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Recent studies suggest that BRAF V600 -mutated melanomas in particular respond to dual anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) immune checkpoint inhibition (ICI). Here we identified an over-representation of interleukin (IL)-17-type 17 helper T (T H 17) gene expression signatures (GES) in BRAF V600 -mutated tumors. Moreover, high baseline IL-17 GES consistently predicted clinical responses in dual-ICI-treated patient cohorts but not in mono anti-CTLA-4 or anti-PD-1 ICI cohorts. High IL-17 GES corresponded to tumor infiltration with T cells and neutrophils. Accordingly, high neutrophil infiltration correlated with clinical response specifically to dual ICI, and tumor-associated neutrophils also showed strong IL-17-T H 17 pathway activity and T cell activation capacity. Both the blockade of IL-17A and the depletion of neutrophils impaired dual-ICI response and decreased T cell activation. Finally, high IL-17A levels in the blood of patients with melanoma indicated a higher global T H 17 cytokine profile preceding clinical response to dual ICI but not to anti-PD-1 monotherapy, suggesting a future role as a biomarker for patient stratification., (© 2023. The Author(s).)

Telomerase reverse transcriptase (TERT) promoter mutations occur frequently in cancer, have been associated with increased TERT expression and cell proliferation, and could potentially influence therapeutic regimens for melanoma. As the role of TERT expression in malignant melanoma and the non-canonical functions of TERT remain understudied, we aimed to extend the current knowledge on the impact of TERT promoter mutations and expression alterations in tumor progression by analyzing several highly annotated melanoma cohorts. Using multivariate models, we found no consistent association for TERT promoter mutations or TERT expression with the survival rate in melanoma cohorts under immune checkpoint inhibition. However, the presence of CD4+ T cells increased with TERT expression and correlated with the expression of exhaustion markers. While the frequency of promoter mutations did not change with Breslow thickness, TERT expression was increased in metastases arising from thinner primaries. As single-cell RNA-sequencing (RNA-seq) showed that TERT expression was associated with genes involved in cell migration and dynamics of the extracellular matrix, this suggests a role of TERT during invasion and metastasis. Co-regulated genes found in several bulk tumors and single-cell RNA-seq cohorts also indicated non-canonical functions of TERT related to mitochondrial DNA stability and nuclear DNA repair. This pattern was also evident in glioblastoma and across other entities. Hence, our study adds to the role of TERT expression in cancer metastasis and potentially also immune resistance., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kuhn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Despite the availability of effective systemic therapies, a significant number of advanced melanoma patients develops brain metastases. This study investigated differences in incidence and time to diagnosis of brain metastasis and survival outcomes dependent on the type of first-line therapy., Methods: Patients with metastatic, non-resectable melanoma (AJCCv8 stage IIIC-V) without brain metastasis at start of first-line therapy (1L-therapy) were identified from the prospective multicenter real-world skin cancer registry ADOREG. Study endpoints were incidence of brain metastasis, brain metastasis-free survival (BMFS), progression-free survival (PFS), and overall survival (OS)., Results: Of 1704 patients, 916 were BRAF wild-type (BRAF wt ) and 788 were BRAF V600 mutant (BRAF mut ). Median follow-up time after start of 1L-therapy was 40.4 months. BRAF wt patients received 1L-therapy with immune checkpoint inhibitors (ICI) against CTLA-4+PD-1 (n=281) or PD-1 (n=544). In BRAF mut patients, 1L-therapy was ICI in 415 patients (CTLA-4+PD-1, n=108; PD-1, n=264), and BRAF+MEK targeted therapy (TT) in 373 patients. After 24 months, 1L-therapy with BRAF+MEK resulted in a higher incidence of brain metastasis compared with PD-1±CTLA-4 (BRAF+MEK, 30.3%; CTLA-4+PD-1, 22.2%; PD-1, 14.0%). In multivariate analysis, BRAF mut patients developed brain metastases earlier on 1L-therapy with BRAF+MEK than with PD-1±CTLA-4 (CTLA-4+PD-1: HR 0.560, 95% CI 0.332 to 0.945, p=0.030; PD-1: HR 0.575, 95% CI 0.372 to 0.888, p=0.013). Type of 1L-therapy, tumor stage, and age were independent prognostic factors for BMFS in BRAF mut patients. In BRAF wt patients, tumor stage was independently associated with longer BMFS; ECOG Performance status (ECOG-PS), lactate dehydrogenase (LDH), and tumor stage with OS. CTLA-4+PD-1 did not result in better BMFS, PFS, or OS than PD-1 in BRAF wt patients. For BRAF mut patients, multivariate Cox regression revealed ECOG-PS, type of 1L-therapy, tumor stage, and LDH as independent prognostic factors for PFS and OS. 1L-therapy with CTLA-4+PD-1 led to longer OS than PD-1 (HR 1.97, 95% CI 1.122 to 3.455, p=0.018) or BRAF+MEK (HR 2.41, 95% CI 1.432 to 4.054, p=0.001), without PD-1 being superior to BRAF+MEK., Conclusions: In BRAF mut patients 1L-therapy with PD-1±CTLA-4 ICI resulted in a delayed and less frequent development of brain metastasis compared with BRAF+MEK TT. 1L-therapy with CTLA-4+PD-1 showed superior OS compared with PD-1 and BRAF+MEK. In BRAF wt patients, no differences in brain metastasis and survival outcomes were detected for CTLA-4+PD-1 compared with PD-1., Competing Interests: Competing interests: All authors declare no conflicts of interest affecting this study. Conflicts of interest outside the submitted work are as following: CF has been on the advisory board or has received honoraria from Bristol Myers Squibb, Immunocore and Novartis and received travel grants from Bristol Myers Squibb, Novartis and Pierre Fabre. PM declares research support from Bristol Myers Squibb, Novartis and Merck Sharp & Dome; speakers and advisory board honoraria from Almirall Hermal, Beiersdorf, Bristol Myers Squibb, Merck Sharp & Dome, Immunocore, Merck Serono, Medac, Novartis, Pierre Fabre, Sanofi Genzyme, Sun Pharma and Roche, and travel support from Bristol Myers Squibb, Merck Sharp & Dohme, Novartis and Pierre Fabre. LB received honoraria from Amgen, Bristol-Myers Squibb and Sun Pharma. FriM has received travel support or/and speaker’s fees or/and advisor’s honoraria by Novartis, Roche, BMS, MSD and Pierre Fabre and research funding from Novartis and Roche. KK has served as consultant or/and has received honoraria from Amgen, Roche, Bristol Myers Squibb, Merck Sharp and Dohme, Pierre Fabre, and Novartis, and received travel support from Amgen, Merck Sharp and Dohme, Bristol Myers Squibb, Amgen, Pierre Fabre, Medac and Novartis. IG declares speakers and advisory board honoraria from Almirall Hermal, Bristol Myers Squibb, Merck Sharp & Dome, Novartis, Pierre Fabre, Sanofi Genzyme, Sun Pharma and Roche. RG: Invited speaker: Roche, BMS, MSD, Novartis, Amgen, Merck Serono, Almirall Hermal, SUN, Sanofi, Pierre-Fabre. Advisory board: BMS, Roche, Novartis, Almirall Hermal, MSD, Amgen, SUN, Sanofi, Pierre-Fabre, 4SC, Bayer, MerckSerono, Pfizer, Immunocore. Research grants: Novartis, Pfizer, Johnson & Johnson, Amgen, Merck-Serono, SUN Pharma, Sanofi. Travel/meeting support: Roche, BMS, SUN, Merck-Serono, Pierre-Fabre. JU is on the advisory board or has received travel support from: Amgen, BMS, GSK, Immunocore, Leo Pharma, MSD, Novartis, Pierre Fabre, Sanofi, Roche. JU has received research support from Novartis; speakers and advisory board honoraria. PT has been on the advisory board or has received honoraria from Almirall, Bristol-Myers Squibb, Novartis, Pierre-Fabre, Merck Serono, Sanofi, Roche, Kyowa Kirin, Biofrontera, and 4SC and received travel grants from Bristol Myers Squibb, and Pierre Fabre. RH reports speakers and advisory board honoraria from Bristol-Myers Squibb (BMS), Immunocore, Novartis, Pierre-Fabre, Roche and SUN pharma outside the submitted work. CP received honoraria (speaker honoraria or honoraria as a consultant) and travel support from: Novartis, BMS, Roche, Merck Serono, MSD, Celgene, AbbVie, SUNPHARMA, UCB, Allergy Therapeutics, Pierre Fabre, Kyowa Kirin and LEO. AF served as consultant to Roche, Novartis, MSD, BMS, Pierre-Fabre; received travel support from Roche, Novartis, BMS, Pierre-Fabre, received speaker fees from Roche, Novartis, BMS, MSD and CeGaT, outside the submitted work. She reports institutional research grants from BMS Stiftung Immunonkologie. FZ declares speakers and advisory board honoraria and/or travel support from BMS, MSD, Roche, Novartis, Pierre Fabre and Sanofi Aventis. FraM (Frank Meiss) served as a consultant and/or has received honoraria from Novartis, BMS, MSD, Pierre Fabre, Sanofi Genzyme, Sun Pharma and travel support from Novartis, Sun Pharma, Roche, Pierre Fabre and MSD. BS is on the advisory board or has received honoraria from Immunocore, Almirall, Pfizer, Sanofi, Novartis, Roche, BMS and MSD, research funding from Novartis and Pierre Fabre and travel support from Novartis, Roche, BMS and Pierre Fabre. AK reports receiving lecture fees and fees for serving on advisory boards from MSD Sharp & Dohme, Almirall, Infectopharm, and Boehringer Ingelheim. JW has been on the advisory board, received honoraria and/or travel grants from Bristol Myers Squibb, Novartis, MSD, and Pierre Fabre. TG has received speakers and/or advisory board honoraria and travel support from BMS, Sanofi-Genzyme, MSD, Novartis Pharma, Roche, Abbvie, Almirall, Janssen, Lilly, Pfizer, Pierre Fabre, Merck-Serono. CL has received speaker’s fees, advisory board honoraria and travel reimbursements from Merck, MSD, Roche, Almirall Hermal, Biontech, Sanofi, Sun Pharma, Kyowa Kirin, Immonocore, BMS, Pierre Fabre, Novartis. LH has received consultancy and speaker fees from Amgen, Biome Dx, BMS, Curevac, Merck, MSD, Myoncare, Novartis, Pierre-Fabre, Roche, Sanofi and SUN. SG has been on the advisory board and/or has received travel support from Bristol Myers Squibb, MSD, Sun Pharma and Novartis and received research support from Novartis and Pierre Fabre. DD has been on the advisory board or has received honoraria from BMS, Kyowa Kirin, MSD, Novartis, Pierre Fabre, Sanofi and received travel grants from Boehringer, Mylan, Pfizer. GS has received honoraria from BMS. GL has received travel support from Sun Pharma. LZ served as consultant and/or has received honoraria from Roche, Bristol-Myers Squibb (BMS), Merck Sharp & Dohme (MSD), Novartis, Pierre Fabre, Sanofi, and Sunpharma and travel support from MSD, BMS, Amgen, Pierre Fabre, Sunpharma, Sanofi and Novartis. EL served as consultant and/or has received honoraria from Amgen, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Medac, Sanofi, Sunpharma and travel support from Medac, Bristol-Myers Squibb, Pierre Fabre, Sunpharma and Novartis. JCB received speaker’s bureau honoraria from Amgen, Pfizer, Recordati and Sanofi, and is a paid consultant/advisory board/DSMB member for Almirall, Boehringer Ingelheim, InProTher, ICON, MerckSerono, Pfizer, 4SC, and Sanofi/Regeneron. His group received research grants from Bristol-Myers Squibb, Merck Serono, HTG, IQVIA, and Alcedis. DS declares relevant financial activities (Roche, Novartis, Bristol-Myers Squibb, Merck Sharp & Dohme, Sanofi, Regeneron, Array, Pierre Fabre, 4SC, Helsinn, Philogen, InFlarX, Merck-Serono, SunPharma, Ultimovacs, Sandoz). SU declares research support from Bristol Myers Squibb and Merck Serono; speakers and advisory board honoraria from Bristol Myers Squibb, Merck Sharp & Dohme, Merck Serono, Novartis and Roche, and travel support from Bristol Myers Squibb, Merck Sharp & Dohme, and Pierre Fabre., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Large genome-scale studies are deposited in various public sequence repositories. However, their access and analysis can be non-trivial to infrequent users. Here, we present a new database connecting whole transcriptomes with clinical data for straight-forward access and analysis of patient-specific samples. Users can perform association tests of survival and gene expression across different cohorts, identify cell-type expressions, or correlate the presence of immune cells. In summary, we present a new data hub for bench scientists to perform replication and discovery studies., (© 2022 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Accurate classification of melanocytic tumors is important for prognostic evaluation, treatment and follow-up protocols of patients. The majority of melanocytic proliferations can be classified solely based on clinical and pathological criteria, however in select cases a definitive diagnostic assessment remains challenging and additional diagnostic biomarkers would be advantageous. We analyzed melanomas, nevi, Spitz nevi and atypical spitzoid tumors using parallel sequencing (exons of 611 genes and 507 gene translocation analysis) and methylation arrays (850k Illumina EPIC). By combining detailed genetic and epigenetic analysis with reference-based and reference-free DNA methylome deconvolution we compared Spitz nevi to nevi and melanoma and assessed the potential for these methods in classifying challenging spitzoid tumors. Results were correlated with clinical and histologic features. Spitz nevi were found to cluster independently of nevi and melanoma and demonstrated a different mutation profile. Multiple copy number alterations and TERT promoter mutations were identified only in melanomas. Genome-wide methylation in Spitz nevi was comparable to benign nevi while the Leukocytes UnMethylation for Purity (LUMP) algorithm in Spitz nevi was comparable to melanoma. Histologically difficult to classify Spitz tumor cases were assessed which, based on methylation arrays, clustered between Spitz nevi and melanoma and in terms of genetic profile or copy number variations demonstrated worrisome features suggesting a malignant neoplasm. Comprehensive sequencing and methylation analysis verify Spitz nevi as an independent melanocytic entity distinct from both nevi and melanoma. Combined genetic and methylation assays can offer additional insights in diagnosing difficult to classify Spitzoid tumors., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Plasma-derived tumour-specific cell-free nucleic acids are increasingly utilized as a minimally invasive, real-time biomarker approach in many solid tumours. Circulating tumour DNA of melanoma-specific mutations is currently the best studied liquid biopsy biomarker for melanoma. However, the combination of hotspot genetic alterations covers only around 80% of all melanoma patients. Therefore, alternative approaches are needed to enable the follow-up of all genotypes, including wild-type., Methods: We identified KPNA2, DTL, BACE2 and DTYMK messenger RNA (mRNA) upregulated in melanoma versus nevi tissues by unsupervised data mining (N = 175 melanoma, N = 20 normal skin, N = 6 benign nevi) and experimentally confirmed differential mRNA expression in vitro (N = 18 melanoma, N = 8 benign nevi). Circulating cell-free RNA (cfRNA) was analysed in 361 plasma samples (collected before and during therapy) from 100 melanoma patients and 18 healthy donors. Absolute cfRNA copies were quantified on droplet digital PCR., Results: KPNA2, DTL, BACE2 and DTYMK cfRNA demonstrated high diagnostic accuracy between melanoma patients' and healthy donors' plasma (AUC > 86%, p < .0001). cfRNA copies increased proportionally with increasing tumour burden independently of demographic variables and even remained elevated in individuals with radiological absence of disease. Re-analysis of single-cell transcriptomes revealed a pan-tumour origin of cfRNA, including endothelial, cancer-associated fibroblasts, macrophages and B cells beyond melanoma cells as cellular sources. Low baseline cfRNA levels were associated with significantly longer progression-free survival (PFS) (KPNA2 HR = .54, p = .0362; DTL HR = .60, p = .0349) and overall survival (KPNA2 HR = .52, p = .0237; BACE2 HR = .55, p = .0419; DTYMK HR = .43, p = .0393). Lastly, we found that cfRNA copies significantly increased during therapy in non-responders compared to responders regardless of therapy and mutational subtypes and that the increase of KPNA2 (HR = 1.73, p = .0441) and DTYMK (HR = 1.82, p = .018) cfRNA during therapy was predictive of shorter PFS., Conclusions: In sum, we identified a new panel of cfRNAs for a pan-tumour liquid biopsy approach and demonstrated its utility as a prognostic, therapy-monitoring tool independent of the melanoma mutational genotype., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Background: Immune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable melanoma, aiming to enhance anti-tumor T cell responses. Switching of melanoma toward a dedifferentiated cell state has recently been linked to T cell and therapy resistance. It remains to be determined whether RIG-I agonists affect melanoma differentiation, potentially leading to T cell resistance., Methods: Patient metastases-derived melanoma cell lines were treated with the synthetic RIG-I agonist 3pRNA, and effects on tumor cell survival, phenotype and differentiation were determined. Transcriptomic data sets from cell lines and metastases were analyzed for associations between RIG-I (DDX58) and melanoma differentiation markers and used to define signaling pathways involved in RIG-I-driven dedifferentiation. The impact of 3pRNA-induced melanoma dedifferentiation on CD8 T cell activation was studied in autologous tumor T cell models., Results: RIG-I activation by 3pRNA induced apoptosis in a subpopulation of melanoma cells, while the majority of tumor cells switched into a non-proliferative cell state. Those persisters displayed a dedifferentiated cell phenotype, marked by downregulation of the melanocytic lineage transcription factor MITF and its target genes, including melanoma differentiation antigens (MDA). Transition into the MITF low /MDA low cell state was JAK-dependent, with some cells acquiring nerve growth factor receptor expression. MITF low /MDA low persisters switched back to the proliferative differentiated cell state when RIG-I signaling declined. Consistent with our in vitro findings, an association between melanoma dedifferentiation and high RIG-I (DDX58) levels was detected in transcriptomic data from patient metastases. Notably, despite their dedifferentiated cell phenotype, 3pRNA-induced MITF low /MDA low persisters were still efficiently targeted by autologous CD8 tumor-infiltrating T lymphocytes (TILs)., Conclusions: Our results demonstrate that RIG-I signaling in melanoma cells drives a transient phenotypic switch toward a non-proliferative dedifferentiated persister cell state. Despite their dedifferentiation, those persisters are highly immunogenic and sensitive toward autologous TILs, challenging the concept of melanoma dedifferentiation as a general indicator of T cell resistance. In sum, our findings support the application of RIG-I agonists as a therapeutic tool for the generation of long-term clinical benefit in non-resectable melanoma., Competing Interests: Competing interests: AP reports research grant support and provision of reagents from Bristol-Myers Squibb (BMS) and Merck Sharp & Dohme (MSD). AS reports personal fees from Novartis Adboard. GH is a co-founder of Rigontec GmbH. DS declares grants, personal fees, and/or nonfinancial support from BMS, Roche, Novartis, Regeneron, Sanofi, MSD, Amgen, 4SC, Merck-EMD, Array, Pierre-Fabre, Philogen, Incyte, and Pfizer., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5B high cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5B high persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination., (© 2022. The Author(s).)

Background: Despite of various therapeutic strategies, treatment of patients with melanoma brain metastasis (MBM) still is a major challenge. This study aimed at investigating the impact of type and sequence of immune checkpoint blockade (ICB) and targeted therapy (TT), radiotherapy, and surgery on the survival outcome of patients with MBM., Method: We assessed data of 450 patients collected within the prospective multicenter real-world skin cancer registry ADOREG who were diagnosed with MBM before start of the first non-adjuvant systemic therapy. Study endpoints were progression-free survival (PFS) and overall survival (OS)., Results: Of 450 MBM patients, 175 (38.9%) received CTLA-4+PD-1 ICB, 161 (35.8%) PD-1 ICB, and 114 (25.3%) BRAF+MEK TT as first-line treatment. Additional to systemic therapy, 67.3% of the patients received radiotherapy (stereotactic radiosurgery (SRS); conventional radiotherapy (CRT)) and 24.4% had surgery of MBM. 199 patients (42.2%) received a second-line systemic therapy. Multivariate Cox regression analysis revealed the application of radiotherapy (HR for SRS: 0.213, 95% CI 0.094 to 0.485, p<0.001; HR for CRT: 0.424, 95% CI 0.210 to 0.855, p=0.016), maximal size of brain metastases (HR for MBM >1 cm: 1.977, 95% CI 1.117 to 3.500, p=0.019), age (HR for age >65 years: 1.802, 95% CI 1.016 to 3.197, p=0.044), and ECOG performance status (HR for ECOG ≥2: HR: 2.615, 95% CI 1.024 to 6.676, p=0.044) as independent prognostic factors of OS on first-line therapy. The type of first-line therapy (ICB vs TT) was not independently prognostic. As second-line therapy BRAF+MEK showed the best survival outcome compared with ICB and other therapies (HR for CTLA-4+PD-1 compared with BRAF+MEK: 13.964, 95% CI 3.6 to 54.4, p<0.001; for PD-1 vs BRAF+MEK: 4.587 95% CI 1.3 to 16.8, p=0.022 for OS). Regarding therapy sequencing, patients treated with ICB as first-line therapy and BRAF+MEK as second-line therapy showed an improved OS (HR for CTLA-4+PD-1 followed by BRAF+MEK: 0.370, 95% CI 0.157 to 0.934, p=0.035; HR for PD-1 followed by BRAF+MEK: 0.290, 95% CI 0.092 to 0.918, p=0.035) compared with patients starting with BRAF+MEK in first-line therapy. There was no significant survival difference when comparing first-line therapy with CTLA-4+PD-1 ICB with PD-1 ICB., Conclusions: In patients with MBM, the addition of radiotherapy resulted in a favorable OS on systemic therapy. In BRAF-mutated MBM patients, ICB as first-line therapy and BRAF+MEK as second-line therapy were associated with a significantly prolonged OS., Competing Interests: Competing interests: The authors declare no competing interests in reference to this work. For conflicts of interest outside the submitted work see list: CF has been on the advisory board or has received honoraria from Bristol Myers Squibb, Immunocore and Novartis and received travel grants from Bristol Myers Squibb, Novartis and Pierre Fabre. PM declares research support from Bristol Myers Squibb, Novartis and Merck Sharp & Dome; speakers and advisory board honoraria from Almirall Hermal, Beiersdorf, Bristol Myers Squibb, Merck Sharp & Dome, Immunocore, Merck Serono, Medac, Novartis, Pierre Fabre, Sanofi Genzyme, Sun Pharma and Roche, and travel support from Bristol Myers Squibb, Merck Sharp & Dohme, Novartis and Pierre Fabre. LB received honoraria from Amgen, Bristol Myers Squibb and Sun Pharma. IG declares speakers and advisory board honoraria from Almirall Hermal, Bristol Myers Squibb, Merck Sharp & Dome, Novartis, Pierre Fabre, Sanofi Genzyme, Sun Pharma and Roche. FM has received travel support or/and speaker’s fees or/and advisor’s honoraria by Novartis, Roche, BMS, MSD and Pierre Fabre and research funding from Novartis and Roche. RH reports speakers and advisory board honoraria from Bristol Myers Squibb (BMS), Immunocore, Novartis, Pierre-Fabre, Roche and SUN pharma. CP received honoraria (speaker honoraria or honoraria as a consultant) and travel support from: Novartis, BMS, Roche, Merck Serono, MSD, Celgene, AbbVie, SUNPHARMA, UCB, Allergy Therapeutics, Pierre Fabre, Kyowa Kirin and LEO. JUl has received research support from Novartis; speakers and advisory board honoraria from Bristol Myers Squibb, Merck Sharp & Dohme, Novartis, Roche, Pierre Fabre, and travel support from Bristol Myers Squibb and medac. PT declares speakers and advisory board honoraria from Almirall, Bristol Myers Squibb, Novartis, Merck Sharp & Dohme, Pierre-Fabre, CureVac, Merck Serono, Sanofi, Roche, Kyowa Kirin, Biofrontera and 4SC; travel support from Bristol Myers Squibb and Pierre-Fabre. DN has received advisory and speaker honoraria from Merck Sharp & Dome, Bristol Myers Squibb, Novartis, Almirall and Sanofi. AF Advisory Board: Roche, Novartis, MSD, BMS, Pierre-Fabre; support for congress participation: Roche, Novartis, BMS, Pierre-Fabre; speaker honoraria: Roche, Novartis, BMS, MSD, CeGaT; institutional research support BMS Stiftung Immunonkologie. JUt is on the advisory board or has received honoraria and travel support from Amgen, Bristol Myers Squibb, GSK, Immunocore, LeoPharma, Merck Sharp and Dohme, Novartis, Pierre Fabre, Roche and Sanofi. TG has received speakers and/or advisory board honoraria and travel support from BMS, Sanofi-Genzyme, MSD, Novartis Pharma, Roche, Abbvie, Almirall, Janssen, Lilly, Pfizer, Pierre Fabre, and Merck-Serono. FM served as consultant and/or has received honoraria from Novartis, Roche, Bristol Myers Squibb, Merck Sharp & Dohme, Pierre Fabre, Sanofi Genzyme and travel support from Novartis, Sunpharma and Bristol Myers Squibb. JW received honoraria from Pierre Fabre, Novartis and Merck Sharp & Dohme. KS has been on the advisory board or has received honoraria from Bristol Myers Squibb, Roche, Merck Sharp & Dome, Pierre Fabre, Novartis and received travel grants from Bristol Myers Squibb, Novartis and Pierre Fabre. IH has been on the advisory board of Ymmunobio. AR reported grants from Novartis, Bristol Myers Squibb, and Adtec; personal fees from Merck Sharp & Dohme; and non-financial support from Amgen, Roche, Merck Sharp & Dohme, Novartis, Bristol Myers Squibb, and Teva. LZ served as consultant and/or has received honoraria from Bristol Myers Squibb, Merck Sharp & Dohme, Novartis, Pierre-Fabre, Sunpharma and Sanofi; research funding to institution: Novartis; travel support from Merck Sharp & Dohme, Bristol Myers Squibb, Amgen, Pierre-Fabre, Sunpharma, Sanofi and Novartis. EL served as consultant and/or has received honoraria from Amgen, Bristol Myers Squibb, Merck Sharp & Dohme, Novartis, Medac, Sanofi, Sunpharma and travel support from Medac, Bristol Myers Squibb, Pierre Fabre, Sunpharma and Novartis. DS: relevant financial activities (Roche, Novartis, Bristol Myers Squibb, Merck Sharp & Dohme, Sanofi, Regeneron, Array, Pierre Fabre, 4SC, Helsinn, Philogen, InFlarX, Merck-Serono, SunPharma, Ultimovacs, and Sandoz). SU declares research support from Bristol Myers Squibb and Merck Serona; speakers and advisory board honoraria from Bristol Myers Squibb, Merck Sharp & Dome, Merck Serono, Novartis and Roche, and travel support from Bristol Myers Squibb, Merck Sharp & Dohme and Pierre Fabre. All other authors have no conflict of interest to declare., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Cutaneous melanoma is notorious for the development of in-transit metastases (ITM). For unknown biological reasons, ITM remain the leading tumour manifestation without progression to distant sites in some patients., Methods: In total, 191 patients with initially unresectable stage III ITM and satellite metastases from 16 skin cancer centres were retrospectively evaluated for their tumour characteristics, survival and therapy response. Three groups according to disease kinetics (no distant progress, slow (>6 months) and fast (<6 months) distant progression) were analysed separately., Results: Median follow-up time was 30.5 (range 0.8-154.0) months from unresectable ITM. Progression to stage IV was observed in 56.5% of cases. Patients without distant metastasis were more often female, older (>70 years) and presented as stage III with lymph node or ITM at initial diagnosis in 45.7% of cases. Melanoma located on the leg had a significantly better overall survival (OS) from time of initial diagnosis compared to non-leg localised primaries (hazard ratio [HR] = 0.61, 95% confidence interval [CI] 0.40-0.91; p = 0.017), but not from diagnosis of unresectable stage III (HR = 0.67, 95% CI 0.45-1.02; p = 0.06). Forty percent of patients received local therapy for satellite and ITM. Overall response rate (ORR) to all local first-line treatments was 38%; disease control rate (DCR) was 49%. In total, 72.3% of patients received systemic therapy for unresectable stage IIIB-D. ORR for targeted therapy (n = 19) was highest with 63.2% and DCR was 84.2% compared to an ORR of 31.4% and a DCR of 54.3% in PD-1 treated patients (n = 70). Patients receiving PD-1 and intralesional talimogene laherparepvec (n = 12) had an ORR of 41.7% and a DCR of 75%., Conclusion: Patients with unresectable ITM and without distant progression are more often female, older, and have a primary on the leg. Response to PD-1 inhibitors in this cohort was lower than expected, but further investigation is required to elucidate the biology of ITM development and the interplay with the immune system., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A.Z.: received travel support from Novartis, Sanofi Genzyme, and Bristol Myers Squibb, outside the submitted work. M.P.: No relevant conflicts of interest. J.C.H.: received honoraria from talks: BMS, MSD, Roche, Novartis, Sun Pharma, Almirall, Sanofi, honoraria from adboards: Sun Pharma, Sanofi, Pierre Fabre, scientific grant: BMS. V.G.: received honoraria from Bristol Myers Squibb (BMS) and reports travel support from Novartis, Pierre Fabre Pharmaceuticals, BMS, Merck Sharp & Dohme (MSD), Sanofi Genzyme and SUN Pharmaceutical Industries outside the submitted work. Z.F.: No relevant conflicts of interest. C.L.: served as consult and and/or has received honoraria and or received travel support from Roche, Novartis, Pierre Fabre, MDS, Merck, BMS, Sanofi, Sun Pharma, Biontech, Kyowa Kirin, Almirall Hermal outside the submitted work. D. R.-S. has received honoraria for talks from Roche and Pierre Fabre and has received travel grants from Novartis, Pierre Fabre and Sanofi Genzyme. R. G.: received honoraria: Roche, BMS, MSD, Novartis, Amgen, Merck Serono, Almirall Hermal, SUN, Sanofi, Pierre Fabr; consultant or advisory role: BMS, Roche, Novartis, Almirall Hermal, MSD, Amgen, SUN, Sanofi, Pierre Fabre, Merck Serono, Bayer, Pfizer; Funding: Novartis, Pfizer, Johnson & Johnson, Amgen, Merck Serono, SUN Pharma, Sanofi; travel, accommodations, expenses: Roche, BMS, SUN, Merck Serono, Pierre Fabre. J. U. is on the advisory board or has received honoraria and travel support from Amgen, Bristol Myers Squibb, GSK, LeoPharma, Merck Sharp and Dohme, Novartis, Pierre Fabre, Roche, Sanofi outside the submitted work. Se.H.: served as consultant and/or has received honoraria from Amgen, Bristol Myers Squibb, Merck Sharp & Dohme, Novartis, Sanofi, Sun Pharma outside the submitted work. L.R.: No relevant conflicts of interest. K.C.K.: served as consultant and/or has received honoraria from Amgen, Roche, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Medac, Sanofi, Sun Pharma and travel support from Amgen, Merck Sharp & Dohme, Bristol Myers Squibb, Amgen, Pierre Fabre, Sun pharma and Novartis, outside the submitted work. C.W.: served as consultant and/or has received honoraria from Amgen, Roche, Bristol Myers Squibb, Kyowa Kirin, Merck Sharp & Dohme, Novartis, Sanofi, Takeda and travel support from Amgen, Bristol Myers Squibb, Curevac, Pierre Fabre and Novartis, outside the submitted work. A.M.: has received speaker or consultant fees from AbbVie, Almirall, Novartis, Bristol Myers Squibb, Pfizer, and Roche, outside of the submitted work; A.M. is employed by Novartis; the work described in this publication was completed prior to his employment at Novartis. K. M.T.: served as consultant and/or has received honoraria from Bristol Myers Squibb, MSD, Roche, Novartis, Pierre Fabre, Sanofi, Sun Pharma, LEO, Galderma, and Candela and travel support from Bristol Myers Squibb, Roche, Novartis, Pierre Fabre, and Candela, outside the submitted work. T.W.: received honoraria and travel support from Amgen, Novartis, Bristol Myers Squibb, Merck Sharp & Dohme, Pierre Fabre, outside the submitted work. C.P.: received honoraria (speaker honoraria or honoraria as a consultant) and travel support from: Novartis, BMS, Roche, Merck Serono, MSD, Celgene, AbbVie, Sanofi ‘Genzyme, Sun Pharma, Amgen and LEO. A. R.: reports non-financial support from Amgen, non-financial support from Roche, personal fees and non-financial support from Merck/MSD, grants and non-financial support from Novartis, grants and non-financial support from BMS, non-financial support from TEVA, grants from Adtec, outside the submitted work. S. U.: declares research support from Bristol Myers Squibb and Merck Serono; speakers and advisory board honoraria from Bristol Myers Squibb, Merck Sharp & Dohme, Merck Serono, Novartis and Roche, and travel support from Bristol Myers Squibb, and Merck Sharp & Dohme. L.Z.: served as consultant and/or has received honoraria from Roche, Bristol Myers Squibb (BMS), Merck Sharp & Dohme (MSD), Novartis, Pierre Fabre, Sanofi, and Sun Pharma and travel support from MSD, BMS, Amgen, Pierre Fabre, Sun Pharma, Sanofi and Novartis, outside the submitted work. N.S.: No relevant conflicts of interest. A.S.: No relevant conflicts of interest. F.K.: No relevant conflicts of interest. L.H.: served as consultant and/or has received honoraria from Amgen, Roche, Bristol Myers Squibb, Curevac, Merck Sharp & Dohme, Novartis, Pierre Fabre, Sanofi, Sun Pharma and a research grant from Novartis, outside the submitted work. Fri.M.: has received travel support or/and speaker's fees or/and advisor's honoraria by Novartis, Roche, BMS, MSD and Pierre Fabre and research funding from Novartis and Roche. Fra.M.: served as consultant and/or has received honoraria from Novartis, Roche, Bristol Myers Squibb, Merck Sharp & Dohme, Pierre Fabre, Sanofi Genzyme and travel support from Novartis, Sun Pharma and Bristol Myers Squibb, outside the submitted work. M.S.: participated in advisory boards of Bristol Myers Squibb, Novartis, MSD, Roche, Pierre Fabre, Kyowa Kirin, Immunocore and Sanofi Genzyme. M.S. received travel accommodation and expenses by Novartis, Pierre Fabre, and Sun Pharma, outside the submitted work. B.S.: received personal honoraria from Bristol Myers Squibb, Merck Sharpe & Dome, Novartis, Pfizer/EMD Serono, Pierre Fabre and Roche; has an advisory role for Bristol Myers Squibb, Merck Sharpe & Dome, Novartis, Pierre Fabre and Roche; and has received research funding from Bristol Myers Squibb, Merck Sharpe & Dome, and Pierre Fabre, all paid to the institute. Su.H.: No relevant conflicts of interest. D.S: reports personal fees and non-financial support from Roche/Genentech, grants, personal fees, non-financial support and other from BMS, personal fees from Merck Sharp & Dohme, personal fees and non-financial support from Merck Serono, grant, personal fees and non-financial support from Amgen, personal fees from Immunocore, personal fees from Incyte, personal fees from 4SC, personal fees from Pierre Fabre, personal fees and non-financial support from Sanofi/Regeneron, personal fees from Array BioPharma, personal fees from Pfizer, personal fees from Philogen, personal fees from Regeneron, personal fees from Nektar, personal fees from Sandoz, grants, personal fees and non-financial support from Novartis, personal fees and non-financial support from Sun Pharma, Replimune, Helsinn, OncoSec and InFlaRx outside the submitted work. E.L.: served as consultant and/or has received honoraria from Amgen, Actelion, Roche, Bristol Myers Squibb, Merck Sharp & Dohme, Novartis, Janssen, Medac, Sanofi, Sun Pharma and travel support from Amgen, Merck Sharp & Dohme, Bristol Myers Squibb, Amgen, Pierre Fabre, Sun Pharma and Novartis, outside the submitted work., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Accurate classification of melanocytic proliferations has important implications for prognostic prediction, treatment and follow-up. Although most melanocytic proliferations can be accurately classified using clinical and pathological criteria, classification (specifically distinction between nevus and melanoma) can be challenging in a subset of cases, including those with spitzoid morphology. Genetic studies have shown that mutation profiles differ between primary melanoma subtypes and Spitz nevi. These differences may aid in distinguishing benign from malignant in some melanocytic tumours. Here, we present a selection of melanocytic proliferations with equivocal histopathological criteria, wherein genetic analysis was requested to help guide classification. In two of four cases, the genetic results offered valuable insights, allowing a definitive diagnosis, indicating the diagnostic value of mutation profiling in a real-world routine clinical setting. Although histopathological assessment remains decisive in melanocytic proliferation classification, we recommend including genetic profiling in cases of borderline or atypical lesion to support accurate classification., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A.Z. received travel support from Novartis, Sanofi Genzyme and Bristol Myers Squibb (BMS), outside the submitted work. G.L. received travel support from Sun Pharma, outside the submitted work. R.M. reports no relevant conflicts of interest. M.P. reports no relevant conflicts of interest. I.C. reports no relevant conflicts of interest. P.J. reports no relevant conflicts of interest. E.C. received travel support from BMS, Merck Sharp & Dohme (MSD) and Novartis, outside the submitted work. C.R. reports no relevant conflicts of interest. B.H. reports no relevant conflicts of interest. J.M. received travel support from BMS, Novartis and Sun Pharmaceutical Industries, outside the submitted work. C.M.T. reports no relevant conflicts of interest. J.K. reports no relevant conflicts of interest. I.M. reports no relevant conflicts of interest. A.S. reports no relevant conflicts of interest. A.P. reports no relevant conflicts of interest. E.L. served as a consultant for and/or has received honoraria from Amgen, Actelion, Roche, BMS, MSD, Novartis, Janssen, Medac, Sanofi and Sun Pharma and reports travel support from Amgen, MSD, BMS, Amgen, Pierre Fabre, Sun Pharma and Novartis, outside the submitted work. L.Z. served as a consultant for and/or has received honoraria from Roche, BMS, MSD, Novartis, Pierre Fabre, Sanofi and Sun Pharma and reports travel support from MSD, BMS, Amgen, Pierre Fabre, Sun Pharma and Novartis, outside the submitted work. S.H. reports no relevant conflicts of interest. D.S. reports grants and other from BMS and personal fees from BMS, during the conduct of the study; personal fees from Amgen; personal fees from Boehringer Ingelheim; personal fees from InflaRx; personal fees and other from Roche; grants, personal fees and other from Novartis; personal fees from Incyte; personal fees and other from Regeneron; personal fees from 4SC; personal fees from Sanofi; personal fees from NeraCare; personal fees from Pierre Fabre; personal fees and other from Merck-EMD; personal fees from Pfizer; personal fees and other from Philogen; personal fees from Array and personal fees and other from MSD, outside the submitted work. E.H. reports no relevant conflicts of interest. K.G. reports no relevant conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: GNAQ and GNA11 mutant nonuveal melanoma represent a poorly characterized rare subgroup of melanoma with a gene mutation profile similar to uveal melanoma., Objectives: To characterize these tumours in terms of clinical behaviour and genetic characteristics., Methods: Patients with nonuveal GNAQ/11 mutated melanoma were identified from the prospective multicentre tumour tissue registry ADOREG, Tissue Registry in Melanoma (TRIM) and additional cooperating skin cancer centres. Extensive data on patient, tumour and treatment characteristics were collected retrospectively. Targeted sequencing was used to determine tumour mutational burden. Immunohistochemistry staining was performed for programmed death-ligand 1 and BRCA1-associated protein (BAP)1. Existing whole-exome cutaneous and uveal melanoma data were analysed for mutation type and burden., Results: We identified 18 patients with metastatic GNAQ/11 mutant nonuveal melanoma. Tumours had a lower tumour mutational burden and fewer ultraviolet signature mutations than cutaneous melanomas. In addition to GNAQ and GNA11 mutations (nine each), six splicing factor 3b subunit 1 (SF3B1), three eukaryotic translation initiation factor 1A X-linked (EIF1AX) and four BAP1 mutations were detected. In contrast to uveal melanoma, GNAQ/11 mutant nonuveal melanomas frequently metastasized lymphatically and concurrent EIF1AX, SF3B1 and BAP1 mutations showed no apparent association with patient prognosis. Objective response to immunotherapy was poor with only one partial response observed in 10 treated patients (10%)., Conclusions: Our findings suggest that GNAQ/11 mutant nonuveal melanomas are a subtype of melanoma that is both clinically and genetically distinct from cutaneous and uveal melanoma. As they respond poorly to available treatment regimens, novel effective therapeutic approaches for affected patients are urgently needed. What is already known about this topic? The rare occurrence of GNAQ/11 mutations in nonuveal melanoma has been documented. GNAQ/11 mutant nonuveal melanomas also harbour genetic alterations in EIF1AX, SF3B1 and BAP1 that are of prognostic relevance in uveal melanoma. What does this study add? GNAQ/11 mutant nonuveal melanomas show metastatic spread reminiscent of cutaneous melanoma, but not uveal melanoma. GNAQ/11 mutant nonuveal melanomas have a low tumour mutational burden that is higher than uveal melanoma, but lower than cutaneous melanoma. What is the translational message? Primary GNAQ/11 mutant nonuveal melanomas are a subtype of melanoma that is clinically and genetically distinct from both cutaneous and uveal melanoma. As metastatic GNAQ/11 mutant nonuveal melanomas respond poorly to available systemic therapies, including immune checkpoint inhibition, novel therapeutic approaches for these tumours are urgently needed. Linked Comment: Rafei-Shamsabadi. Br J Dermatol 2020; 183:806-807., (© 2020 The Authors. British Journal of Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.)

Melanoma cells shift between epigenetic-metabolic states to adapt to stress and, particularly, to drugs. Here, we unraveled the metabolome of an H3K4 demethylase (KDM5B/JARID1B)-driven melanoma cell phenotype that is known to be multidrug resistant. We set up a fast protocol for standardized, highly sensitive liquid chromatography-high resolution mass spectrometry analyzing stably controlled KDM5B expression by RNAi or doxycycline-induced overexpression. Within the KDM5B-dependent metabolome, we found significant and highly specific regulation of 11 intracellular metabolites. Functionally, overexpression of KDM5B in melanoma cells led to broadening of their oxidative metabolism from mainly glutamine-dependent to additionally glucose- and fatty acid-utilizing, upregulation of the pentose phosphate pathway as a source of antioxidant NADPH, and maintenance of a high ratio of reduced to oxidized glutathione. Histone lysine demethylase inhibition (GSK-J1, 2,4-PDCA) decreased colony formation and invasion in three-dimensional models. Thus, targeting KDM5B could represent an alternative way of modulating the metabolome and malignant cell behavior in melanoma., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Treatment of patients with metastatic melanoma is hampered by drug-resistance and often requires combination with radiotherapy as last-resort option. However, also after radiotherapy, clinical relapses are common., Methods & Results: Our preclinical models indicated a higher rate of tumour relapse when melanoma cells were first treated with BRAF V600E inhibition (BRAFi) followed by radiotherapy as compared to the reverse sequence. Accordingly, retrospective follow-up data from 65 stage-IV melanoma patients with irradiated melanoma brain metastases confirmed a shortened duration of local response of mitogen-activated protein kinase (MAPK)-inhibitor-pretreated compared with MAPK-inhibitor-naïve intracranial metastases. On the molecular level, we identified JARID1B/KDM5B as a cellular marker for cross-resistance between BRAFi and radiotherapy. JARID1B high cells appeared more frequently under upfront BRAFi as compared with upfront radiation. JARID1B favours cell survival by transcriptional regulation of genes controlling cell cycle, DNA repair and cell death., Conclusion: The level of cross-resistance between combined MAPK inhibition and radiotherapy is dependent on the treatment sequence. JARID1B may represent a novel therapy-overarching resistance marker., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown., Experimental Design and Results: In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma. NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively. Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples., Conclusions: Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma. RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type. BRAF and KIT mutations were rare with frequencies below 10% each. Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors.

Melanoma treatment has been revolutionized by antibody-based immunotherapies. IFNγ secretion by CD8 + T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on tumour cells. Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models. Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity. JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ. Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma. Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies. Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions. Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making.

Melanoma often recurs after a latency period of several years, presenting a T cell-edited phenotype that reflects a role for CD8(+) T cells in maintaining metastatic latency. Here, we report an investigation of a patient with multiple recurrent lesions, where poorly immunogenic melanoma phenotypes were found to evolve in the presence of autologous tumor antigen-specific CD8(+) T cells. Melanoma cells from two of three late recurrent metastases, developing within a 6-year latency period, lacked HLA class I expression. CD8(+) T cell-resistant, HLA class I-negative tumor cells became clinically apparent 1.5 and 6 years into stage IV disease. Genome profiling by SNP arrays revealed that HLA class I loss in both metastases originated from a shared chromosome 15q alteration and independently acquired focal B2M gene deletions. A third HLA class I haplotype-deficient lesion developed in year 3 of stage IV disease that acquired resistance toward dominant CD8(+) T-cell clonotypes targeting stage III tumor cells. At an early stage, melanoma cells showed a dedifferentiated c-Jun(high)/MITF(low) phenotype, possibly associated with immunosuppression, which contrasted with a c-Jun(low)/MITF(high) phenotype of T cell-edited tumor cells derived from late metastases. In summary, our work shows how tumor recurrences after long-term latency evolve toward T-cell resistance by independent genetic events, as a means for immune escape and immunotherapeutic resistance. Cancer Res; 76(15); 4347-58. ©2016 AACR., (©2016 American Association for Cancer Research.)

Melanocytic tumors originating in the central nervous system (MT-CNS) are rare tumors that generally have a favorable prognosis, however malignant tumors do occur. Pathogenetically MT-CNS are not well characterized. Similar to uveal melanoma and blue nevi, they frequently harbor activating GNAQ or GNA11 mutations. Rare NRAS mutations have also been reported. Other mutations have not yet been described. We analyzed 19 MT-CNS, 7 uveal melanomas and 19 cutaneous melanomas using a targeted next generation sequencing approach analyzing 29 genes known to be frequently mutated in other melanocytic tumors (in particular uveal and cutaneous melanomas). In concordance with previous studies, cutaneous melanoma samples showed frequent NRAS or BRAF mutations, as well as mutations in other genes (e.g. NF1, RAC1, PIK3CA, ARID1A). Metastasized uveal melanomas exhibited mutations in GNAQ, GNA11 and BAP1. In contrast, MT-CNS almost exclusively demonstrated mutations in GNAQ (71 %) or GNA11 (12 %). Interestingly both GNA11 mutations identified were detected in MT-CNS diagnosed as intermediate grade melanocytomas which also recurred. One of these recurrent cases also harbored an inactivating BAP1 mutation and was found to have lost one copy of chromosome 3. Our findings show that while MT-CNS do have GNAQ or GNA11 mutations, they rarely harbor other recurrent mutations found in uveal or cutaneous melanomas. Considering chromosome 3 and BAP1 loss are robust markers of poor prognosis in uveal melanoma, it will prove interesting to determine whether these genomic alterations are also of prognostic significance in MT-CNS.

The multifunctional Ig-like carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is neo-expressed in the majority of malignant melanoma lesions. CEACAM1 acts as a driver of tumor cell invasion, and its expression correlates with poor patient prognosis. Despite its importance in melanoma progression, how CEACAM1 expression is regulated is largely unknown. Here, we show that CEACAM1 expression in melanoma cell lines and melanoma tissue strongly correlates with that of the microphthalmia-associated transcription factor (MITF), a key regulator of melanoma proliferation and invasiveness. MITF is revealed as a direct and positive regulator for CEACAM1 expression via binding to an M-box motif located in the CEACAM1 promoter. Taken together, our study provides novel insights into the regulation of CEACAM1 expression and suggests an MITF-CEACAM1 axis as a potential determinant of melanoma progression., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The key metabolic enzyme lactate dehydrogenase A (LDHA) is overexpressed in many cancers, and several preclinical studies have shown encouraging results of targeted inhibition. However, the mechanistic importance of LDHA in melanoma is largely unknown and hitherto unexplored in brain metastasis., Methods: We investigated the spatial, temporal, and functional features of LDHA expression in melanoma brain metastasis across multiple in vitro assays, in a robust and predictive animal model employing MRI and PET imaging, and in a unique cohort of 80 operated patients. We further assessed the genomic and proteomic landscapes of LDHA in different cancers, particularly melanomas., Results: LDHA expression was especially strong in early and small brain metastases in vivo and related to intratumoral hypoxia in late and large brain metastases in vivo and in patients. However, LDHA expression in human brain metastases was not associated with the number of tumors, BRAF(V600E) status, or survival. Moreover, LDHA depletion by small hairpin RNA interference did not affect cell proliferation or 3D tumorsphere growth in vitro or brain metastasis formation or survival in vivo. Integrated analyses of the genomic and proteomic landscapes of LDHA indicated that LDHA is present but not imperative for tumor progression within the CNS, or predictive of survival in melanoma patients., Conclusions: In a large patient cohort and in a robust animal model, we show that although LDHA expression varies biphasically during melanoma brain metastasis formation, tumor progression and survival seem to be functionally independent of LDHA., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Recently, recurrent mutations in regulatory DNA regions, such as promoter mutations in the TERT gene were identified in melanoma. Subsequently, Weinhold et al. reported SDHD promoter mutations occurring in 10% of melanomas and being associated with a lower overall survival rate. Our study analyzes the mutation rate and clinico-pathologic associations of SDHD promoter mutations in a large cohort of different melanoma subtypes., Methods: 451 melanoma samples (incl. 223 non-acral cutaneous, 38 acral, 33 mucosal, 43 occult, 43 conjunctival and 51 uveal melanoma) were analyzed for the presence of SDHD promoter mutations by Sanger-sequencing. Statistical analysis was performed to screen for potential correlations of SDHD promoter mutation status with various clinico-pathologic criteria., Results: The SDHD promoter was successfully sequenced in 451 tumor samples. ETS binding site changing SDHD promoter mutations were identified in 16 (4%) samples, of which 5 mutations had not been described previously. Additionally, 5 point mutations not located in ETS binding elements were identified. Mutations in UV-exposed tumors were frequently C>T. One germline C>A SDHD promoter mutation was identified. No statistically significant associations between SDHD promoter mutation status and various clinico-pathologic variables or overall patient survival were observed., Conclusions: Melanomas harbor recurrent SDHD promoter mutations, which occur primarily as C>T alterations in UV-exposed melanomas. In contrast to the initial report and promoter mutations in the TERT gene, our analysis suggests that SDHD promoter mutations are a relatively rare event in melanoma (4% of tumors) of unclear clinical and prognostic relevance.

Acquired or intrinsic resistance to apoptotic and necroptotic stimuli is considered a major hindrance of therapeutic success in malignant melanoma. Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptotic and necroptotic cell death mediated by numerous cell death signalling platforms. In this report we investigated the impact of IAPs for cell death regulation in malignant melanoma. Suppression of IAPs strongly sensitized a panel of melanoma cells to death ligand-induced cell death, which, surprisingly, was largely mediated by apoptosis, as it was completely rescued by addition of caspase inhibitors. Interestingly, the absence of necroptosis signalling correlated with a lack of receptor-interacting protein kinase-3 (RIPK3) mRNA and protein expression in all cell lines, whereas primary melanocytes and cultured nevus cells strongly expressed RIPK3. Reconstitution of RIPK3, but not a RIPK3-kinase dead mutant in a set of melanoma cell lines overcame CD95L/IAP antagonist-induced necroptosis resistance independent of autocrine tumour necrosis factor secretion. Using specific inhibitors, functional studies revealed that RIPK3-mediated mixed-lineage kinase domain-like protein (MLKL) phosphorylation and necroptosis induction critically required receptor-interacting protein kinase-1 signalling. Furthermore, the inhibitor of mutant BRAF Dabrafenib, but not Vemurafenib, inhibited necroptosis in melanoma cells whenever RIPK3 is present. Our data suggest that loss of RIPK3 in melanoma and selective inhibition of the RIPK3/MLKL axis by BRAF inhibitor Dabrafenib, but not Vemurafenib, is critical to protect from necroptosis. Strategies that allow RIPK3 expression may allow unmasking the necroptotic signalling machinery in melanoma and points to reactivation of this pathway as a treatment option for metastatic melanoma.

Purpose: CD8(+) T lymphocytes can kill autologous melanoma cells, but their activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here, we analyzed three consecutive melanoma lesions obtained within one year of developing stage IV disease for their recognition by autologous T cells., Experimental Design: One skin (Ma-Mel-48a) and two lymph node (Ma-Mel-48b, Ma-Mel-48c) metastases were analyzed for T-cell infiltration. Melanoma cell lines established from the respective lesions were characterized, determining the T-cell-stimulatory capacity, expression of surface molecules involved in T-cell activation, and specific genetic alterations affecting the tumor-T-cell interaction., Results: Metastases Ma-Mel-48a and Ma-Mel-48b, in contrast with Ma-Mel-48c, were infiltrated by T cells. The T-cell-stimulatory capacity was found to be strong for Ma-Mel-48a, lower for Ma-Mel-48b, and completely abrogated for Ma-Mel-48c cells. The latter proved to be HLA class I-negative due to an inactivating mutation in one allele of the beta-2-microglobulin (B2M) gene and concomitant loss of the other allele by a deletion on chromosome 15q. The same deletion was already present in Ma-Mel-48a and Ma-Mel-48b cells, pointing to an early acquired genetic event predisposing to development of β2m deficiency. Notably, the same chronology of genetic alterations was also observed in a second β2m-deficient melanoma model., Conclusion: Our study reveals a progressive loss in melanoma immunogenicity during the course of metastatic disease. The genetic evolvement of T-cell resistance suggests screening tumors for genetic alterations affecting immunogenicity could be clinically relevant in terms of predicting patient responses to T-cell-based immunotherapy., (©2014 American Association for Cancer Research.)

Cutaneous melanoma occurs in both familial and sporadic forms. We investigated a melanoma-prone family through linkage analysis and high-throughput sequencing and identified a disease-segregating germline mutation in the promoter of the telomerase reverse transcriptase (TERT) gene, which encodes the catalytic subunit of telomerase. The mutation creates a new binding motif for Ets transcription factors and ternary complex factors (TCFs) near the transcription start and, in reporter gene assays, caused up to twofold increase in transcription. We then screened the TERT promoter in sporadic melanoma and observed recurrent ultraviolet signature somatic mutations in 125 of 168 (74%) of human cell lines derived from metastatic melanomas, 45 of 53 corresponding metastatic tumor tissues (85%), and 25 of 77 (33%) primary melanomas. The majority of those mutations occurred at two positions in the TERT promoter and also generated binding motifs for Ets/TCF transcription factors.