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Background: Melanomas lacking mutations in BRAF, NRAS and NF1 are frequently referred to as "triple wild-type" (tWT) melanomas. They constitute 5-10 % of all melanomas and remain poorly characterized regarding clinical characteristics and response to therapy. This study investigates the largest multicenter collection of tWT-melanomas to date., Methods: Targeted next-generation sequencing of the TERT promoter and 29 melanoma-associated genes were performed on 3109 melanoma tissue samples of the prospective multicenter study ADOREG/TRIM of the DeCOG revealing 292 patients suffering from tWT-melanomas. Clinical characteristics and mutational patterns were analyzed. As subgroup analysis, we analyzed 141 tWT-melanoma patients receiving either anti-CTLA4 plus anti-PD1 or anti PD1 monotherapy as first line therapy in AJCC stage IV., Results: 184 patients with cutaneous melanomas, 56 patients with mucosal melanomas, 34 patients with acral melanomas and 18 patients with melanomas of unknown origin (MUP) were included. A TERT promoter mutation could be identified in 33.2 % of all melanomas and 70.5 % of all tWT-melanomas harbored less than three mutations per sample. For the 141 patients with stage IV disease, mPFS independent of melanoma type was 6.2 months (95 % CI: 4-9) and mOS was 24.8 months (95 % CI: 14.2-53.4) after first line anti-CTLA4 plus anti-PD1 therapy. After first-line anti-PD1 monotherapy, mPFS was 4 months (95 %CI: 2.9-8.5) and mOS was 29.18 months (95 % CI: 17.5-46.2)., Conclusions: While known prognostic factors such as TERT promoter mutations and TMB were equally distributed among patients who received either anti-CTLA4 plus anti-PD1 combination therapy or anti-PD1 monotherapy as first line therapy, we did not find a prolonged mPFS or mOS in either of those. For both therapy concepts, mPFS and mOS were considerably shorter than reported for melanomas with known oncogene mutations., Competing Interests: Declaration of Competing Interest All other authors declare no conflicts of interest for the submitted work., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Around 50% of cutaneous melanomas harbour therapeutically targetable BRAF V600 mutations. Reliable clinical biomarkers predicting duration of response to BRAF-targeted therapies are still lacking. Recent in vitro studies demonstrated that BRAF-MEK inhibitor therapy response is associated with tumour TERT promoter mutation status. We assessed this potential association in a clinical setting., Methods: The study cohort comprised 232 patients with metastatic or unresectable BRAF V600-mutated melanoma receiving combined BRAF/MEK inhibitor treatment, including a single-centre retrospective discovery cohort (N = 120) and a prospectively collected multicenter validation cohort (N = 112). Patients were excluded if they received BRAF or MEK inhibitors in an adjuvant setting, as monotherapy, or in combination with immunotherapy. Kaplan-Meier and univariate/multivariate Cox regression analyses were performed as appropriate., Results: median age at first diagnosis was 54 years (range 16-84 years). The majority of patients were men 147/232 (63.4%). Most tumours harboured TERT promoter mutations (72%, N = 167). A survival advantage was observed in both progression-free survival (PFS) and overall survival (OS) for patients with TERT promoter-mutant versus wild-type tumours in both the discovery cohort (mPFS of 9.6 months [N = 87] vs 5.0 months [N = 33]; hazard ratio [HR] = 0.56 [95% confidence interval {CI} 0.33-0.96] and mOS of 33.6 months vs 15.0 months; HR = 0.47 [95%CI 0.32-0.70]) as well as the validation cohort (mPFS of 7.3 months [N = 80] vs 5.8 months [N = 32]; HR = 0.67 [95%CI 0.41-1.10] and mOS of 51.1 months vs 15.0 months; HR = 0.33 [95%CI 0.18-0.63]). In the pooled cohort of TERT promoter-mutant (N = 167) versus wild-type (N = 65) tumours, respectively, PFS was 8.9 versus 5.5 months, (HR = 0.62; 95%CI 0.45-0.87; P = 0.004), and OS was 33.6 versus 17.0 months, (HR = 0.51; 95%CI 0.35-0.75, P = 0.0001)., Conclusions: In patients with melanoma receiving BRAF/MEK-targeted therapies, TERT promoter mutations are associated with longer survival. If validated in larger studies, TERT promoter mutation status should be included as a predictive biomarker in treatment algorithms for advanced melanoma., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: C.M.T. reported no relevant conflicts of interest. J.M. declares travel support from Bristol Myers Squibb, Novartis and Sun Pharmaceutical Industries, outside the submitted work. A.Z. declares travel support from Novartis, Sanofi Genzyme and Bristol-Myers Squibb, outside the submitted work. R.M. reported no relevant conflicts of interest. E.C. reported no relevant conflicts of interest. G.L. declares travel support from Sun Pharma, outside the submitted work. P.J. reported no relevant conflicts of interest. R.H. declares speakers and advisory board honoraria from BMS, MSD, Novartis, Pierre Fabre, Roche and SUN-Pharma, outside the submitted work. P.T. declares honoraria from Bristol-Myers Squibb, Novartis, Merck Sharp & Dohme, Pierre-Fabre, CureVac, Merck Serono, Sanofi, Roche, Kyowa Kirin and Biofrontera; travel support from Bristol-Myers Squibb and Pierre-Fabre, outside the submitted work. J.U. declares advisory board or has received honoraria and travel support from Amgen, Bristol Myers Squibb, GSK, LeoPharma, Merck Sharp and Dohme, Novartis, Pierre Fabre, Roche and Sanofi, outside the submitted work. C.P. served as consultant and/or has received honoraria from Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Sanofi, Sun Pharma, Pierre Fabre, AbbVie, Kyona Kirin and Amgen and received travel support from Amgen, Merck Sharp & Dohme, Bristol-Myers Squibb, Pierre Fabre, Sun Pharma and Novartis, outside the submitted work. J.Ul declares travel support: Medac, Sun Pharma; consulting: Bristol-Myers Squibb, Sun Pharma; lectures: Bristol-Myers Squibb, MSD, Merck, Novartis, Roche, Sanofi, Sun Pharma; grants: Novartis, outside the submitted work. A.K. reported no relevant conflicts of interest. P.M. declares research support (to institution): Bristol-Myers Squibb, Novartis, MSD. Honoraria for lectures (personally): Roche Pharma, Bristol-Myers Squibb, Novartis, MSD, Almirall-Hermal, Amgen, Merck-Serono, Bayer, Pierre-Fabre, Sanofi. Honoraria for advisory boards: Bayersdorf, Roche Pharma, Bristol-Myers Squibb, Novartis, MSD, Almirall-Hermal, Amgen, Pierre-Fabre, Merck-Serono, SUN, Merck-Serono, Sanofi, outside the submitted work. R.G. 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, Merck Serono, Pfizer, Immunocore. Research grants: Novartis, Pfizer, Johnson & Johnson, Amgen, Merck-Serono, SUN Pharma, Sanofi. Travel/meeting support: Roche, BMS, SUN, Merck-Serono, Pierre-Fabre, outside the submitted work. F.M. declares 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, outside the submitted work. E.D. reported no relevant conflicts of interest. M.W. reported no relevant conflicts of interest. J.K. reported no relevant conflicts of interest. I.M. reported no relevant conflicts of interest. A.S. reported no relevant conflicts of interest. A.P. reported no relevant conflicts of interest. E.L. served as a consultant and/or has received honoraria from Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Medac, Sanofi, Sun Pharma and travel support from Amgen, Merck Sharp & Dohme, Bristol-Myers Squibb, Pierre Fabre, Sun Pharma and Novartis, outside the submitted work. L.Z. served as consultant and/or has received honoraria from Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Pierre-Fabre, Sun Pharma and Sanofi; Research funding to institution: Novartis; travel support from Merck Sharp & Dohme, Bristol-Myers Squibb, Amgen, Pierre-Fabre, Sanofi, Sun Pharma and Novartis, outside the submitted work.E.H. reported no relevant conflicts of interest. 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, outside the submitted work. 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. K.G.: No relevant conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Combined inhibition of BRAF/MEK is an established therapy for melanoma. In addition to its canonical mode of action, effects of BRAF/MEK inhibitors on antitumor immune responses are emerging. Thus, we investigated the effect of these on adaptive immune responses., Patients, Methods and Results: Sequential tumor biopsies obtained before and during BRAF/MEK inhibitor treatment of four (n = 4) melanoma patients were analyzed. Multiplexed immunofluorescence staining of tumor tissue revealed an increased infiltration of CD4 + and CD8 + T cells upon therapy. Determination of the T-cell receptor repertoire usage demonstrated a therapy induced increase in T-cell clonotype richness and diversity. Application of the Grouping of Lymphocyte Interactions by Paratope Hotspots algorithm revealed a pre-existing immune response against melanoma differentiation and cancer testis antigens that expanded preferentially upon therapy. Indeed, most of the T-cell clonotypes found under BRAF/MEK inhibition were already present in lower numbers before therapy. This expansion appears to be facilitated by induction of T-bet and TCF7 in T cells, two transcription factors required for self-renewal and persistence of CD8 + memory T cells., Conclusions: Our results suggest that BRAF/MEK inhibition in melanoma patients allows an increased expansion of pre-existing melanoma-specific T cells by induction of T-bet and TCF7 in these.

Despite markedly improved treatment options for metastatic melanoma, resistance to targeted therapies such as BRAF inhibitors (BRAFi) or BRAFi plus MEK inhibitors (MEKi) remains a major problem. Our aim was to characterize progression on BRAFi therapy and outcome of subsequent treatment. One hundred and eighty patients with BRAF-mutant metastatic melanoma who had progressed on treatment with single-agent BRAFi from February 2010 to April 2015 were included in a retrospective data analysis focused on patterns of progression, treatment beyond progression (TBP) and subsequent treatments after BRAFi therapy. Analysis revealed that 51.1% of patients progressed with both new and existing metastases opposed to progression of only preexisting (28.3%) or only new (20.6%) metastases. Exclusive extracranial progression occurred in 50.6% of patients compared to both extra- and intracranial (29.4%) or sole cerebral progression (20%). Multivariable analyses demonstrated that single site progression and primary response to BRAFi were associated with improved progression-free survival. Progression with exclusively new or only existing metastases and a baseline Eastern Cooperative Oncology Group (ECOG) of 0 were associated with prolonged overall survival (OS). TBP had no significant impact on OS. Other subsequent treatments showed low efficacy with the exception of anti-PD-1 antibodies. In conclusion we identified specific patterns of progression which significantly correlate with further prognosis after progression on BRAFi treatment. In contrast to previously published data, we could not demonstrate a significant survival benefit for BRAFi TBP. Subsequent therapies had strikingly low efficacy except for PD-1 inhibitors., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Targeted therapies with BRAF plus MEK inhibitors (BRAFi; MEKi) represent the major treatment strategy for patients with BRAF-mutated metastatic melanoma (MM). Previous analyses suggested a correlation between programmed death-ligand 1 (PD-L1) expression in tumour tissues and the outcome of targeted therapies. This study investigated PD-L1 as a potential predictive biomarker of BRAFi-based targeted therapies in MM patients., Patients and Methods: We analysed two independent cohorts of BRAF V600-mutated MM patients undergoing BRAFi-based therapies for PD-L1 expression in pre-treatment tumour tissues. The oligocentre cohort 1 included 83 patients whose tumour tissues were analysed retrospectively with the anti-PD-L1 antibody clone E1L3N. The multicentre cohort 2 included 58 patients whose tumour tissues were analysed prospectively within the framework of the "Registry of the Arbeitsgemeinschaft Dermatologische Onkologie" (ADOREG) and "Tissue Registry in Melanoma" (TRIM) project using the anti-PD-L1 antibody clone 28-8., Results: PD-L1 expression in pre-treatment tumour tissue did not correlate with response or survival to BRAFi-based therapies in both MM patient cohorts. This finding was not influenced by retrospective versus prospective immunohistochemistry analyses, oligocentre versus multicentre cohorts or the different anti-PD-L1 antibody clones used. In cohort 1, PD-L1 positivity was detected in tumour tissue of 41.0% and 18.1% of patients (cut-off 1% and 5%, respectively). In cohort 2, 58.6% and 39.7% of patients showed PD-L1 positivity (cut-off 1% and 5%, respectively)., Conclusion: In two independent cohorts including a total of 141 MM patients, PD-L1 expression in tumour tissue did not correlate with the outcome of BRAFi-based treatment. Therefore, PD-L1 cannot be recommended for the use as a predictive biomarker of BRAFi-based therapy in BRAF V600-mutated MM., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Importance: BRAF inhibitors have been licensed for the therapy of BRAF-mutated melanoma. Recently, inflammatory skin lesions clinically resembling erythema nodosum have been reported as therapy side-effects that may lead to treatment discontinuation., Objective: To identify and characterize cases with BRAF inhibitor-associated erythema nodosum-like inflammatory skin lesions and development of an algorithm for their management., Design and Setting: Retrospective chart review of melanoma patients treated with BRAF inhibitors in 14 departments of Dermatology in Germany and Austria and PubMed search for cases in the literature., Results: Sixteen patients were identified who developed erythema nodosum-like lesions under BRAF inhibitor therapy; 14 had received vemurafenib and two dabrafenib plus trametinib. The most frequently involved body sites were the legs. Histopathology was performed in five cases and revealed panniculitis in three and vasculitis in two patients respectively. Arthralgia and fever were associated symptoms in 44% and 31% of patients respectively. Inflammatory symptoms led to discontinuation of treatment in three patients, while in the majority of cases symptomatic management was sufficient. Skin lesions finally resolved despite continued BRAF inhibitor therapy in seven patients. In the literature, 19 additional patients with similar cutaneous appearance under BRAF inhibitors could be identified. An algorithm for the management of such lesions is proposed., Conclusion: Erythema nodosum-like skin lesions histologically correspond to panniculitis and/or vasculitis. Symptomatic treatment may be sufficient. However, additional work-up and interruption of BRAF inhibitor therapy may be necessary in severe cases which are commonly associated with systemic symptoms., (© 2015 European Academy of Dermatology and Venereology.)

Importance: A frequent adverse effect of mutation-specific BRAF inhibitor therapy is the induction of epithelial proliferations including cutaneous squamous cell carcinomas. To date, the only factor identified contributing to their development is the activation of the mitogen-activated signal transduction cascade by mutations in the RAS genes. However, these mutations explain only 60% of the tumors; hence, it is important to identify what is causing the remaining tumors., Objective: To test for the presence of human papillomaviruses (HPVs) and the recently identified human polyomaviruses (HPyVs), Merkel cell polyomavirus (MCPyV), and trichodysplasia spinulosa-associated polyomavirus (TSPyV), as well as HPyV-6, HPyV-7, HPyV-9, and HPyV-10, in epithelial proliferations occurring after BRAF inhibitor therapy to determine whether these oncogenic viruses may contribute to BRAF inhibitor-induced skin tumors., Design, Setting, and Participants: Retrospective study at a university hospital in Austria of epithelial proliferations that developed in patients with melanoma after initiation of treatment with the BRAF inhibitor vemurafenib. Samples were analyzed for (1) presence of the most frequently observed RAS mutations by SNaPshot technology, (2) detection of the viruses by real-time polymerase chain reaction, and (3) presence of capsid proteins of the most abundantly detected virus by immunohistochemical analysis., Main Outcomes and Measures: RAS mutational status, as well as HPV and HPyV presence, in BRAF inhibitor-induced epithelial proliferations., Results: Eighteen biopsy samples from 6 patients were retrieved from our hospital's archive. We identified RAS mutations in 10 (62%) of the 16 samples with clear results. DNA of HPyV-9, HPyV-10, and TSPyV were virtually absent in the samples. MCPyV DNA was present in 13 of 18 samples, and HPV, HPyV-6, and HPyV-7 DNA were present in all samples. In general, the amount of DNA encoding the latter viruses was rather low, with the exception of HPyV-6 in several samples of 1 individual patient. Notably, the relevance of the presence of HPyV-6 in the epithelial proliferation was underlined by immunohistochemical detection of the core protein VP1 of HPyV-6., Conclusions and Relevance: The presence of both high HPyV-6 DNA load and VP1 protein suggests that polyomaviruses may contribute to the epithelial proliferations observed in patients receiving BRAF inhibitor therapy, albeit the relative impact as compared with that of RAS mutations appears circumstantial.

Background: BRAF and MEK inhibitors are new targeted therapies which are used in the treatment of malignancies, in particular of malignant melanoma., Side Effects: Cutaneous side effects are common during the treatment with both types of inhibitors. These side effects include inflammatory reactions such as maculopapular and papulopustular exanthema, hand-foot syndrome, panniculitis, paronychia, photo- and radio-sensitization. As a class effect, BRAF-inhibitors induce proliferative disorders of keratinocytes and melanocytes, such as palmoplantar hyperkeratosis (as part of the hand-foot syndrome), verruciform and acanthoma-like lesions, follicular and Grover disease-like hyperkeratoses, keratoacanthomas, squamous cell carcinomas and atypical melanocytic nevi with transition to secondary melanomas. Furthermore, hair alterations and xerosis are possible., Conclusions: Treatment with BRAF and MEK inhibitors requires close dermatologic monitoring of the patient. This manuscript summarizes the most frequent cutaneous side effects and their management.

Most patients with BRAF(V600)-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF(V600)-mutant metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a "long tail" of new mitogen-activated protein kinase (MAPK) pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAF(V600)-mutant melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies.

BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients. BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi. A total of 23 melanoma patients (six females, 17 males, aged 47-80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2-23.2) and subsequent for BRAFi 4.5 (1.2-15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed. Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In melanoma, the RAS/RAF/MEK/ERK signalling pathway is an area of great interest, because it regulates tumor cell proliferation and survival. A varying mutation rate has been reported for B-RAF and N-RAS, which has been largely attributed to the differential source of tumor DNA analyzed, e.g., fixed tumor tissues or in vitro propagated melanoma cells. Notably, this variation also interfered with interpreting the impact of these mutations on the clinical course of the disease. Consequently, we investigated the mutational profile of B-RAF and N-RAS in biopsies and corresponding cell lines from metastatic tumor lesions of 109 melanoma patients (AJCC stage III/IV), and its respective impact on survival. 97 tissue biopsies and 105 biopsy-derived cell lines were screened for B-RAF and N-RAS mutations by PCR single strand conformation polymorphism and DNA sequencing. Mutations were correlated with patient survival data obtained within a median follow-up time of 31 months. B-RAF mutations were detected in 55% tissues and 51% cell lines, N-RAS mutations in 23% tissues and 25% cell lines, respectively. There was strong concordance between the mutational status of tissues and corresponding cell lines, showing a differing status for B-RAF in only 5% and N-RAS in only 6%, respectively. Patients with tumors carrying mutated B-RAF showed an impaired median survival (8.0 versus 11.8 months, p = 0.055, tissues; 7.1 versus 9.3 months, p = 0.068, cell lines), whereas patients with N-RAS-mutated tumors presented with a favorable prognosis (median survival 12.5 versus 7.9 months, p = 0.084, tissues; 15.4 versus 6.8 months, p = 0.0008, cell lines), each in comparison with wildtype gene status. Multivariate analysis qualified N-RAS (p = 0.006) but not B-RAF mutation status as an independent prognostic factor of overall survival. Our findings demonstrate that B-RAF and N-RAS mutations are well preserved during short term in vitro propagation and, most importantly, differentially impact the outcome of melanoma patients.

The molecular biology of metastatic potential in melanoma has been studied many times previously and changes in the expression of many genes have been linked to metastatic behaviour. What is lacking is a systematic characterization of the regulatory relationships between genes whose expression is related to metastatic potential. Such a characterization would produce a molecular taxonomy for melanoma which could feasibly be used to identify epigenetic mechanisms behind changes in metastatic behaviour. To achieve this we carried out three separate DNA microarray analyses on a total of 86 cultures of melanoma. Significantly, multiple testing correction revealed that previous reports describing correlations of gene expression with activating mutations in BRAF or NRAS were incorrect and that no gene expression patterns correlate with the mutation status of these MAPK pathway components. Instead, we identified three different sample cohorts (A, B and C) and found that these cohorts represent melanoma groups of differing metastatic potential. Cohorts A and B were susceptible to transforming growth factor-beta (TGFbeta)-mediated inhibition of proliferation and had low motility. Cohort C was resistant to TGFbeta and demonstrated high motility. Meta-analysis of the data against previous studies linking gene expression and phenotype confirmed that cohorts A and C represent transcription signatures of weakly and strongly metastatic melanomas, respectively. Gene expression co-regulation suggested that signalling via TGFbeta-type and Wnt/beta-catenin pathways underwent considerable change between cohorts. These results suggest a model for the transition from weakly to strongly metastatic melanomas in which TGFbeta-type signalling upregulates genes expressing vasculogenic/extracellular matrix remodelling factors and Wnt signal inhibitors, coinciding with a downregulation of genes downstream of Wnt signalling.

We studied global gene expression in three melanoma cell lines with the most common and potent V600E mutation in the B-RAF gene-four cell lines with a common Q61R mutation in the N-RAS gene and three cell lines with no mutations using human HG-U133A 2.0 micro-arrays with 22 277 transcripts. Data analysis using stringent criteria revealed several upregulated and downregulated genes in cell lines with B-RAF and N-RAS mutations compared with cell lines without mutations. We found 29 genes specifically upregulated and 32 genes downregulated in cell lines with B-RAF mutations, whereas 70 genes were upregulated and 39 downregulated in cell lines with N-RAS mutations; 11 genes showed overlapping upregulation and 45 downregulation. The micro-array data for nine selected genes were validated by the real-time PCR technique. Expression of a large number of genes, that encode members or regulators of the RAS/RAF/MEK/ERK pathways or are involved in metastasis or invasion, was affected in cell lines with mutations in B-RAF and N-RAS. Upregulated genes in cell lines with mutations included dual-specificity phosphatase 6 (DUSP6), sprouty 2 (SPRY2), v-akt murine thymoma viral oncogene homolog 3 (AKT3) and matrix metalloproteinase 14 (MMP14); downregulated genes included interleukin 18 (IL18), Krüppel-like factor 5 (KLF5) and inhibitor of DNA binding 2 (ID2). Our results, though carried on cell lines, provide a novel insight into the effect of mutations in the B-RAF and N-RAS genes on global gene expression in melanoma and highlight the complexity of mechanisms involved in tumour initiation and maintenance.