- Email: [email protected]
489 Investigation of the immune infiltrate of metastatic melanoma under immune checkpoint inhibition
ABSTRACTS | Melanoma and Other Skin Cancers 489
490
Investigation of the immune infiltrate of metastatic melanoma under immune checkpoint inhibition JC Hassel1, M Flossdorf2, S Ha¨nzelmann3, J Winkler1, L Appel2, E Streit1, N Halama4, M Faryna5 and I Poschke6 1 Dermatology and NCT, University Hospital Heidelberg, Heidelberg, Germany, 2 Div. of Theoretical Systems Biology, DKFZ, Heidelberg, Germany, 3 TRON, Mainz, Germany, 4 Medical Oncology, NCT, Heidelberg, Germany, 5 BioNTech, Mainz, Germany and 6 Div. Molecular Oncology of Gastrointestinal Tumors, DKFZ, Heidelberg, Germany Tumor infiltrating lymphocytes (TIL) play a crucial role in the therapeutic impact of immune checkpoint blocker (ICB). We investigated tumor samples as well as peripheral blood from 16 patients with metastatic melanoma before and during treatment with ICB. Aims were (i) the identification of biomarkers for clinical response through immunohistochemical analysis, (ii) the investigation of TILs through T-cell receptor (TCR) repertoire profiling and (iii) analysis of the transcriptome. Immunohistochemical analysis revealed no significant differences between responder and non-responder in pretreatment samples, but a tendency to more CD8+ T-cells and tumor associated macrophages (TAM) in posttreatment samples of responder. In addition, responder tended to have a higher increase in the number of TAM (CD163) and TIL (CD3, FoxP3, CD20) under treatment. Using TCR repertoire profiling by deep TCR sequencing we found that responding metastases develop more new TIL clones compared to non-responder (p¼0.012). In contrast pretreatment clones were found posttreatment in a similar extent (7.8 vs 13.1; p¼0.609). In addition, clone size of the top 10 clones was generally smaller in responder indicating a more diverse repertoire. RNA sequencing revealed differential expression profiles between responder and non-responder pretreatment as well as during the treatment. In conclusion, in contrast to previous reports and despite a differential RNA expression profile the number of TIL pretreatment did not correlate with response but responding metastases tended to have a higher increase in TIL and TAM. In addition, responder were characterized by expansion of more new TIL clones under treatment with a more diverse repertoire instead of an expansion of preexisting clones as previously reported.
The effect of Dsg2 in the SSC cells on exosome contents, release, and effects on recipient fibroblasts A Overmiller1, J Pierluissi1, P Wermuth1, U Martinez-Outschoorn1, A Luginbuhl1, J Curry1, L Harshyne1, J Wahl2, A South1 and MG Mahoney1 1 Thomas Jefferson University, Philadelphia, PA and 2 University of Nebraska, Lincoln, NE Exosomes are nanoscale membrane-derived vesicles (<150 nm) that serve as intercellular messengers carrying lipids, proteins, and genetic material. Substantial evidence has shown that cancer-derived exosomes, secreted by tumor cells into the blood and other bodily fluids, play a critical role in modulating the tumor microenvironment and affecting the pathogenesis of cancer. Here, exosomes were isolated from squamous cell carcinoma (SCC) keratinocytes by sequential ultracentrifugation or one-step polymer precipitation methods and analyzed by Nanoparticle Tracking Analysis. Western blotting showed positivity for exosome markers including the tetraspanins CD9 and CD63. We demonstrate for the first time that SCC exosomes were enriched with the 65 kDa C-terminal fragment (CTF) of desmoglein 2 (Dsg2), a desmosomal cadherin often overexpressed in skin malignancies. Dsg2 has been shown to activate mitogenic signaling, enhance cell growth and migration and promote oncogenesis. Expression of Dsg2-GFP further confirmed the presence of a distinct GFP-labeled Dsg2 CTF in exosomes. In normal and SCC keratinocytes, Dsg2 increased exosome release and mitogenic content including EGFR and c-Src. SCC exosomes were internalized by primary human fibroblasts, demonstrating the intercellular signaling axis between epidermal keratinocytes and nearby stromal cells. SCC exosomes enhanced fibroblast proliferation in a dose-dependent manner and exosomes from cells overexpressing Dsg2 had a greater pro-proliferative effect. Localized expansion, invasion, and microenvironment manipulation by malignant cells is important for tumor progression. While Dsg2 has low basal expression in the normal oral epithelium, we show that it was upregulated in the highly malignant head and neck SCCs, and exosomes isolated from SCC patients’ sera were enriched in Dsg2 CTF. This study defines a mechanism by which Dsg2 expression in cancer cells can modulate the tumor microenvironment.
491
492
Cellular response patterns to single MEK inhibition correlate with the efficacy of combined MEK/CDK4,6 targeting in melanoma C Posch3, M Sanlorenzo1, J Ma2, ST Kim2, M Zekhtser2, K Rappersberger3 and S Ortiz-Urda2 1 Department of Medical Sciences, University of Turin, Turin, Italy, 2 Dermatology, University of California San Francisco, San Francisco, CA and 3 Dermatology, The Rudolfstiftung Hospital, Vienna, Austria Treatment of NRAS mutant melanoma is challenging. The discovery that co-targeting of MEK + CDK4,6 has antitumor activity created excitement for patients and clinicians; however, first clinical results did not met pre-clinical expectations. In this study we investigate the response patterns of NRAS mutant melanoma cells in vitro and in vivo when challenged with inhibitors of MEK, CDK4,6 and their combination. Data revealed, that in vitro growth response patterns of cells treated with the MEK/CDK4,6 combination can be used to predict the in vivo efficacy of co-targeting in a xenograft model of NRAS mutant melanoma. In addition, signaling changes after single MEK inhibition also correlated with the response to the MEK/CDK4,6 combination: Cells displaying activation of the cell cycle pathway after MEK inhibition evidenced by elevated pRb levels, showed more effective growth reduction with MEK/CDK4,6 co-targeting compared to single MEK inhibitor treatment. In contrast, MEK/CDK4,6 and single MEK inhibitor treatment were equally effective in cells that responded with unchanged or decreased protein levels of pRb after single MEK inhibition. Cells characterized by these criteria, showed induction of apoptosis in vivo. This pattern is not limited to NRAS mutant cells, but can be applied to BRAF mutant cells and cells that are wild-type for these mutations. This study shows that the MEK/CDK4,6 combination effectively reduces growth of a subset of NRAS mutant melanoma cells. Further, MEK/CDK4,6 has antitumor potential in cells with genetic driving alterations other than NRAS mutations, and might thus offer a new treatment strategy for an extended cohort of patients with melanoma. Results suggest that the efficacy of the MEK/CDK4,6 combination can be predicted by in vitro viability assays and by the changes of pRb levels of cells after single MEK inhibition.
Non-cell autonomous suppression of melanoma by the epidermal polarity protein Par3 through control of direct keratinocyte-melanocyte interactions M Mescher1, P Jeong1, M Ru¨bsam1, M Kranen1, J Landsberg2, M Schlaak3, C Mauch3, T Tueting4, CM Niessen3 and S Iden1 1 CECAD, University of Cologne, Cologne, Germany, 2 University of Bonn, Bonn, Germany, 3 Department of Dermatology, University of Cologne, Cologne, Germany and 4 Department of Dermatology, University Hospital Magdeburg, Magdeburg, Germany Melanoma, an aggressive skin malignancy with increasing lifetime risk, originates from melanocytes that are in close contact with surrounding epidermal keratinocytes. Though keratinocyte-melanocyte cross-talk is essential for skin photoprotection it is poorly understood how the epidermal microenvironment controls melanomagenesis. Here we identify an unexpected non-cell autonomous role of epidermal polarity proteins, molecular determinants of cyto-architecture, in malignant melanoma. Epidermal Par3 inactivation in mice promotes melanocyte dedifferentiation, motility and hyperplasia and in an autochthonous melanoma model results in increased tumor formation and lung metastasis. Keratinocyte-specific Par3 loss upregulates surface P-cadherin that is essential to promote melanocyte proliferation and phenotypic switch towards dedifferentiation. In agreement, low epidermal PAR3 and high PCadherin expression correlate with human melanoma progression, implying that this mechanism also drives human disease. Collectively, our data show that reduced keratinocyte Par3 function fosters a permissive P-cadherin-dependent niche for melanocyte transformation, invasion and metastasis. This reveals a previously unrecognized extrinsic tumor suppressive mechanism, whereby epithelial polarity proteins dictate the cyto-architecture and fate of other tissue-resident cells to suppress their malignant outgrowth.
493
494
Characterization and modulation of CC-chemokine Receptor 6 (CCR6) mediated immunosurveillance in malignant melanoma D Martin-Garcia, A Enk and A Lonsdorf Department of Dermatology, University Hospital Heidelberg, Heidelberg, Germany Chemokine ligand 20 (CCL20) and the antimicrobial peptide ß-defensin expressed in the epidermis are a potent impetus for the recruitment of subsets of dendritic cells (DC), B-cells and memory T cells expressing chemokine receptor 6 (CCR6), the exclusive receptor. In addition to its constitutive expression in the epidermis, CCL20 and a corresponding CCR6expressing immune cell infiltrate have been detected in several malignancies, including melanoma. Yet, the functional contribution of the CCR6/CCL20 axis for the immune control of melanoma remains controversial. The characterization of CCR6-guided immune cell subsets and their functional contribution for the immune control of melanoma comprises the focus of this project. We evaluated the homeostatic and inducible secretion of CCL20 by different murine and human melanoma cutaneous cell lines by enzyme-linked Immunoabsorbent assay (ELISA). Both, murine (B16, Ret) and human (A375, C32) melanoma cell lines are capable of secreting CCL20 and show significant differences in the magnitude of inducible CCL20 expression upon stimulation with pro-inflammatory cytokines (i.e. TNF-a, IL-1a, IL-1b and TGF-b) in vitro. In order to determine the functional relevance of CCR6 on local tumor growth, metastasis and the tumor microenvironment, B16 melanoma cells retrovirally transduced with luciferase (B16-luc) were injected subcutaneously into the flank of wild type C57BL/6 (WT) and congenic CCR6eknockout (CCR6KO) mice. While mice in both groups developed local tumors, we observed a trend towards slower tumor growth in CCR6KO mice inoculated with CCL20-expressing B16-luc melanoma cells, suggesting that the capability of recruiting CCR6 immune cells to the tumor site may be an essential factor during tumor progression. Additional experimental approaches will evaluate the effects of stably enhanced (by injection of retrovirally transduced B16 cells overexpressing CCL20) and reduced (by local injection of a CCL20-neutralizing antibody) CCL20 expression in the tumor microenvironment in both WT and CCR6KO mice.
S244 Journal of Investigative Dermatology (2016), Volume 136
Heterogeneous mutational status of melanomas in multiple primary melanoma patients C Pellegrini1, C Martorelli1, G Cipolloni2, L Di Nardo1, A Antonini1, M Maturo1 and M Fargnoli1 1 Dept. of Dermatology, University of L’Aquila, L’Aquila, Italy and 2 Dept. of Pathology, University of L’Aquila, L’Aquila, Italy Multiple primary melanomas (MPM) develop in about 5% of sporadic melanoma patients and in up to 19% of melanoma patients with a positive family history. Many genetic alterations, including predisposing and/or somatic mutations, may contribute to the development of MPM and data on the genetic diversity of MPMs are limited. We aimed to assess the frequency and distribution of BRAF, NRAS and TERT promoter somatic mutations in subsequent melanomas of the same patient and evaluate the association of somatic alterations with germline mutational profile of MPM patients. Sixty-six synchronous/asynchronous FFPE melanoma tissues from 31 patients were analyzed by Real-Time PCR, Sanger Sequencing and SNaPshot molecular methods for BRAF, NRAS and TERT promoter mutations. Germline DNA was screened by Sanger sequencing for mutations in CDKN2A, CDK4, POT1, MITF, MC1R genes and TERT promoter. BRAF V600 mutations were found in 46.2% of melanomas, NRAS mutations in 10.6% and TERT promoter rs2853669 polymorphism in 64.2%. Mutational somatic profile (BRAF, NRAS) was concordant between first and subsequent primary tumors in 65.4% of patients. Somatic mutational status was not associated with any of the clinical characteristics of patients or melanomas, including age, melanoma thickness and body site. At the germline level, MPM patients were wild-type for mutations in CDKN2A, CDK4, POT1 and MITF genes. TERT-promoter polymorphisms (rs2853669 and rs35226131) were identified in 46.1% of patients and MC1R allelic variants in 73.1%. Carriers of MC1R changes were diagnosed more frequently with melanoma on the trunk compared to wild-type patients (p¼0.02), but no association between MC1R status and somatic mutations was identified in our patients. In conclusion, our results support the heterogeneity of molecular profiles in MPM of the same patient with implication in clinical practice due to the difficulties in molecularly classifying patients with discrepant primary melanomas.
490
Investigation of the immune infiltrate of metastatic melanoma under immune checkpoint inhibition JC Hassel1, M Flossdorf2, S Ha¨nzelmann3, J Winkler1, L Appel2, E Streit1, N Halama4, M Faryna5 and I Poschke6 1 Dermatology and NCT, University Hospital Heidelberg, Heidelberg, Germany, 2 Div. of Theoretical Systems Biology, DKFZ, Heidelberg, Germany, 3 TRON, Mainz, Germany, 4 Medical Oncology, NCT, Heidelberg, Germany, 5 BioNTech, Mainz, Germany and 6 Div. Molecular Oncology of Gastrointestinal Tumors, DKFZ, Heidelberg, Germany Tumor infiltrating lymphocytes (TIL) play a crucial role in the therapeutic impact of immune checkpoint blocker (ICB). We investigated tumor samples as well as peripheral blood from 16 patients with metastatic melanoma before and during treatment with ICB. Aims were (i) the identification of biomarkers for clinical response through immunohistochemical analysis, (ii) the investigation of TILs through T-cell receptor (TCR) repertoire profiling and (iii) analysis of the transcriptome. Immunohistochemical analysis revealed no significant differences between responder and non-responder in pretreatment samples, but a tendency to more CD8+ T-cells and tumor associated macrophages (TAM) in posttreatment samples of responder. In addition, responder tended to have a higher increase in the number of TAM (CD163) and TIL (CD3, FoxP3, CD20) under treatment. Using TCR repertoire profiling by deep TCR sequencing we found that responding metastases develop more new TIL clones compared to non-responder (p¼0.012). In contrast pretreatment clones were found posttreatment in a similar extent (7.8 vs 13.1; p¼0.609). In addition, clone size of the top 10 clones was generally smaller in responder indicating a more diverse repertoire. RNA sequencing revealed differential expression profiles between responder and non-responder pretreatment as well as during the treatment. In conclusion, in contrast to previous reports and despite a differential RNA expression profile the number of TIL pretreatment did not correlate with response but responding metastases tended to have a higher increase in TIL and TAM. In addition, responder were characterized by expansion of more new TIL clones under treatment with a more diverse repertoire instead of an expansion of preexisting clones as previously reported.
The effect of Dsg2 in the SSC cells on exosome contents, release, and effects on recipient fibroblasts A Overmiller1, J Pierluissi1, P Wermuth1, U Martinez-Outschoorn1, A Luginbuhl1, J Curry1, L Harshyne1, J Wahl2, A South1 and MG Mahoney1 1 Thomas Jefferson University, Philadelphia, PA and 2 University of Nebraska, Lincoln, NE Exosomes are nanoscale membrane-derived vesicles (<150 nm) that serve as intercellular messengers carrying lipids, proteins, and genetic material. Substantial evidence has shown that cancer-derived exosomes, secreted by tumor cells into the blood and other bodily fluids, play a critical role in modulating the tumor microenvironment and affecting the pathogenesis of cancer. Here, exosomes were isolated from squamous cell carcinoma (SCC) keratinocytes by sequential ultracentrifugation or one-step polymer precipitation methods and analyzed by Nanoparticle Tracking Analysis. Western blotting showed positivity for exosome markers including the tetraspanins CD9 and CD63. We demonstrate for the first time that SCC exosomes were enriched with the 65 kDa C-terminal fragment (CTF) of desmoglein 2 (Dsg2), a desmosomal cadherin often overexpressed in skin malignancies. Dsg2 has been shown to activate mitogenic signaling, enhance cell growth and migration and promote oncogenesis. Expression of Dsg2-GFP further confirmed the presence of a distinct GFP-labeled Dsg2 CTF in exosomes. In normal and SCC keratinocytes, Dsg2 increased exosome release and mitogenic content including EGFR and c-Src. SCC exosomes were internalized by primary human fibroblasts, demonstrating the intercellular signaling axis between epidermal keratinocytes and nearby stromal cells. SCC exosomes enhanced fibroblast proliferation in a dose-dependent manner and exosomes from cells overexpressing Dsg2 had a greater pro-proliferative effect. Localized expansion, invasion, and microenvironment manipulation by malignant cells is important for tumor progression. While Dsg2 has low basal expression in the normal oral epithelium, we show that it was upregulated in the highly malignant head and neck SCCs, and exosomes isolated from SCC patients’ sera were enriched in Dsg2 CTF. This study defines a mechanism by which Dsg2 expression in cancer cells can modulate the tumor microenvironment.
491
492
Cellular response patterns to single MEK inhibition correlate with the efficacy of combined MEK/CDK4,6 targeting in melanoma C Posch3, M Sanlorenzo1, J Ma2, ST Kim2, M Zekhtser2, K Rappersberger3 and S Ortiz-Urda2 1 Department of Medical Sciences, University of Turin, Turin, Italy, 2 Dermatology, University of California San Francisco, San Francisco, CA and 3 Dermatology, The Rudolfstiftung Hospital, Vienna, Austria Treatment of NRAS mutant melanoma is challenging. The discovery that co-targeting of MEK + CDK4,6 has antitumor activity created excitement for patients and clinicians; however, first clinical results did not met pre-clinical expectations. In this study we investigate the response patterns of NRAS mutant melanoma cells in vitro and in vivo when challenged with inhibitors of MEK, CDK4,6 and their combination. Data revealed, that in vitro growth response patterns of cells treated with the MEK/CDK4,6 combination can be used to predict the in vivo efficacy of co-targeting in a xenograft model of NRAS mutant melanoma. In addition, signaling changes after single MEK inhibition also correlated with the response to the MEK/CDK4,6 combination: Cells displaying activation of the cell cycle pathway after MEK inhibition evidenced by elevated pRb levels, showed more effective growth reduction with MEK/CDK4,6 co-targeting compared to single MEK inhibitor treatment. In contrast, MEK/CDK4,6 and single MEK inhibitor treatment were equally effective in cells that responded with unchanged or decreased protein levels of pRb after single MEK inhibition. Cells characterized by these criteria, showed induction of apoptosis in vivo. This pattern is not limited to NRAS mutant cells, but can be applied to BRAF mutant cells and cells that are wild-type for these mutations. This study shows that the MEK/CDK4,6 combination effectively reduces growth of a subset of NRAS mutant melanoma cells. Further, MEK/CDK4,6 has antitumor potential in cells with genetic driving alterations other than NRAS mutations, and might thus offer a new treatment strategy for an extended cohort of patients with melanoma. Results suggest that the efficacy of the MEK/CDK4,6 combination can be predicted by in vitro viability assays and by the changes of pRb levels of cells after single MEK inhibition.
Non-cell autonomous suppression of melanoma by the epidermal polarity protein Par3 through control of direct keratinocyte-melanocyte interactions M Mescher1, P Jeong1, M Ru¨bsam1, M Kranen1, J Landsberg2, M Schlaak3, C Mauch3, T Tueting4, CM Niessen3 and S Iden1 1 CECAD, University of Cologne, Cologne, Germany, 2 University of Bonn, Bonn, Germany, 3 Department of Dermatology, University of Cologne, Cologne, Germany and 4 Department of Dermatology, University Hospital Magdeburg, Magdeburg, Germany Melanoma, an aggressive skin malignancy with increasing lifetime risk, originates from melanocytes that are in close contact with surrounding epidermal keratinocytes. Though keratinocyte-melanocyte cross-talk is essential for skin photoprotection it is poorly understood how the epidermal microenvironment controls melanomagenesis. Here we identify an unexpected non-cell autonomous role of epidermal polarity proteins, molecular determinants of cyto-architecture, in malignant melanoma. Epidermal Par3 inactivation in mice promotes melanocyte dedifferentiation, motility and hyperplasia and in an autochthonous melanoma model results in increased tumor formation and lung metastasis. Keratinocyte-specific Par3 loss upregulates surface P-cadherin that is essential to promote melanocyte proliferation and phenotypic switch towards dedifferentiation. In agreement, low epidermal PAR3 and high PCadherin expression correlate with human melanoma progression, implying that this mechanism also drives human disease. Collectively, our data show that reduced keratinocyte Par3 function fosters a permissive P-cadherin-dependent niche for melanocyte transformation, invasion and metastasis. This reveals a previously unrecognized extrinsic tumor suppressive mechanism, whereby epithelial polarity proteins dictate the cyto-architecture and fate of other tissue-resident cells to suppress their malignant outgrowth.
493
494
Characterization and modulation of CC-chemokine Receptor 6 (CCR6) mediated immunosurveillance in malignant melanoma D Martin-Garcia, A Enk and A Lonsdorf Department of Dermatology, University Hospital Heidelberg, Heidelberg, Germany Chemokine ligand 20 (CCL20) and the antimicrobial peptide ß-defensin expressed in the epidermis are a potent impetus for the recruitment of subsets of dendritic cells (DC), B-cells and memory T cells expressing chemokine receptor 6 (CCR6), the exclusive receptor. In addition to its constitutive expression in the epidermis, CCL20 and a corresponding CCR6expressing immune cell infiltrate have been detected in several malignancies, including melanoma. Yet, the functional contribution of the CCR6/CCL20 axis for the immune control of melanoma remains controversial. The characterization of CCR6-guided immune cell subsets and their functional contribution for the immune control of melanoma comprises the focus of this project. We evaluated the homeostatic and inducible secretion of CCL20 by different murine and human melanoma cutaneous cell lines by enzyme-linked Immunoabsorbent assay (ELISA). Both, murine (B16, Ret) and human (A375, C32) melanoma cell lines are capable of secreting CCL20 and show significant differences in the magnitude of inducible CCL20 expression upon stimulation with pro-inflammatory cytokines (i.e. TNF-a, IL-1a, IL-1b and TGF-b) in vitro. In order to determine the functional relevance of CCR6 on local tumor growth, metastasis and the tumor microenvironment, B16 melanoma cells retrovirally transduced with luciferase (B16-luc) were injected subcutaneously into the flank of wild type C57BL/6 (WT) and congenic CCR6eknockout (CCR6KO) mice. While mice in both groups developed local tumors, we observed a trend towards slower tumor growth in CCR6KO mice inoculated with CCL20-expressing B16-luc melanoma cells, suggesting that the capability of recruiting CCR6 immune cells to the tumor site may be an essential factor during tumor progression. Additional experimental approaches will evaluate the effects of stably enhanced (by injection of retrovirally transduced B16 cells overexpressing CCL20) and reduced (by local injection of a CCL20-neutralizing antibody) CCL20 expression in the tumor microenvironment in both WT and CCR6KO mice.
S244 Journal of Investigative Dermatology (2016), Volume 136
Heterogeneous mutational status of melanomas in multiple primary melanoma patients C Pellegrini1, C Martorelli1, G Cipolloni2, L Di Nardo1, A Antonini1, M Maturo1 and M Fargnoli1 1 Dept. of Dermatology, University of L’Aquila, L’Aquila, Italy and 2 Dept. of Pathology, University of L’Aquila, L’Aquila, Italy Multiple primary melanomas (MPM) develop in about 5% of sporadic melanoma patients and in up to 19% of melanoma patients with a positive family history. Many genetic alterations, including predisposing and/or somatic mutations, may contribute to the development of MPM and data on the genetic diversity of MPMs are limited. We aimed to assess the frequency and distribution of BRAF, NRAS and TERT promoter somatic mutations in subsequent melanomas of the same patient and evaluate the association of somatic alterations with germline mutational profile of MPM patients. Sixty-six synchronous/asynchronous FFPE melanoma tissues from 31 patients were analyzed by Real-Time PCR, Sanger Sequencing and SNaPshot molecular methods for BRAF, NRAS and TERT promoter mutations. Germline DNA was screened by Sanger sequencing for mutations in CDKN2A, CDK4, POT1, MITF, MC1R genes and TERT promoter. BRAF V600 mutations were found in 46.2% of melanomas, NRAS mutations in 10.6% and TERT promoter rs2853669 polymorphism in 64.2%. Mutational somatic profile (BRAF, NRAS) was concordant between first and subsequent primary tumors in 65.4% of patients. Somatic mutational status was not associated with any of the clinical characteristics of patients or melanomas, including age, melanoma thickness and body site. At the germline level, MPM patients were wild-type for mutations in CDKN2A, CDK4, POT1 and MITF genes. TERT-promoter polymorphisms (rs2853669 and rs35226131) were identified in 46.1% of patients and MC1R allelic variants in 73.1%. Carriers of MC1R changes were diagnosed more frequently with melanoma on the trunk compared to wild-type patients (p¼0.02), but no association between MC1R status and somatic mutations was identified in our patients. In conclusion, our results support the heterogeneity of molecular profiles in MPM of the same patient with implication in clinical practice due to the difficulties in molecularly classifying patients with discrepant primary melanomas.