23.8% (97/408) at position −124[C>T]; 33.3% (136/408) at position −146[C>T]. BRAF or NRAS mutations were identified in 53.2% (217/408) of tumors. Co-occurring TERT and BRAF, or TERT and NRAS mutations were identified in 56.2% (131/233) of TERTmutant cases. Tumors lacking any of the BRAF, NRAS or TERT somatic mutations comprised 20.6% (84/408) of cases. Six samples failed to yield a clear TERT mutational profile, and two of the samples that carried one of the TERT promoter mutations failed BRAF analyses (Table 1). The −245[T>C] (rs2853669) germline variant was identified in 47.5% (194/408) of patients (Table 1). Fifty-five point one percent of these patients (107/194) had a tumor with one of the TERT somatic promoter mutations, either −124[C>T] or −146[C>T]. Among the 50.7% (207/408) of patients with the germline consensus sequence, 60.9% (126/207) had one of the somatic promoter mutations. For technical reasons, the germline variant could not be determined in 7/408 (1.7%) cases.

Associations between clinicopathological variables and TERT, BRAF and NRAS sequence variants

TERT−124[C>T] was statistically significantly associated with multiple clinicopathologic variables associated with aggressive tumor phenotypes compared to TERT−124[C]. These tumor phenotypes of TERT−124[C>T] included, thicker tumors (OR=1.52 [95% CI 1.16–1.98], p=0.002), ulceration (OR=2.63 [95% CI 1.53–4.52], p<0.001), presence of any mitoses (OR=2.00 [95% CI 1.19–3.34], p=0.009), nodular histotype (OR=1.82 [95% CI 1.12–2.94] p=0.015), de novo histology (i.e. lack of associated nevus) (OR=2.08 [95% CI 1.19–3.57], p=0.010), anatomical location on the head and neck (OR=2.11 [95% CI 1.12–3.96], p=0.021), CNS (Central Nervous System) involvement (OR=5.29 [95% CI 2.10–13.37], p,0.001) and recurrence (OR=3.47 [95% CI 1.91–6.32], p<0.001) (Table 2). To further explore the relationship between these clinicopathologic variables and TERT−124[C>T] (e.g are the univariable associations primarily accounted for by an association between TERT−124[C>T] and tumor thickness) we conducted a multivariable analysis of TERT−124[C>T] in relation to these variables. We observed that none of the clinicopathological variables were independently associated with TERT−124[C>T] (Supplementary Table S2)

Table 2:

BRAF, NRAS, TERT −124 C>T Associations
BRAF Associations NRAS Associations TERT C228T(−124[C>T]) Associations
Characteristic OR (95%CI) p-value OR (95%CI) p-value OR (95%CI) p-value
Age 0.98(0.97–0.99) <0.001 1.03(1.01–1.05) <0.001 1.01(0.99–1.02) 0.227
Sex
 F vs M 0.93(0.62–1.41) 0.743 0.77(0.47–1.27) 0.304 0.64(0.40–1.03) 0.066
Anatomic Site
 Head or Neck vs Extremity 0.73(0.38–1.39) 0.334 0.69(0.32–1.49) 0.345 2.02(1.08–3.78) 0.028
 Trunk vs Extremity 1.04(0.67–1.62) 0.863 0.70(0.41–1.19) 0.191 0.72(0.43–1.21) 0.217
Thickness (continuous)
 Thickness 0.92(0.80–1.05) 0.207 1.05(0.93–1.20) 0.421 1.15(1.02–1.30) 0.023
 log(thickness) 0.76(0.59–0.98) 0.033 1.33(1.00–1.77) 0.047 1.52(1.16–1.98) 0.002
Ulceration
 Present vs Absent 0.67(0.38–1.18) 0.168 1.41(0.78–2.57) 0.260 2.63(1.53–4.52) <0.001
Mitoses
 Present vs Absent 0.71(0.46–1.08) 0.113 2.05(1.17–3.60) 0.012 2.00(1.19–3.34) 0.009
 Continuous(1/mm2) 0.97(0.92–1.03) 0.341 1.06(1.01–1.12) 0.029 1.06(1.01–1.11) 0.030
Histotype
 SSM vs NM 2.08(1.26–3.44) 0.004 0.48(0.29–0.81) 0.006 0.55(0.34–0.89) 0.015
Regression
 Present vs Absent 1.06(0.64–1.76) 0.824 0.97(0.53–1.78) 0.913 0.60(0.32–1.12) 0.111
TILs
 Present vs Absent 1.01(0.62–1.62) 0.983 0.58(0.34–0.99) 0.046 1.55(0.88–2.75) 0.132
TIL Characteristics
 Brisk vs Non-Brisk 0.95(0.55–1.64) 0.849 0.95(0.48–1.85) 0.869 0.81(0.45–1.46) 0.477
Satellites
 Present vs Absent 0.83(0.21–3.27) 0.791 1.61(0.42–6.23) 0.488 1.28(0.33–4.93) 0.720
Solar Elastosis
 Present vs Absent 0.64(0.42–0.98) 0.040 1.25(0.75–2.07) 0.389 0.94(0.59–1.52) 0.815
Associated Nevus
 Present vs Absent 2.01(1.29–3.11) 0.002 1.42(0.85–2.38) 0.180 0.48(0.28–0.84) 0.010
Lymphovascular Invasion
 Present vs Absent 0.90(0.36–2.27) 0.831 1.10(0.40–3.05) 0.850 2.05(0.87–4.86) 0.101
Lymph Node Positive
 Yes vs No 1.45(0.72–2.90) 0.300 0.60(0.22–1.58) 0.298 1.39(0.66–2.92) 0.389
CNS Involvement
 Yes vs No 1.50(0.62–3.65) 0.372 0.96(0.31–2.94) 0.947 5.29(2.10–13.37) <0.001
Recurred
 Yes vs No 1.15(0.63–2.10) 0.658 1.48(0.77–2.88) 0.243 3.47(1.91–6.32) <0.001
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The reference category for TERT−124[C>T] is TERT−124[C].

In contrast, TERT−146[C>T] was only statistically significantly associated with older age (OR=1.02 [95% CI 1.00–1.03], p=0.007) (Supplementary Table S3). NRAS mutations were statistically significantly associated some markers of aggressive tumor phenotypes such as thicker tumors (OR=1.33 [95% CI 1.00–1.77], p=0.047), presence of mitoses (OR=2.05 [95% CI 1.17–3.60], p=0.012), nodular subtype (OR=2.08 [95% CI 1.23–3.44], p=0.006), absence of tumor infiltrating lymphocytes (TILs) (OR=1.72 [95% CI 1.01–2.94], p.0.046), and older age (OR=1.03 [95% CI 1.01–1.05], p<0.001) (Table 2). Conversely, BRAFV600 mutations were statistically significantly associated less aggressive tumor phenotypes such as thinner tumors (OR=0.76 [95% CI 0.59–0.98], p=0.033), superficial spreading subtype (OR=2.08 [95% CI 1.26–3.44], p=0.004), and associated nevus (OR=2.01 [95% CI 1.29–3.11], p=0.002). BRAFV600 mutations were statistically significantly associated inversely with age (OR=0.98 [95% CI 0.97–0.99], p<0.001) (i.e. associated with younger age), and less solar elastosis (OR=0.64 [95% CI 0.42–0.98], p=0.04) (Table 2). The germline −245[T>C] rs2853669 variant was statistically significantly associated with nevus-associated histology (OR=1.53 [95% CI 1.00 – 2.34], p=0.05) (Supplementary Table S3).

Associations between the TERT germline rs2853669 and somatic −124[C>T] sequence variants, clinicopathological characteristics

Due to the potential impact of the germline rs2853669 variant on TERT transcription (Dratwa et al., 2020, Hsu et al., 2006, Ko et al., 2016), we investigated possible associations between the germline variant, the somatic TERT−124[C>T] mutation, and clinicopathological variables. In a univariate analysis we unexpectedly found a modest, yet statistically significant association between the TERT−124[C>T] mutation and the superficial spreading histological subtype among patients carrying the germline variant, compared to individuals lacking the germline variant (OR=2.36 [95% CI 1.00 – 5.55], p<0.05) (Supplementary Table S4). This contrasted with the statistically significant association between the TERT−124[C>T] mutation and the nodular histotype when the germline variant was not considered (Table 2). We examined the distributions of tumor thicknesses between SSM and NM according to patients’ germline status and the TERT−124[C>T] mutation. Although there were no differences in thickness across groups in NM, patients with SSM and TERT−124[C>T] mutant tumors who inherited the germline variant had significantly thicker tumors than those who didn’t (Supplementary Figure S1). We further explored the association between the presence of the germline variant and the TERT−124[C>T] mutation among the SSM and NM histological subtypes in the survival analysis below.

Survival analysis of the TERT germline rs2853669 and somatic −124[C>T] sequence variants

Several clinicopathological variables were associated with shorter recurrence free and overall survival (RFS and OS). These variables were: tumor thickness, ulceration, nodular histotype, mitoses (continuous/mm2), satellites, lymphovascular invasion and lymph node involvement (all p<0.001 for RFS and OS). Sex differences were also observed, notably the male sex was associated with shorter RFS (p=0.011) and OS (p=0.037). In addition, clinical stage (p<0.001) and de novo histology (p=0.023) were associated with shorter RFS. No associations were found between RFS or OS, and either the TERT−146[C>T], BRAF or NRAS mutations (Table 3). However, patients whose tumors harbored the TERT−124[C>T] mutation had both a reduced RFS (HR=2.94 [1.69–5.14], p<0.001) and OS (HR=3.30 [1.48–7.36], p=0.004) compared to patients with TERT−124[C] (Figure 1 and Table 3).

Table 3.

Univariate Survival Analysis

RFS OS
Characteristic HR (95%CI) p-value HR (95%CI) p-value
TERT−124[C>T] vs TERT−124[C] 2.94 (1.69 – 5.14) <0.001 3.30 (1.48 – 7.36) 0.004
TERT−146[C>T] vs TERT−146[C] 0.91 (0.50 – 1.68) 0.767 0.44 (0.15 – 1.30) 0.138
TERT Status
 Either TERT mutation vs WT 2.54 (1.32 – 4.86) 0.005 1.53 (0.66 – 3.58) 0.324
TERT polymorphism
 −245 [T>C] vs consensus 1.01 (0.58 – 1.77) 0.969 1.28 (0.55 – 2.96) 0.565
BRAF Status
 BRAF vs WT 1.09 (0.61 – 1.95) 0.763 0.67 (0.28 – 1.60) 0.369
NRAS Status
 NRAS vs WT 1.25 (0.65 – 2.38) 0.502 1.51 (0.64 – 3.59) 0.351
Age 1.01 (1.00 – 1.03) 0.166 1.02 (1.00 – 1.05) 0.093
Sex
 F vs M 0.45 (0.24 – 0.83) 0.011 0.38 (0.15 – 0.94) 0.037
Thickness (continuous)
 Thickness 1.39 (1.31 – 1.48) <0.001 1.48 (1.36 – 1.62) <0.001
 Log(Thickness) 5.08 (3.71 – 6.97) <0.001 7.20 (4.40 – 11.77) <0.001
Ulceration
 Present vs Absent 8.94 (5.13 – 15.56) <0.001 19.20 (7.67 – 48.04) <0.001
Histotype
 SSM vs NM 0.11 (0.06 – 0.20) <0.001 0.06 (0.02 – 0.17) <0.001
Mitoses (continuous) 1.14 (1.10 – 1.18) <0.001 1.16 (1.11 – 1.21) <0.001
Regression
 Present vs Absent 0.84 (0.41 – 1.73) 0.642 0.71 (0.25 – 2.07) 0.536
TILs
 Present vs Absent 1.08 (0.57 – 2.07) 0.806 0.89 (0.37 – 2.12) 0.784
TIL Characteristics
 Brisk vs Non-Brisk 0.49 (0.24 – 1.01) 0.053 0.41 (0.13 – 1.29) 0.126
Satellites
 Present vs Absent 21.53 (9.93 – 46.68) <0.001 25.25 (10.34 – 61.64) <0.001
Solar Elastosis
 Present vs Absent 0.64 (0.36 – 1.15) 0.135 0.68 (0.31 – 1.49) 0.334
Associated Nevus
 Present vs Absent 0.43 (0.21 – 0.89) 0.023 §
Lymphovascular Invasion
 Present vs Absent 13.65 (7.48 – 24.91) <0.001 17.94 (8.24 – 39.04) <0.001
Lymph Node Positive
 Yes vs No 16.86 (9.73 – 29.23) <0.001 19.74 (8.93 – 43.61) <0.001
AJCC Staging
 II vs I 5.86 (2.53 – 13.59) <0.001 §
 III vs I 41.96 (18.96 – 92.88) <0.001 §
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§

indicates category contained less than 4 events

The reference category for TERT−124[C>T] is TERT−124[C], and the reference category for TERT−146[C>T] is TERT−146[C].

Figure 1: Kaplan-Meier plots of Recurrence Free Survival (RFS) & Overall Survival (OS) for the complete cohort (n=408).

Figure 1:

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Recurrence free survival analysis for patients with positive TERT−124[C>T] tumors (a), patients with positive TERT−146[C>T] tumors (b) and patients who are carriers of the −245 [T>C] germline variant rs2853669 (c). Overall survival analysis for TERT−124[C>T] positives (d), TERT−146[C>T] positives (e) and −245 [T>C] germline variant carriers (f). In the figure −124 wild-type refers to TERT−124[C]. Similarly, −146 wild-type refers to TERT−146[C].

Interestingly, patients who developed SSM in which the TERT−124[C>T] mutation was co-occurring with the germline variant had worse RFS compared to patients with the TERT−124[C>T] mutation alone (p=0.008) (Figures 2A vs. 2B, and Supplementary Figure S2). Among NM patients, TERT−124[C>T] mutation plus germline variant resulted in both reduced RFS (HR=4.51 [1.26–16.15], p=0.03)) and OS (HR=2.93, p=0.011), (Figures 2C vs. 2D, and Supplementary Figures S2, S3 and S4). When we assessed the SSM and NM patients together as a single group, we found that the TERT−124[C>T] mutation was associated with worse survival regardless of the presence of the germline variant (Figures 2E vs. 2F). This may be due to the imbalance in the numbers of patients in each subgroup category. Specifically, among the patients who inherited the consensus sequence, there were 24 TERT−124[C>T] mutant NMs, 28 TERT−124[C>T] mutant SSMs, 37 TERT wild type NMs, but 118 TERT wild type SSMs. Although the p-values are not significant in panels B and D, the blue line corresponding to TERT−124[C>T] is above the gold line in SSM, and below the gold line in NM. Hence, the effect sizes in the different histopathologic subgroups are trending in opposite directions.

Figure 2: Kaplan-Meier plots of Recurrence Free Survival among histopathological subgroups and TERT promoter alterations.

Figure 2:

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RFS analysis for SSM patients who inherited the TERT germline variant −245[T>C] (T234C) with positive TERT−124[C>T] tumors (a), for SSM patients who inherited the consensus sequence with positive TERT−124[C>T] tumors (b), for NM patients who inherited the TERT germline variant −245[T>C] (T234C) with positive TERT−124[C>T] tumors (c) and for NM patients who inherited the consensus sequence with positive TERT−124[C>T] tumors (d). RFS analysis for all patients who inherited the germline variant −245[T>C] (T234C) with positive TERT−124[C>T] (e) and for all patients who inherited the consensus sequence with positive TERT−124[C>T] tumors (f). In the figure −124 wild-type refers to TERT−124[C].

Additionally, we conducted multivariable modeling to assess the independent prognostic value of the TERT−124[C>T] mutations in the context of AJCC stage (Table 4). In a model combining TERT−124[C>T] and AJCC stage, we found that TERT−124[C>T] was an independent prognostic factor for RFS (HR=2.58 [95% CI =1.47–4.53], p=0.001), and OS (HR=2.47 [95% CI=1.10–5.56], p=0.029).

Table 4.

Multivariate Cox PH Survival Analysis

RFS OS
Variables HR (95%CI) p-value p-value of test for PH HR (95%CI) p-value p-value of test for PH
Age + Sex + AJCC + TERT−124[C>T]
Age 1.01(1.00,1.03) 0.135 0.503 1.03(1.00,1.07) 0.039 0.398
Sex F 0.54(0.29,1.01) 0.053 0.63(0.24,1.63) 0.342
AJCC Stage II 4.80(2.04,11.29) <0.001 15.14(1.86,123.63) 0.011
AJCC Stage III 41.77(18.65,93.55) <0.001 146.09(19.16,1113.70) <0.001
TERT−124[C>T] 2.49(1.42,4.39) 0.002 2.54(1.10,5.86) 0.028
AJCC + TERT−124[C>T]
AJCC Stage II 4.89 (2.08 – 11.49) <0.001 0.213 16.50 (2.02 –134.64) 0.009 0.361
AJCC Stage III 39.36 (17.68 – 87.61) <0.001 132.91 (17.56 – 1006.19) <0.001
TERT−124[C>T] 2.58 (1.47 – 4.53) 0.001 2.47 (1.10 – 5.56) 0.029
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The reference category for TERT−124[C>T] is TERT−124[C].

DISUCUSSION

In this study, we assessed 408 primary tumors from a prospective cohort of melanoma patients encompassing stages I, II and III, for common TERT, BRAF and NRAS genetic variants and their associations with clinicopathological features and survival. We found that TERT−124[C>T], and not TERT−146[C>T], was statistically significantly associated with aggressive histopathological features such as nodular subtype, thicker, mitotically active, ulcerated tumors, and clinical recurrence, including a higher rate of CNS involvement. Additionally, in a multivariable analysis, which included current staging criteria, age and sex, we identified TERT−124[C>T] as an independent predictor of survival compared to patients with TERT−124[C]. We also explored the relationship between TERT−124[C>T] and the germline rs2853669 variant. Although TERT−124[C>T] was associated with NM when inheritance of the variant was not considered, the somatic mutation was associated with SSM in patients carrying the rs2853669 variant. The survival analysis revealed that when the TERT−124[C>T] mutation co-occurred with the germline variant, both SSM and NM patients had worse RFS, and NM patients had a shorter OS. However, when SSM and NM patients were assessed together, the presence of the germline variant had little to no effect on survival when tumors were mutated for TERT−124[C>T]. The discrepancy between the results in each histological subgroup and the overall group might be due to the sizable imbalance of the subgroups and the opposite direction of the effect sizes (Figures 2B and 2D). Overall, these observations suggest that in the presence of the somatic mutation and the germline variant, SSM and NM tumors may develop a more aggressive phenotype leading to worse survival. However, additional investigations including mechanistic studies, are needed to further define the potential cooperation between the somatic mutation and the germline variant.

Multiple studies have examined the association between TERT promoter mutations and survival in melanoma. Although the results vary between studies, promoter mutations are generally associated with thicker tumors and poor survival as described in a recent meta-analysis (Gandini et al., 2021). In most of these survival analyses TERT promoter mutations were grouped together. In contrast, our study identified 97 TERT−124[C>T] and 136 TERT−146[C>T] patients out of 408, providing us with a sufficient sample size to support separate analyses. Similar work was conducted in two mostly overlapping melanoma patient cohorts from Spain (Andres-Lencina et al., 2019, Nagore et al., 2016). In agreement with our results, they showed that −124[C>T] mutation, and not −146[C>T], was associated with worse outcomes. They did note an even stronger association between poor survival and the rare tandem mutation −138/−139 [CC>TT] (detected in 5% of patients) (Andres-Lencina et al., 2019). In contrast to our findings, they observed that the effect of the TERT mutation on survival outcomes was limited to patients with the germline consensus sequence −245[T], not the −245[T>C] rs2853669 variant (Nagore et al., 2016). One particular difference between our study and the Spanish cohort was that our cohort had a 10% higher percentage of NMs among the tumor histotypes, and a 10% higher percentage of patients with the germline rs2853669 variant. Since these factors were associated with survival outcomes, the proportional differences could contribute to differences between our studies. Interestingly, 47.5% of our patients had the germline rs2853669 variant, which is higher than the European average (30.5%) based on NCBI ALFA (L. Phan, 2020). Visual inspection of Figure 2, panels A-D, suggests there is a significant association between shorter survival and TERT−124[C>T] among carriers of the germline rs2853669 variant in SSM, but no evidence of a similar association among the non-carriers of the germline rs2853669 variant in SSM. In contrast, a significant association between shorter survival and TERT−124[C>T] exists among carriers of the germline rs2853669 variant in NM, and a similar association may also exist to a lesser extent among the non-carriers of the germline rs2853669 variant in NM. Although, the interaction term for these variables was not significant (p=0.70), the effect sizes in panels B (SSM) and D (NM) trended in opposite directions. In addition, from Supplementary Figure S1, the significantly greater tumor thicknesses for SSM (but not NM) patients who inherited the germline variant rs2853669, and acquired the TERT−124[C>T] mutation, would be consistent with a model in which these histotypes are biologically distinct (Elder et al., 2020, Greenwald et al., 2012, Lattanzi et al., 2019, Poliseno et al., 2012, Salhi et al., 2015).

Overall the variability between all published studies (Gandini et al., 2021) may be due to a combination of factors that may alter how TERT impacts survival. Notably, most studies assessing the germline variant have combined the TERT mutants instead of assessing them individually. Most have also analyzed all histological subtypes together, including those in which TERT mutations are rare (i.e. acral). Combining confounding variables, where the effect of TERT on survival is subtype-specific, may result in different survival outcomes.

The TERT promoter mutations have been associated with poor survival in many other cancers including, bladder, thyroid, hepatocellular carcinoma, renal cell carcinoma and gliomas (Casuscelli et al., 2019, Lee et al., 2017, Liu C. et al., 2016, Liu et al., 2014, Shen et al., 2017, Vuong et al., 2017). These associations may be conditional on the presence or absence of the germline variant rs2853699 (reviewed in Shen et al., 2017), suggesting a complex, tissue-specific role (Hsu et al., 2006, Mosrati et al., 2015, Rachakonda et al., 2013, Shen et al., 2017, Spiegl-Kreinecker et al., 2015). Our data suggest that the impact of the −124[C>T] mutation on survival is modulated by the germline rs2853669 variant and other melanoma subtype-specific factors, yet to be identified.

Functionally, these TERT promoter sequence variants have been shown to activate TERT transcription in melanoma, as well as other cancers (Cancer Genome Atlas, 2015, Ko et al., 2016, Li et al., 2015, Panebianco et al., 2019, Shaughnessy et al., 2020, Yuan et al., 2019). The mutations create de novo consensus binding motifs for E-twenty-six (ETS) transcription factors and can promote a switch to active chromatin (Stern et al., 2015). Both −124[C>T] and −146[C>T] have been shown to increase TERT expression in a set of melanoma cell lines (Shaughnessy et al., 2020); however, TCGA analysis of melanoma tumors showed that increased expression was only associated with TERT −124[C>T] and not −146[C>T] (Cancer Genome Atlas, 2015). The germline rs2853669 variant is located proximally to an E2F1 binding site and is known to disrupt a non-canonical ETS2 site (Heidenreich and Kumar, 2017, Ko et al., 2016). Interestingly, the presence of the germline variant was shown to increase TERT expression in hepatocellular carcinoma, an effect which seemed to double in the presence of the −124[C>T] mutant but not −146[C>T] (Ko et al., 2016). In contrast, Li and colleagues demonstrated that TERT expression could be induced via a non-canonical NF-κB signaling pathway among glioblastoma cell lines mutated at position −146[C>T] (but not −124[C>T]). In that study, the effect of the germline variant was not described (Li et al., 2015). Taken together, the −124[C>T] and −146[C>T] promoter mutations seem to be functionally distinct. Despite evidence that each mutant appears to require different upstream signaling pathways to induce TERT expression, the precise mechanism by which the −124[C>T] but not the −146[C>T] mutation contributes to worse prognosis in melanoma is unknown. Indeed, some studies in other cancers have used a variety of techniques to examine how TERT over-expression may directly affect cell survival independently from its role in telomere maintenance. These non-canonical roles encompass a wide array of functions including: reduction of reactive oxygen species; promotion of epithelial mesenchymal transition, angiogenesis and metastasis; and DNA methylation of tumor suppressor genes (Indran et al., 2011, Rosen et al., 2020, Sherman et al., 2021, Yuan et al., 2019).

Among the limitations of our study, we did not investigate the impact of other TERT promoter sequence variants; we focused our analyses on the most commonly found variants in melanoma. We also limited our analysis to the two most common histological subtypes, due to the high frequency of TERT mutations. However, this does not eliminate the potential impact of TERT mutations on the other subtypes. As the primary purpose of this investigation was to assess these variants as potential biomarkers associated with clinicopathological features and patient survival we did not examine how changes in the TERT promoter affected TERT expression. Another limiting factor in our study was the relatively low rates of recurrence and mortality events, which precluded us from performing stage specific sub-analyses. Additionally, using overall, rather than melanoma specific survival limits the analysis, as it is unknown whether patients with TERT−124[C>T] or rs2853669 are more like to die of other diseases that are not related to melanoma.

In conclusion, the current report represents the largest analysis of prospectively accrued, non-acral and non-lentigo maligna melanoma patients to date, and validates several prior studies demonstrating the specific adverse prognostic impact of the −124[C>T] mutation (Andres-Lencina et al., 2019, Gandini et al., 2021, Griewank et al., 2014a). The protocol-driven clinical follow up (compared to more common retrospective studies) is a strength of the study design. As such, it adds high-level evidence to support the prognostic role of the −124[C>T] mutation in melanoma pathogenesis and justify additional studies to advance its potential as a biomarker (along with the germline rs2853669 variant) in melanoma.

MATERIALS & METHODS

Patients

We studied 408 melanoma patients prospectively enrolled between 2003 and 2013 in the New York University Interdisciplinary Melanoma Cooperative Group (IMCG), an IRB-approved (IRB #10362), clinicopathological biospecimen database (Wich et al., 2009). All eligible patients provided written informed consent. Patients diagnosed with stage I, II or III (AJCC 8th edition) superficial spreading (SSM) or nodular melanoma (NM) were included in the study (Amin et al., 2017). Clinicopathological characteristics were obtained through medical records and pathology reports and stored in a RedCap database with several layers of protection of patients’ personal health information (PHI). All patients had protocol-driven follow up to collect clinical data. Only patients with a minimum of 3 years follow-up or until evidence of recurrence were included. Median follow up time was 78 months.

Mutational analysis

Hematoxylin and eosin (H&E) stained slides were reviewed for tumor content and only cases with >10% tumor content were selected for mutational analysis. Up to eight 5μm FFPE tissue sections per tumor were scraped into 1.5ml microcentrifuge tubes. DNA was extracted using the Mo Bio Biostic FFPE tissue DNA isolation kit (MoBio, Carlsbad, CA) as previously described (Chang et al., 2020). DNA concentrations were measured using QuBit 2.0 Fluorometer (Invitrogen, Carlsbad, CA.). Multiplex SNaPshot assays (Applied Biosystems, Foster City, CA) were used to detect BRAFV600E/K/M, NRASQ61K/L/R, TERT promoter mutations −124[C>T] (C228T) and −146[C>T] (C250T), as well as the TERT promoter rs2853669 germline polymorphism −245[T>C] (T235C)as previously described (Chang et al., 2020, Corless et al., 2019). Probe information is described in Supplementary Text. Laboratory personnel were blinded to patient clinical data.

Statistical Analysis

Patient and tumor characteristics were analyzed using descriptive statistics. Association between clinicopathologic variables and TERT germline/somatic variants were assessed by univariate and/or multivariate logistic regression. Survival outcomes were further assessed by Kaplan–Meier estimate, log-rank test and Cox Proportional Hazard model. Statistical analyses for TERT mutations used the following reference categories: For TERT−124[C>T], the reference category was all tumors with TERT−124[C]. For TERT−146[C>T], the reference category was all tumors with TERT−146[C]. The p-values for testing proportional hazard assumption are also provided. Statistical significance was attained when p-values (two-sided) reached 0.05 or lower.

Supplementary Material

Supp.Materials

FUNDING SOURCES:

This work was supported by the National Cancer Institute at the National Institutes of Health (grant numbers R21 CA198495, P50 CA225450 and P30 CA016087) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases at the National Institutes of Health (grant number T32 AR064184).

Footnotes

CONFLICT OF INTEREST STATEMENT:

- Yilong Zhang is an employee of Merck & Co., Inc.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

DATA AVAILABILITY STATEMENT:

No datasets were generated or analyzed during the current study.

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Associated Data

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Supplementary Materials

Supp.Materials
NIHMS1816625-supplement-Supp_Materials.pdf (1.2MB, pdf)

Data Availability Statement

No datasets were generated or analyzed during the current study.

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