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Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer with a tripled incidence in the US and Europe over the past decade. Around 80% of MCC is linked to Merkel cell polyomavirus, but the cell of origin remains unknown. We stably introduced Merkel cell polyomavirus (MCPyV)-sT) and LT antigens to MCC13 and REH cell lines, analyzing DNA methylation and gene transcriptional regulation. Gene ontology analysis assessed MCPyV effects, and integrative analysis correlated gene expression and methylation. Expression patterns were compared with 15 previously sequenced primary MCCs. We found that MCPyV-LT induces DNA methylation changes in both cell lines, while MCPyV-sT only affected REH cells. Greater gene expression changes are observed in MCC13 cells, with upregulated genes associated with cellular components and downregulated genes related to biological processes. Integrative analysis of differentially expressed genes (DEG) and differentially methylated regions (DMR) of REH cell lines revealed that no genes were commonly methylated and differentially expressed. The study compared DEGs and DMG in MCC13 and REH cells to overlapping genes in MCPyV-positive cell lines (MKL1, MKL2, and WaGa), identifying hypomethylated genes in the gene body and hypermethylated genes at TSS1500. GO analysis of the two cell lines showed that MCPyV-TAs can downregulate genes in MHC-I pathways; this downregulation offers a target that can be used to create novel and efficient MCC immunotherapy approaches. Finally, it was confirmed that MCPyV-LT controls gene expression in MCC tissues using an integrative investigation of DNA methylation and gene expression., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Background and Aim: Angiotropism and extravascular migratory metastasis (EVMM) are an important alternative means of melanoma spread. In EVMM, melanoma cells migrate along the external surfaces of vascular channels to distant sites and demonstrate microscopic angiotropism, i.e. melanoma cells arrayed along the external surfaces of vascular endothelium. Pertinent to EVMM are the origin of melanocytes from the neural crest (NC) and strong analogies of EVMM with NC cell migration. Our aim is to elucidate the molecular mechanisms underlying angiotropism and EVMM., Methods: Frozen primary melanomas, previously utilised for gene expression profiling, were analysed for angiotropism as a differential marker. From the results of this new microarray analysis, we sought to identify genes which were directly relevant to the basic mechanisms underlying EVMM., Results: Among 66 melanomas from patients who developed metastases or remained disease-free at 4 years of follow-up, 26 melanomas were angiotropic while 35 were not, and five were equivocal. The new microarray analysis identified 128 genes differentially expressed in angiotropic versus non-angiotropic melanomas. Among these 128 genes, 15 genes were potentially directly involved in EVMM, based their respective expressions in the NC (seven genes), in other malignant tumours of NC origin (three genes), in cell motility and/or migration (four genes) and in neurotropism (one gene)., Conclusion: The detection of these 15 genes provides additional support for the importance of angiotropism and the mechanism of EVMM in melanoma. Ongoing studies on this new profile of 15 genes may potentially identify new targets for controlling melanoma metastasis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

With the aim to correlate BRAF mutation status with gene expression in human primary cutaneous melanomas, and thus to get more insight on the consequences of BRAF mutation on cell biology, we analyzed all expression data obtained in melanomas from which DNA was extracted from the same tissue slides that were used for the expression study. A cohort of 69 frozen primary melanoma whose oligonucleotide micro-array expression data were available, were genotyped for BRAF and NRAS genes. The expression data from these melanomas were re-analyzed according to BRAF mutational status. A set of 250 probes representing 209 genes that were significantly (raw P< or =0.001) associated with BRAF mutation status was identified and 17 of these were previously shown to be implicated in cutaneous melanoma progression or pigmentation pathway-associated genes driven by the microphthalmia transcription factor (MITF). The list of 34 top probes contained no more than 1% of false discoveries with a probability of 0.95. Among the genes that differentiated most strongly between BRAF mutated and non-mutated melanomas, there were those involved in melanoma immune response such as MAGE-D2, CD63, and HSP70. These findings support the immunogenicity of BRAF(V600E), eliciting patients T-cell responses in various in vitro assays. The genes whose expression is associated with BRAF mutations are not simply restricted to the MAPK/ERK signaling but also converge to enhanced immune responsiveness, cell motility and melanosomes processing involved in the adaptative UV response.

To obtain insight into the mechanisms of tumor progression in cutaneous malignant melanoma (CMM), and in search of new prognostic markers, gene expression profiling on a series of CMMs with known follow-up was performed. We identified an expression signature of 254 genes with prognostic significance, with a predictive accuracy comparable to that obtainable from tumor thickness and ulceration. Most of these genes were correlated with thickness, thereby stressing the importance of thickness in the prognosis of CMM. On immunohistochemistry, eight new prognostic markers were identified, of which three (mini-chromosome maintenance proteins 3, 4 and 6) had independent prognostic value. These data provide new information regarding CMM progression. As several of the progression-related genes encode molecules that are the target of established or experimental therapies, our results may hopefully contribute to the treatment of end-stage CMM patients.

Cutaneous Malignant Melanoma (CMM) is the most malignant skin tumour in humans, the incidence of which is rising rapidly in most fair-skinned populations, without apparent decline in mortality. Both hereditary, constitutional and environmental factors play a role in its etiology. CMM arises from melanocytes in the epidermis, and proceeds through discrete steps of tumor-progression that consist histologically of the radial growth phase (RGP), vertical growth phase (VGP) and metastatic phase. The underlying molecular mechanisms that govern the transition between these growth phases are hardly known. The prognosis of patients with VGP melanoma depends on several clinical and histological parameters; the latter include thickness, mitotic activity, presence or absence of ulceration and regression, and pattern of lymphocytic host response. However, there is still need for new prognostic parameters. To obtain insight in the molecular mechanisms of tumor progression in CMM, and in search of new prognostic markers, we performed global gene-expression profiling using 44K oligonucleotide micro-arrays on a unique retrospective series of 83 frozen primary MM with VGP, 9 metastases and 23 benign nevi. Unsupervised analysis allowed us o identify clusters of melanoma patients with different outcome based on their gene expression profile only. Supervised analysis resulted in the identification of a genomic signature of 254 genes with prognostic significance. The large majority of the 254 enes was correlated with thickness, thereby stressing the importance of thickness in he prognosis of CMM. This signature was validated on a separate series of melanoma patients, and proved to have a predictive accuracy comparable to what can be obtained by tumour thickness and ulceration. On an immunohistochemical level, we identified 8 new markers that may help in the prognostication of melanoma patients; three of these markers, i.e. the mini-chromosome maintenance (mcm) proteins mcm3, 4 and 6, hat are involved in DNA-replication, had independent prognostic value. Additionally, upervised analysis showed similarities in gene expression profile between primary CMM and their metastases. In conclusion, our data provide new information regarding the molecules that are operative in the progression of CMM. CMM is notorious for its resistance to chemotherapy, and disseminated CMM is a uniformly fatal disease. As several of the progression-related genes, encode molecules that have been the target of established or xperimental cancer therapies, our results may hopefully contribute to the treatment of end-stage CMM-patients.

Background: Gene expression profiling data for human primary cutaneous melanomas are scarce because of the lack of retrospective collections of frozen tumors. To identify differentially expressed genes that may be involved in melanoma progression and prognosis, we investigated the relationship between gene expression profiles and clinical outcome in a cohort of patients with primary melanoma., Methods: Labeled complementary RNA (cRNA) from each tissue sample was hybridized to a pangenomic 44K 60-mer oligonucleotide microarray. Class comparison and class prediction analyses were performed to identify genes whose expression in primary melanomas was associated with 4-year distant metastasis-free survival among 58 patients with at least 4 years of follow-up, distant metastasis, or death. Results were validated immunohistochemically at the protein level in 176 independent primary melanomas from patients with a median clinical follow-up of 8.5 years. Survival was analyzed with a Cox multivariable model and stratified log-rank test. All statistical tests were two-sided., Results: We identified 254 genes that were associated with distant metastasis-free survival of patients with primary melanoma. These 254 genes include genes involved in activating DNA replication origins, such as minichromosome maintenance genes and geminin. Twenty-three of these genes were studied at the protein level; expression of five (MCM4, P = .002; MCM3, P = .030; MCM6, P = .004; KPNA2, P = .021; and geminin, P = .004) was statistically significantly associated with overall survival in the validation set. In a multivariable Cox model adjusted for tumor thickness, ulceration, age, and sex, expression of MCM4 (hazard ratio [HR] of death = 4.04, 95% confidence interval [CI] = 1.39 to 11.76; P = .010) and MCM6 (HR of death = 7.42, 95% CI = 1.99 to 27.64; P = .003) proteins was still statistically significantly associated with overall survival., Conclusion: We identified 254 genes whose expression was associated with metastatic dissemination of cutaneous melanomas. These genes may shed light on the molecular mechanisms underlying poor prognosis in melanoma patients.