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Association between the Germline MC1R Variants and Somatic BRAF/NRAS Mutations in Melanoma Tumors

      Abbreviations

      ALM
      acral lentigeous melanoma
      LMM
      lentigeous malignant melanoma
      MAP
      mitogen-activated protein
      NM
      nodular melanoma
      PKA
      protein kinase A
      RHC
      red hair color
      SSM
      superficial spreading melanoma
      TO THE EDITOR
      Sporadic melanoma is associated with high frequencies of somatic mutations in BRAF, a serine–threonine kinase acting in the mitogen-activated protein (MAP) kinase pathway (
      • Michaloglou C.
      • Vredeveld L.C.
      • Mooi W.J.
      • et al.
      BRAF (E600) in benign and malignant human tumours.
      ). Mutations in the gene arise mainly in tumors that develop on skin with intermittent sun exposure (
      • Dhomen N.
      • Marais R.
      New insight into BRAF mutations in cancer.
      ). The most frequent mutation in BRAF, an A-to-T transversion at nucleotide 1799, results in a valine to glutamic acid change at codon 600 (
      • Michaloglou C.
      • Vredeveld L.C.
      • Mooi W.J.
      • et al.
      BRAF (E600) in benign and malignant human tumours.
      ). BRAFV600E is intrinsically activated with a several-hundred-fold increase in basal kinase activity. The possible association between the BRAFV600E mutation and germline variants in the pigmentation-related melanocortin receptor 1 (MC1R) gene has been the focus of several studies in recent years. MC1R is the major contributor to pigmentation diversity in humans (
      • Rees J.L.
      Genetics of hair and skin color.
      ). The receptor gene is highly polymorphic, with more than 100 variants, many being nonsynonymous (
      • Garcia-Borron J.C.
      • Sanchez-Laorden B.L.
      • Jimenez-Cervantes C.
      Melanocortin-1 receptor structure and functional regulation.
      ). The importance of MC1R is accentuated by the association of its variants with skin pigmentation variation, skin cancer risk, and the penetrance of germline CDKN2A mutations (
      • Box N.F.
      • Duffy D.L.
      • Chen W.
      • et al.
      MC1R genotype modifies risk of melanoma in families segregating CDKN2A mutations.
      ;
      • Sulem P.
      • Gudbjartsson D.F.
      • Stacey S.N.
      • et al.
      Genetic determinants of hair, eye and skin pigmentation in Europeans.
      ;
      • Scherer D.
      • Nagore E.
      • Bermejo J.L.
      • et al.
      Melanocortin receptor 1 variants and melanoma risk: a study of 2 European populations.
      ). So far, studies on the association between germline MC1R variants and frequency of somatic BRAF mutations in melanoma tumors have produced inconsistent results—some researchers have suggested that the presence of MC1R variants increases the frequency of BRAF mutations; others found no association (
      • Landi M.T.
      • Bauer J.
      • Pfeiffer R.M.
      • et al.
      MC1R germline variants confer risk for BRAF-mutant melanoma.
      ;
      • Fargnoli M.C.
      • Pike K.
      • Pfeiffer R.M.
      • et al.
      MC1R variants increase risk of melanomas harboring BRAF mutations.
      ;
      • Hacker E.
      • Hayward N.K.
      • Dumenil T.
      • et al.
      The association between MC1R genotype and BRAF mutation status in cutaneous melanoma: findings from an Australian population.
      ;
      • Thomas N.E.
      • Kanetsky P.A.
      • Edmiston S.N.
      • et al.
      Relationship between germline MC1R variants and BRAF-mutant melanoma in a North Carolina population-based study.
      ).
      In the present study, we investigated the correlation between MC1R variants and the most common somatic BRAF/NRAS mutations in 202 paraffin-embedded primary melanoma tissues. Mutations in BRAF and NRAS were detected using single-strand conformation polymorphism and confirmed by sequencing. The MC1R gene was screened in three fragments by direct sequencing. Out of all 202 tumors, 63 (31%) were superficial spreading melanoma (SSM), 84 (42%) were nodular melanoma (NM), 20 (10%) were lentigeous malignant melanoma (LMM), 13 (6%) were acral lentigeous melanoma (ALM), and 22 (11%) were histologically not specified.
      Screening for MC1R failed in 29 samples, for BRAF in 23 samples, and for NRAS in 17 samples. Overall, 82.7% of 173 genotyped tumors carried at least one MC1R variant; 59.0% carried one MC1R variant, 22.0% carried two, and 1.7% carried three variants; the red hair color (RHC) variants (D84E, R142H, R151C, R160W, and D294H) were observed in 57.2% of tumors (Table 1).
      Table 1MC1R variant and BRAF/NRAS mutation frequencies in primary melanoma tumors according to histological melanoma subtype
      Histotype
      GeneAllSSMNMLLMALMNOS
      MC1R
      ≥1 variant82.7% (143)35.0% (50)37.0% (53)9.8% (14)6.3% (9)11.9% (17)
       1 variant59.0% (102)36.3% (37)37.2% (38)8.8% (9)5.9% (6)11.8% (12)
       2 variants22.0% (38)28.9% (11)39.5% (15)10.5% (4)7.9% (3)13.2% (5)
       3 variants1.7% (3)66.6% (2)33.4% (1)
       RHC variants57.2% (99)34.3% (34)38.4% (38)10.1% (10)7.1% (7)10.1% (10)
       non-RHC variants25.5% (44)36.4% (16)34.1% (15)9.1% (4)4.5% (2)15.9% (7)
      BRAF
       Total20.7% (37)43.2% (16)43.2% (16)2.7% (1)2.7% (1)8.2% (3)
       V600E17.3% (31)41.9% (13)48.4% (15)3.2% (1)6.5% (2)
       V600K3.0% (5)40.0% (2)20.0% (1)20.0% (1)20.0% (1)
       L597R<1% (1)100.0% (1)
      NRAS
       Total12.4% (23)17.4% (4)60.9% (14)4.3% (1)4.3% (1)13.1% (3)
       Q61K9.1% (16)25.0% (4)50.0% (8)6.3% (1)6.3% (1)12.5% (2)
       Q61R3.2% (5)80.0% (4)20.0% (1)
       Q61L1.1% (2)100.0% (2)
      Abbreviations: ALM, acral lentigeous melanoma; LMM, lentigeous malignant melanoma; MC1R, melanocortin receptor 1; mut, mutated; NM, nodular melanoma; NOS, not specified; RHC, red hair color variant; SSM, superficial spreading melanoma; wt, wild type.
      Somatic mutations in the BRAF gene were detected in 20.7% of screened melanoma tumors (Table 1). All but one of the mutations were observed at the “hot spot” codon 600, with a T1799A mutation (V600E) in 31 tumors and a GT1798-99AA tandem mutation (V600K) in five tumors. One tumor had a T1792G mutation in codon 597, resulting in a lysine-to-arginine switch. In exon 2 of the NRAS gene, mutations at codon 61 were detected in 23 (12.4%) tumors (Table 1). BRAF and NRAS mutations were mutually exclusive.
      Of 132 tumors with at least one MC1R variant, 22 (16.7%) carried a somatic BRAF mutation. In contrast, of 24 samples with the MC1R consensus sequence, 11 (45.8%) carried a BRAF mutation. The odds ratio for the probability of the acquisition of somatic BRAF mutations in tumors from individuals with the variant MC1R genotype was 0.24 (95% CI, 0.09–0.60; P=0.001; Table 2). The correlation remained significant for RHC (OR, 0.23; 95% CI, 0.09–0.61; P=0.002) and non-RHC variants (OR, 0.25; 95% CI, 0.08–0.79; P=0.015) separately (Table 2). In contrast, only 2 (8.0%) of 25 tumors with the MC1R consensus sequence carried somatic NRAS mutations, whereas of 127 tumors with MC1R variants 17 (13.4%) revealed an NRAS mutation (OR, 1.78; 95% CI, 0.38–8.23; P=0.46; Table 2).
      Table 2Distribution of somatic mutations in the BRAF (a) and NRAS (b) genes in MC1R carriers and noncarriers according to histological melanoma subtype
      HistotypeMC1R consensusMC1R carrierRHC
      Two individuals were homozygous for the R151C variants and two were homozygous for the R160W variant. All other individuals were heterozygous for RHC variants.
      Non-RHC
      (a)
      All
      BRAF
       wt54.2% (13)83.3% (110)83.7% (77)82.5% (33)
       mut45.8% (11)16.7% (22)16.3% (15)17.5% (7)
       OR (95% CI)ref0.24 (0.09–0.60)0.23 (0.09–0.61)0.25 (0.08–0.79)
      P0.0010.0020.015
      SSM
      BRAF
       wt80.0% (4)70.8% (34)75.8% (25)60.0% (9)
       mut20.0% (1)29.2% (14)24.2% (8)40.0% (6)
       OR (95% CI)ref1.65 (0.17–16.07)1.28 (0.12–13.17)2.67 (0.24–30.07)
      P0.670.840.42
      NM
       BRAF
       wt42.9% (6)87.2% (41)81.8% (27)100.0% (14)
       mut57.1% (8)12.8% (6)18.2% (6)0
       OR (95% CI)ref0.11 (0.03–0.43)0.17 (0.04–0.66)NA
      P<0.0010.008
      LMM
      BRAF
       wt100.0% (1)92.3% (12)90.0% (9)100.0% (3)
       mut07.7% (1)10.0% (1)0
       OR (95% CI)refNANANA
      P
      ALM
      BRAF
       wt66.7% (2)100.0% (9)100.0% (7)100.0% (2)
       mut33.3 (1)000
       OR (95% CI)refNANANA
      P
      All
      Individuals whose tumors were positive for NRAS mutations were excluded.
      BRAF
       wt50.0% (11)80.7% (92)81.5% (66)78.8% (26)
       mut50.0% (11)19.3% (22)18.5% (15)21.2% (7)
       OR (95% CI)ref0.24 (0.09–0.62)0.23 (0.08–0.62)0.27 (0.08–0.88
      P0.0020.0030.026
      All
      Individuals whose tumors were either of the ALM type or not specified were excluded.
      BRAF
       wt55.0% (11)80.6% (87)80.3% (61)81.3% (26)
       Mut45.0% (9)19.4% (21)19.7% (15)18.7% (6)
       OR (95% CI)ref0.30 (0.11–0.80)0.24 (0.08–0.66)0.25 (0.08–0.81)
      P0.0130.0200.042
      (b)
      All
      NRAS
       wt92.0% (23)86.6% (110)88.5% (77)82.5% (33)
       mut8.0% (2)13.4% (17)11.5% (10)17.5% (7)
       OR (95% CI)ref1.78 (0.38–8.23)1.49 (0.31–7.31)2.44 (0.46–12.82)
      P0.460.620.28
      All
      Individuals whose tumors were positive for BRAF mutations were excluded.
      NRAS
       wt86.7% (13)84.3% (91)86.5% (64)79.4% (27)
       mut13.3% (2)15.7% (17)13.5% (10)20.6% (7)
       OR (95% CI)ref1.21 (0.25–5.87)1.02 (0.20–5.19)1.69 (0.31–9.27)
      P0.810.990.55
      Abbreviations: ALM, acral lentigeous melanoma; CI, confidence interval; LMM, lentigeous malignant melanoma; MC1R, melanocortin receptor 1; mut, mutated; NA, not analyzed; NM, nodular melanoma; OR, odds ratio; RHC, red hair color variant; SSM, superficial spreading melanoma; wt, wild type.
      1 Two individuals were homozygous for the R151C variants and two were homozygous for the R160W variant. All other individuals were heterozygous for RHC variants.
      2 Individuals whose tumors were positive for NRAS mutations were excluded.
      3 Individuals whose tumors were either of the ALM type or not specified were excluded.
      4 Individuals whose tumors were positive for BRAF mutations were excluded.
      Stratification of the data according to histological melanoma subtype showed that the observed correlation between MC1R variants and oncogenic BRAF mutations was restricted to the subset of 61 NM. Of 47 NM samples that had at least one MC1R variant, only six (12.8%) tumors revealed a BRAF mutation compared with eight (57.1%) in which no MC1R variant was present (OR, 0.11; 95% CI, 0.03–0.43; P<0.001; Table 2). This significant observation held for RHC variants as well (OR, 0.17; 95% CI, 0.04–0.66; P=0.008; Table 2). In contrast, for 53 analyzed SSM tumors we did not observe this correlation between MC1R variants and BRAF mutations (OR, 1.65; 95% CI, 0.17–16.07; P=0.67). Owing to the low numbers of ALM and LMM, no separate analysis was performed for those subtypes.
      The cross-talk between the pigmentation and the MAP kinase pathways is well known; however, the effect of MC1R variants on somatic mutations has remained unresolved (
      • Dhomen N.
      • Marais R.
      New insight into BRAF mutations in cancer.
      ;
      • Fargnoli M.C.
      • Pike K.
      • Pfeiffer R.M.
      • et al.
      MC1R variants increase risk of melanomas harboring BRAF mutations.
      ;
      • Michaloglou C.
      • Vredeveld L.C.
      • Mooi W.J.
      • et al.
      BRAF (E600) in benign and malignant human tumours.
      ;
      • Hacker E.
      • Hayward N.K.
      • Dumenil T.
      • et al.
      The association between MC1R genotype and BRAF mutation status in cutaneous melanoma: findings from an Australian population.
      ). We observed that the frequency of somatic BRAF mutations was significantly lower in carriers of MC1R variants than in noncarriers. However, the observed correlation was restricted to NM, which is the second most common subtype of melanoma. NM lacks the radial growth phase and is generally detected in its later stages. Involvement of distinct pathways in the development of SSM and NM is indicated by differences in gene expression patterns (
      • Jaeger J.
      • Koczan D.
      • Thiesen H.J.
      • et al.
      Gene expression signatures for tumor progression, tumor subtype, and tumor thickness in laser-microdissected melanoma tissues.
      ;
      • Warycha M.A.
      • Christos P.J.
      • Mazumdar M.
      • et al.
      Changes in the presentation of nodular and superficial spreading melanomas over 35 years.
      ).
      One plausible explanation for the observed correlation between germline MC1R variants and somatic BRAF mutations in our study could be that in normal melanocytes NRAS signaling is usually directed through BRAF, which is a stronger activator of the downstream signaling than CRAF (
      • Dumaz N.
      • Hayward R.
      • Martin J.
      • et al.
      In melanoma, RAS mutations are accompanied by switching signaling from BRAF to CRAF and disrupted cyclic AMP signaling.
      ). Mutated NRAS seems to use intrinsically weak CRAF instead of BRAF for downstream signaling (
      • Dumaz N.
      • Hayward R.
      • Martin J.
      • et al.
      In melanoma, RAS mutations are accompanied by switching signaling from BRAF to CRAF and disrupted cyclic AMP signaling.
      ). In normal melanocytes, CRAF is, in general, inactivated through protein kinase A (PKA), which is a downstream effecter of elevated cAMP levels due to signaling from MC1R ligand binding (
      • Michaloglou C.
      • Vredeveld L.C.
      • Mooi W.J.
      • et al.
      BRAF (E600) in benign and malignant human tumours.
      ). Most of the loss-of-function MC1R variants are associated with decreased cAMP levels and consequently reduced PKA function. It is reasonable to infer that the presence of MC1R variants results in diminished suppression of CRAF facilitating signaling by mutant NRAS. The presence of MC1R variants might contribute to the selection of clones with mutant NRAS. Our findings in this study are in accord with those observations. Two previous studies, however, showed increased frequency of BRAF mutations in the carriers of MC1R variants (
      • Landi M.T.
      • Bauer J.
      • Pfeiffer R.M.
      • et al.
      MC1R germline variants confer risk for BRAF-mutant melanoma.
      ;
      • Fargnoli M.C.
      • Pike K.
      • Pfeiffer R.M.
      • et al.
      MC1R variants increase risk of melanomas harboring BRAF mutations.
      ); two other studies found no association (
      • Hacker E.
      • Hayward N.K.
      • Dumenil T.
      • et al.
      The association between MC1R genotype and BRAF mutation status in cutaneous melanoma: findings from an Australian population.
      ;
      • Thomas N.E.
      • Kanetsky P.A.
      • Edmiston S.N.
      • et al.
      Relationship between germline MC1R variants and BRAF-mutant melanoma in a North Carolina population-based study.
      ). Further studies with larger numbers of tumor samples are imperative to validate the true correlation between the germline MC1R variants and somatic oncogenic mutations in melanoma, with an emphasis on separation of tumor subtypes.

      ACKNOWLEDGMENTS

      SA was supported by a guest fellowship awarded by the German Cancer Research Center, Heidelberg, during her stay at the Division of Molecular Genetic Epidemiology.

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