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Among skin epithelial tumors, recurrent mutations in the APC/CTNNB1 genes resulting in activation of the Wnt/ß-catenin pathway have been reported predominantly in neoplasms with matrical differentiation. In the present study, we describe the morphologic, immunohistochemical, and genetic features of 16 primary cutaneous carcinomas harboring mutations activating the Wnt/ß-catenin pathway without evidence of matrical differentiation, as well as four combined tumors in which a similar Wnt/ß-catenin activated carcinoma component was associated with Merkel cell carcinoma or pilomatrical carcinoma. Among the pure tumor cases, 6/16 patients were female with a median age of 80 years (range: 58-98). Tumors were located on the head and neck (n=7, 44%), upper limb (n=4, 25%), trunk (n=3, 18%), and leg (n=2, 13%). Metastatic spread was observed in 4 cases resulting in death from disease in one patient. Microscopically, all cases were poorly differentiated neoplasms infiltrating the dermis and/or subcutaneous tissue. In 13 cases, solid "squamoid" areas were associated with a basophilic component characterized by rosette/pseudoglandular formation resulting in a biphasic appearance. Three specimens consisted only of poorly differentiated carcinoma lacking rosette formation. Immunohistochemical studies showed frequent expression of EMA (100%), BerEP4 (100%), cytokeratin 7 (94%), chromogranin A (44%), synaptophysin (82%) and cytokeratin 20 (69%). Complete loss of Rb expression was observed in all but one case. Nuclear ß-catenin and CDX2 expressions were detected in all cases. Recurrent pathogenic somatic mutations were observed in APC (60%), CTNNB1 (40%) and RB1 (n=47%). Global methylation analysis confirmed that cases with rosette formation constituted a homogenous tumor group distinct from established skin tumor entities (pilomatrical carcinoma, Merkel cell carcinoma and squamous cell carcinoma) while the 3 other cases lacking such morphologic features did not. In addition, we identified four combined neoplasms in which there was a component showing a similar poorly differentiated rosette forming carcinoma demonstrating Rb loss and beta-catenin activation associated with either Merkel cell carcinoma (n=3) or pilomatrical carcinoma (n=1). In conclusion, we describe a distinctive neoplasm, for which we propose the term "Wnt/ß-catenin activated rosette-forming carcinoma", morphologically characterized by the association of rosette formation, squamous and/or neuroendocrine differentiation, diffuse CDX2 expression, Rb loss, and mutations in CTNNB1/APC genes.
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Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.
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Antígenos Transformantes de Poliomavirus , Antígenos Virais de Tumores , Poliomavírus das Células de Merkel , Células-Tronco Neoplásicas , Proteínas de Ligação a Retinoblastoma , Humanos , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/virologia , Carcinoma de Célula de Merkel/genética , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Poliomavírus das Células de Merkel/genética , Células-Tronco Neoplásicas/virologia , Células-Tronco Neoplásicas/metabolismo , Neurônios/virologia , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Neoplasias Cutâneas/virologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismoRESUMO
Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by genomic integration of the Merkel cell polyomavirus (MCPyV). MCPyV-negative cases often present as combined MCCs, which represent a distinctive subset of tumors characterized by association of an MCC with a second tumor component, mostly squamous cell carcinoma. Up to now, only exceptional cases of combined MCC with neuroblastic differentiation have been reported. Herein we describe two additional combined MCCs with neuroblastic differentiation and provide comprehensive morphologic, immunohistochemical, transcriptomic, genetic and epigenetic characterization of these tumors, which both arose in elderly men and appeared as an isolated inguinal adenopathy. Microscopic examination revealed biphasic tumors combining a poorly differentiated high-grade carcinoma with a poorly differentiated neuroblastic component lacking signs of proliferation. Immunohistochemical investigation revealed keratin 20 and MCPyV T antigen (TA) in the MCC parts, while neuroblastic differentiation was confirmed in the other component in both cases. A clonal relation of the two components can be deduced from 20 and 14 shared acquired point mutations detected by whole exome analysis in both combined tumors, respectively. Spatial transcriptomics demonstrated a lower expression of stem cell marker genes such as SOX2 and MCM2 in the neuroblastic component. Interestingly, although the neuroblastic part lacked TA expression, the same genomic MCPyV integration and the same large T-truncating mutations were observed in both tumor parts. Given that neuronal transdifferentiation upon TA repression has been reported for MCC cell lines, the most likely scenario for the two combined MCC/neuroblastic tumors is that neuroblastic transdifferentiation resulted from loss of TA expression in a subset of MCC cells. Indeed, DNA methylation profiling suggests an MCC-typical cellular origin for the combined MCC/neuroblastomas. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Antígenos Virais de Tumores , Carcinoma de Célula de Merkel , Transdiferenciação Celular , Poliomavírus das Células de Merkel , Neoplasias Cutâneas , Humanos , Carcinoma de Célula de Merkel/patologia , Carcinoma de Célula de Merkel/virologia , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/metabolismo , Masculino , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/virologia , Neoplasias Cutâneas/metabolismo , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Poliomavírus das Células de Merkel/genética , Pontos de Checagem do Ciclo Celular/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Idoso de 80 Anos ou mais , Idoso , Neoplasias Complexas Mistas/patologia , Neoplasias Complexas Mistas/genética , Neoplasias Complexas Mistas/metabolismo , Neuroblastoma/patologia , Neuroblastoma/genética , Neuroblastoma/metabolismoRESUMO
Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high mortality rate. Merkel cell polyomavirus causes 80% of MCCs, encoding the viral oncogenes small T and truncated large T (tLT) antigens. These proteins impair the RB1-dependent G1/S checkpoint blockade and subvert the host cell epigenome to promote cancer. Whole-proteome analysis and proximal interactomics identified a tLT-dependent deregulation of DNA damage response (DDR). Our investigation revealed, to our knowledge, a previously unreported interaction between tLT and the histone methyltransferase EHMT2. T antigen knockdown reduced DDR protein levels and increased the levels of the DNA damage marker γH2Ax. EHMT2 normally promotes H3K9 methylation and DDR signaling. Given that inhibition of EHMT2 did not significantly change the MCC cell proteome, tLT-EHMT2 interaction could affect the DDR. With tLT, we report that EHMT2 gained DNA damage repair proximal interactors. EHMT2 inhibition rescued proliferation in MCC cells depleted for their T antigens, suggesting impaired DDR and/or lack of checkpoint efficiency. Combined tLT and EHMT2 inhibition led to altered DDR, evidenced by multiple signaling alterations. In this study, we show that tLT hijacks multiple components of the DNA damage machinery to enhance tolerance to DNA damage in MCC cells, which could explain the genetic stability of these cancers.
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Immunotherapy has achieved tremendous success in melanoma. However, only around 50% of advanced melanoma patients benefit from immunotherapy. Cyclin-dependent kinase inhibitor 2A (CDKN2A), encoding the two tumor-suppressor proteins p14ARF and p16INK4a, belongs to the most frequently inactivated gene loci in melanoma and leads to decreased T cell infiltration. While the role of p16INK4a has been extensively investigated, knowledge about p14ARF in melanoma is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF in melanoma cell lines diminished their recognition and killing by melanoma differentiation antigen (MDA)-specific T cells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. Immunopeptidomic analyses revealed that antigen presentation via human leukocyte antigen class I (HLA-I) molecules was enhanced upon p14ARF downregulation in general, but absolute and relative expression of cognate peptides was decreased. However, this phenotype is associated with a favorable outcome for melanoma patients. Limiting Wnt5a signaling reverted this phenotype, suggesting an involvement of non-canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA-specific T cell responses by decreasing both absolute and relative MDA-peptide presentation in melanoma.
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Merkel cell carcinoma is a rare, aggressive skin cancer that mainly occurs in elderly and immunocompromised patients. Due to the success of immune checkpoint inhibition in MCC, the importance of immunotherapy and vaccines in MCC has increased in recent years. In this article, we aim to present the current progress and perspectives in the development of vaccines for this disease. Here, we summarize and discuss the current literature and ongoing clinical trials investigating vaccines against MCC. We identified 10 articles through a PubMed search investigating a vaccine against MCC. From the international clinical trial database Clinical.Trials.gov, we identified nine studies on vaccines for the management of MCC, of which seven are actively recruiting. Most of the identified studies investigating a vaccine against MCC are preclinical or phase 1/2 trials. The vaccine principles mainly included DNA- and (synthetic) peptide-based vaccines, but RNA-based vaccines, oncolytic viruses, and the combination of vaccines and immunotherapy are also under investigation for the treatment of MCC. Although the management of MCC is changing, when compared to times before the approval of immune checkpoint inhibitors, it will still take some time before the first MCC vaccine is ready for approval.
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Elevated levels of peripheral blood and tumor tissue neutrophils are associated with poorer clinical response and therapy resistance in melanoma. The underlying mechanism and the role of neutrophils in targeted therapy is still not fully understood. Serum samples of patients with advanced melanoma were collected and neutrophil-associated serum markers were measured and correlated with response to targeted therapy. Blood neutrophils from healthy donors and patients with advanced melanoma were isolated, and their phenotypes, as well as their in vitro functions, were compared. In vitro functional tests were conducted through nonadherent cocultures with melanoma cells. Protection of melanoma cell lines by neutrophils was assessed under MAPK inhibition. Blood neutrophils from advanced melanoma patients exhibited lower CD16 expression compared to healthy donors. In vitro, both healthy-donor- and patient-derived neutrophils prevented melanoma cell apoptosis upon dual MAPK inhibition. The effect depended on cell-cell contact and melanoma cell susceptibility to treatment. Interference with protease activity of neutrophils prevented melanoma cell protection during treatment in cocultures. The negative correlation between neutrophils and melanoma outcomes seems to be linked to a protumoral function of neutrophils. In vitro, neutrophils exert a direct protective effect on melanoma cells during dual MAPK inhibition. This study further hints at a crucial role of neutrophil-related protease activity in protection.
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Merkel cell carcinoma (MCC) is a rare skin cancer characterized by neuroendocrine differentiation. Its carcinogenesis is based either on the integration of the Merkel cell polyomavirus or on ultraviolet (UV) mutagenesis, both of which lead to high immunogenicity either through the expression of viral proteins or neoantigens. Despite this immunogenicity resulting from viral or UV-associated carcinogenesis, it exhibits highly aggressive behavior. However, owing to the rarity of MCC and the lack of epidemiologic registries with detailed clinical data, there is some uncertainty regarding the spontaneous course of the disease. Historically, advanced MCC patients were treated with conventional cytotoxic chemotherapy yielding a median response duration of only 3 months. Starting in 2017, four programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors-avelumab, pembrolizumab, nivolumab (utilized in both neoadjuvant and adjuvant settings), and retifanlimab-have demonstrated efficacy in treating patients with disseminated MCC on the basis of prospective clinical trials. However, generating clinical evidence for rare cancers, such as MCC, is challenging owing to difficulties in conducting large-scale trials, resulting in small sample sizes and therefore lacking statistical power. Thus, to comprehensively understand the available clinical evidence on various immunotherapy approaches for MCC, we also delve into the epidemiology and immune biology of this cancer. Nevertheless, while randomized studies directly comparing immune checkpoint inhibitors and chemotherapy in MCC are lacking, immunotherapy shows response rates comparable to those previously reported with chemotherapy but with more enduring responses. Notably, adjuvant nivolumab has proven superiority to the standard-of-care therapy (observation) in the adjuvant setting.
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Carcinoma de Célula de Merkel , Inibidores de Checkpoint Imunológico , Neoplasias Cutâneas , Carcinoma de Célula de Merkel/terapia , Carcinoma de Célula de Merkel/epidemiologia , Carcinoma de Célula de Merkel/imunologia , Carcinoma de Célula de Merkel/diagnóstico , Humanos , Neoplasias Cutâneas/terapia , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Poliomavírus das Células de Merkel/imunologiaRESUMO
AIMS: Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV). Characteristic for these virus-positive (VP) MCC is MCPyV integration into the host genome and truncation of the viral oncogene Large T antigen (LT), with full-length LT expression considered as incompatible with MCC growth. Genetic analysis of a VP-MCC/trichoblastoma combined tumour demonstrated that virus-driven MCC can arise from an epithelial cell. Here we describe two further cases of VP-MCC combined with an adnexal tumour, i.e. one trichoblastoma and one poroma. METHODS AND RESULTS: Whole-genome sequencing of MCC/trichoblastoma again provided evidence of a trichoblastoma-derived MCC. Although an MCC-typical LT-truncating mutation was detected, we could not determine an integration site and we additionally detected a wildtype sequence encoding full-length LT. Similarly, Sanger sequencing of the combined MCC/poroma revealed coding sequences for both truncated and full-length LT. Moreover, in situ RNA hybridization demonstrated expression of a late region mRNA encoding the viral capsid protein VP1 in both combined as well as in a few cases of pure MCC. CONCLUSION: The data presented here suggest the presence of wildtype MCPyV genomes and VP1 transcription in a subset of MCC.
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Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Poroma , Neoplasias Cutâneas , Neoplasias das Glândulas Sudoríparas , Humanos , Carcinoma de Célula de Merkel/metabolismo , Poliomavírus das Células de Merkel/genética , Infecções por Polyomavirus/complicações , Neoplasias Cutâneas/patologia , GenômicaRESUMO
Poroma is a benign sweat gland tumour showing morphological features recapitulating the superficial portion of the eccrine sweat coil. A subset of poromas may transform into porocarcinoma, its malignant counterpart. Poroma and porocarcinoma are characterised by recurrent gene fusions involving YAP1, a transcriptional co-activator, which is controlled by the Hippo signalling pathway. The fusion genes frequently involve MAML2 and NUTM1, which are also rearranged in other cutaneous and extracutaneous neoplasms. We aimed to review the clinical, morphological and molecular features of this category of adnexal neoplasms with a special focus upon emerging differential diagnoses, and discuss how their systematic molecular characterisation may contribute to a standardisation of diagnosis, more accurate classification and, ultimately, refinement of their prognosis and therapeutic modalities.
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Porocarcinoma Écrino , Poroma , Neoplasias Cutâneas , Neoplasias das Glândulas Sudoríparas , Humanos , Poroma/genética , Poroma/metabolismo , Poroma/patologia , Porocarcinoma Écrino/genética , Porocarcinoma Écrino/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias das Glândulas Sudoríparas/diagnóstico , Pele/patologia , Fatores de Transcrição/genéticaRESUMO
INTRODUCTION: Merkel cell carcinoma (MCC) is a rare, highly aggressive form of skin cancer with neuroendocrine features. The origin of this cancer is still unclear, but research in the last 15 years has demonstrated that MCC arises via two distinct etiologic pathways, i.e. virus and UV-induced. Considering the high mortality rate and the limited therapeutic options available, this review aims to highlight the significance of MCC research and the need for advancement in MCC treatment. AREAS COVERED: With the advent of the immune checkpoint inhibitor therapies, we now have treatment options providing a survival benefit for patients with advanced MCC. However, the issue of primary and acquired resistance to these therapies remains a significant concern. Therefore, ongoing efforts seeking additional therapeutic targets and approaches for MCC therapy are a necessity. Through a comprehensive literature search, we provide an overview on recent preclinical and clinical studies with respect to MCC therapy. EXPERT OPINION: Currently, the only evidence-based therapy for MCC is immune checkpoint blockade with anti-PD-1/PD-L1 for advanced patients. Neoadjuvant, adjuvant and combined immune checkpoint blockade are promising treatment options.
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Carcinoma de Célula de Merkel , Neoplasias Cutâneas , Humanos , Carcinoma de Célula de Merkel/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Imunoterapia/métodosRESUMO
BACKGROUND: The mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival. METHODS: RSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses. RESULTS: In line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing. CONCLUSIONS: Collectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.
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Melanoma , Proteínas Quinases S6 Ribossômicas 90-kDa , Humanos , Animais , Camundongos , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Proto-Oncogênicas B-raf , Evasão da Resposta Imune , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ciclo Celular , Melanoma Maligno CutâneoRESUMO
Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV), and MCPyV-positive tumor cells depend on expression of the virus-encoded T antigens (TA). Here, we identify 4-[(5-methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone (PHT)-a reported inhibitor of Aurora kinase A-as a compound inhibiting growth of MCC cells by repressing noncoding control region (NCCR)-controlled TA transcription. Surprisingly, we find that TA repression is not caused by inhibition of Aurora kinase A. However, we demonstrate that ß-catenin-a transcription factor repressed by active glycogen synthase kinase 3 (GSK3)-is activated by PHT, suggesting that PHT bears a hitherto unreported inhibitory activity against GSK3, a kinase known to function in promoting TA transcription. Indeed, applying an in vitro kinase assay, we demonstrate that PHT directly targets GSK3. Finally, we demonstrate that PHT exhibits in vivo antitumor activity in an MCC xenograft mouse model, suggesting a potential use in future therapeutic settings for MCC.
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Merkel cell carcinoma (MCC) is an aggressive skin cancer for which Merkel cell polyomavirus integration and expression of viral oncogenes small T and Large T have been identified as major oncogenic determinants. Recently, a component of the PRC2 complex, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) that induces H3K27 trimethylation as a repressive mark has been proposed as a potential therapeutic target in MCC. Because divergent results have been reported for the levels of EZH2 and trimethylation of lysine 27 on histone 3, we analyzed these factors in a large MCC cohort to identify the molecular determinants of EZH2 activity in MCC and to establish MCC cell lines' sensitivity to EZH2 inhibitors. Immunohistochemical expression of EZH2 was observed in 92% of MCC tumors (156 of 170), with higher expression levels in virus-positive than virus-negative tumors (P = 0.026). For the latter, we showed overexpression of EZHIP, a negative regulator of the PRC2 complex. In vitro, ectopic expression of the large T antigen in fibroblasts led to the induction of EZH2 expression, whereas the knockdown of T antigens in MCC cell lines resulted in decreased EZH2 expression. EZH2 inhibition led to selective cytotoxicity on virus-positive MCC cell lines. This study highlights the distinct mechanisms of EZH2 induction between virus-negative and -positive MCC.
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Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Neoplasias Cutâneas , Humanos , Carcinoma de Célula de Merkel/patologia , Histonas/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neoplasias Cutâneas/patologia , Poliomavírus das Células de Merkel/genética , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismoRESUMO
The best characterized polyomavirus family member, i.e., simian virus 40 (SV40), can cause different tumors in hamsters and can transform murine and human cells in vitro. Hence, the SV40 contamination of millions of polio vaccine doses administered from 1955-1963 raised fears that this may cause increased tumor incidence in the vaccinated population. This is, however, not the case. Indeed, up to now, the only polyomavirus family member known to be the most important cause of a specific human tumor entity is Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma (MCC). MCC is a highly deadly form of skin cancer for which the cellular origin is still uncertain, and which appears as two clinically very similar but molecularly highly different variants. While approximately 80% of cases are found to be associated with MCPyV the remaining MCCs carry a high mutational load. Here, we present an overview of the multitude of molecular functions described for the MCPyV encoded oncoproteins and non-coding RNAs, present the available MCC mouse models and discuss the increasing evidence that both, virus-negative and -positive MCC constitute epithelial tumors.
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AIMS: Recently, YAP1 fusion genes have been demonstrated in eccrine poroma and porocarcinoma, and the diagnostic use of YAP1 immunohistochemistry has been highlighted in this setting. In other organs, loss of YAP1 expression can reflect YAP1 rearrangement or transcriptional repression, notably through RB1 inactivation. In this context, our objective was to re-evaluate the performance of YAP1 immunohistochemistry for the diagnosis of poroma and porocarcinoma. METHODS AND RESULTS: The expression of the C-terminal part of the YAP1 protein was evaluated by immunohistochemistry in 543 cutaneous epithelial tumours, including 27 poromas, 14 porocarcinomas and 502 other cutaneous tumours. Tumours that showed a lack of expression of YAP1 were further investigated for Rb by immunohistochemistry and for fusion transcripts by real-time PCR (YAP1::MAML2 and YAP1::NUTM1). The absence of YAP1 expression was observed in 24 cases of poroma (89%), 10 porocarcinoma (72%), 162 Merkel cell carcinoma (98%), 14 squamous cell carcinoma (SCC) (15%), one trichoblastoma and one sebaceoma. Fusions of YAP1 were detected in only 16 cases of poroma (n = 66%), 10 porocarcinoma (71%) all lacking YAP1 expression, and in one sebaceoma. The loss of Rb expression was detected in all cases except one of YAP1-deficient SCC (n = 14), such tumours showing significant morphological overlap with porocarcinoma. In-vitro experiments in HaCat cells showed that RB1 knockdown resulted in repression of YAP1 protein expression. CONCLUSION: In addition to gene fusion, we report that transcriptional repression of YAP1 can be observed in skin tumours with RB1 inactivation, including MCC and a subset of SCC.
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Carcinoma , Porocarcinoma Écrino , Poroma , Neoplasias Cutâneas , Neoplasias das Glândulas Sudoríparas , Humanos , Poroma/genética , Poroma/metabolismo , Poroma/patologia , Neoplasias das Glândulas Sudoríparas/diagnóstico , Porocarcinoma Écrino/genética , Porocarcinoma Écrino/patologia , Neoplasias Cutâneas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismoRESUMO
Defects in DNA repair pathways have been associated with an improved response to immune checkpoint inhibition (ICI). In particular, patients with the nucleotide excision repair (NER) defect disease Xeroderma pigmentosum (XP) responded impressively well to ICI treatment. Recently, in melanoma patients, pretherapeutic XP gene expression was predictive for anti-programmed cell death-1 (PD-1) ICI response. The underlying mechanisms of this finding are still to be revealed. Therefore, we used CRISPR/Cas9 to disrupt XPA in A375 melanoma cells. The resulting subclonal cell lines were investigated by Sanger sequencing. Based on their genetic sequence, candidates from XPA exon 1 and 2 were selected and further analyzed by immunoblotting, immunofluorescence, HCR and MTT assays. In XPA exon 1, we established a homozygous (c.19delG; p.A7Lfs*8) and a compound heterozygous (c.19delG/c.19_20insG; p.A7Lfs*8/p.A7Gfs*55) cell line. In XPA exon 2, we generated a compound heterozygous mutated cell line (c.206_208delTTG/c.208_209delGA; p.I69_D70delinsN/p.D70Hfs*31). The better performance of the homozygous than the heterozygous mutated exon 1 cells in DNA damage repair (HCR) and post-UV-C cell survival (MTT), was associated with the expression of a novel XPA protein variant. The results of our study serve as the fundamental basis for the investigation of the immunological consequences of XPA disruption in melanoma.
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Melanoma , Proteína de Xeroderma Pigmentoso Grupo A , Xeroderma Pigmentoso , Sistemas CRISPR-Cas/genética , Dano ao DNA , Reparo do DNA/genética , Éxons/genética , Humanos , Inibidores de Checkpoint Imunológico , Melanoma/genética , Receptor de Morte Celular Programada 1/metabolismo , Raios Ultravioleta , Xeroderma Pigmentoso/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
MCC (Merkel cell carcinoma) is an aggressive neuroendocrine cutaneous neoplasm. Integration of the Merkel cell polyomavirus (MCPyV) is observed in about 80% of the cases, while the remaining 20% are related to UV exposure. Both MCPyV-positive and -negative MCCs-albeit by different mechanisms-are associated with RB1 inactivation leading to overexpression of SOX2, a major contributor to MCC biology. Moreover, although controversial, loss of RB1 expression seems to be restricted to MCPyV-negative cases.The aim of the present study was to assess the performances of RB1 loss and SOX2 expression detected by immunohistochemistry to determine MCPyV status and to diagnose MCC, respectively.Overall, 196 MCC tumors, 233 non-neuroendocrine skin neoplasms and 70 extra-cutaneous neuroendocrine carcinomas (NEC) were included. SOX2 and RB1 expressions were assessed by immunohistochemistry in a tissue micro-array. Diagnostic performances were determined using the likelihood ratio (LHR).RB1 expression loss was evidenced in 27% of the MCC cases, 12% of non-neuroendocrine skin tumors and 63% of extra-cutaneous NEC. Importantly, among MCC cases, RB1 loss was detected in all MCPyV(-) MCCs, while MCPyV( +) cases were consistently RB1-positive (p < 0.001). SOX2 diffuse expression was observed in 92% of the MCC cases and almost never observed in non-neuroendocrine skin epithelial neoplasms (2%, p < 0.0001, LHR + = 59). Furthermore, SOX2 diffuse staining was more frequently observed in MCCs than in extra-cutaneous NECs (30%, p < 0.001, LHR + = 3.1).These results confirm RB1 as a robust predictor of MCC viral status and further suggest SOX2 to be a relevant diagnostic marker of MCC.