Novel cellular systems unveil mucosal melanoma initiating cells and a role for PI3K/Akt/mTOR pathway in mucosal melanoma fitness.

BACKGROUND: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. METHODS: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. RESULTS: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. CONCLUSIONS: Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Molecular investigation of some DNA viruses in mucosal melanoma: Case-control study.

The association between viral infections and both cutaneous and mucosal melanoma (MM) has not been fully investigated. Here, we assessed the prevalence of the DNA of a broad range of viruses in 31 MMs and 15 biopsies of healthy mucosa (HM) using molecular methods. The parvoviruses CuV and B19V, herpesviruses HSV1, HSV2, EBV, HHV6, and HHV8, polyomavirus MCPyV, and α-HPVs were not detected, or rarely found, in MMs, and in HM, of the digestive, respiratory, and female genital tract. The overall prevalence of ß-HPV in MMs was not significantly higher compared to that in HM (70.9% and 53.3% respectively; p = 0.514). However, the number of MMs positive for ß-HPV types belonging to Species 3 and 5 and for some viral types belonging to Species 1, 2, 3, and 5 were significantly higher compared with HM (p < 0.05). Moreover, compared to HM, the MM samples contained a significantly higher number of ß-HPV types, mainly belonging to Species 1, 3, and 5 (p < 0.05). Our data, although suggesting a role for certain ß-HPV types in MM oncogenesis, require additional investigation in larger populations to support this hypothesis.

Current Trends in Mucosal Melanomas: An Overview.

Primary mucosal melanomas (MMs) are uncommon tumors originating from melanocytes located in the mucous membranes at various anatomic sites within the body. MM significantly differs from cutaneous melanoma (CM) regarding epidemiology, genetic profile, clinical presentation, and response to therapies. Despite these differences, that have important implications for both disease diagnosis and prognosis, MMs are usually treated in the same way as CM but exhibit a lower response rate to immunotherapy leading to a poorer survival rate. Furthermore, a high inter-patient variability can be observed in relation to therapeutic response. Recently, novel "omics" techniques have evidenced that MM lesions have different genomic, molecular, and metabolic landscapes as compared with CM lesions, thus explaining the heterogeneity of the response. Such specific molecular aspects might be useful to identify new biomarkers aimed at improving the diagnosis and selection of MM patients who could benefit from immunotherapy or targeted therapy. In this review, we have focused on relevant molecular and clinical advancements for the different MM subtypes in order to describe the updated knowledge relating to main diagnostic, clinical, and therapeutic implications as well as to provide hints on likely future directions.

Metabolic heterogeneity protects metastatic mucosal melanomas cells from ferroptosis.

Cancer heterogeneity has been proposed to be one of the main causes of metastatic dissemination and therapy failure. However, the underlying mechanisms of this phenomenon remain poorly understood. Melanoma is an aggressive malignancy with a high heterogeneity and metastatic potential. Therefore, the present study investigated the possible association between cancer heterogeneity and metastasis in melanoma. In total, two novel Chinese oral mucosal melanoma (COMM) cell lines, namely COMM­1 and COMM­2, were established for exploring methods into preventing the loss of cellular heterogeneity caused by long­term cell culture. Each cell line was grown under two different models of culture, which yielded two subtypes, one exhibited an adhesive morphology (COMM­AD), whereas the other was grown in suspension (COMM­SUS). Compared with the COMM­AD cells, the COMM­SUS cells exhibited higher metastatic capacities and autofluorescence. Further investigations indicated that the COMM­SUS cells exhibited metabolic reprogramming by taking up lactate produced by COMM­AD cells at increased levels to accumulate NADH through monocarboxylate transporter 1, whilst also increasing NADPH levels through the pentose phosphate pathway (PPP). Additionally, increased NADH and NADPH levels in the COMM­SUS cells, coupled with the upregulation of the anti­ferroptotic proteins, glutathione peroxidase 4 and ferrop-tosis suppressor protein 1, enabled them to resist ferroptotic cell death induced by oxidative stress during hematogenous dissemination. The inhibition of ferroptosis was found to substantially increase the metastatic capacity of COMM­AD cells. Furthermore, suppressing lactate uptake and impairing PPP activation significantly decreased the metastatic potential of the COMM­SUS cells. Thus, the present study on metabolic heterogeneity in COMM cells potentially provides a novel perspective for exploring this mechanism underlying cancer metastasis.

Expression of mitochondrial dynamics markers during melanoma progression: Comparative study of head and neck cutaneous and mucosal melanomas.

BACKGROUND: Head and neck mucosal melanomas (MMs) are rare tumors with adverse outcomes and poorer prognoses than their more common cutaneous counterparts (cutaneous melanomas-CMs). Few studies have compared the expression of mitochondrial dynamic markers in these tumors. This study aimed to assess the correlations of mitochondrial markers with melanoma progression and their potential as predictors of lymph node involvement and distant metastasis. METHODS: Immunohistochemistry against anti-mitochondrial (AMT), dynamin-related protein 1 (DRP1), mitochondrial fission protein 1 (FIS1), mitofusin-1 (MFN1), and mitofusin-2 (MFN2) antibodies was performed in 112 cases of head and neck CM and MM. A Cox regression multivariate model was used to assess the correlation of AMT, FIS1, and MFN2 expressions considering the risk for nodal and distant metastasis. RESULTS: All markers studied presented higher staining in tumor cells than normal adjacent tissues. Higher mitochondrial content was observed in MM than in CM, and it was significantly associated with nodal metastasis in oral melanomas. Both FIS1 and DRP1 expressions were related to advanced Clark's levels in CM, and they were overexpressed in oral melanomas. Moreover, increased immunoexpression of MFN2 was significantly associated with a higher risk of metastasis in CM, and it was also overexpressed in sinonasal melanomas. CONCLUSIONS: Our results suggest that mitochondrial fission and fusion processes can play an important role during multiple stages of tumorigenesis and the development of nodal and distant metastasis in cutaneous and mucosal melanomas.

Prognostic importance of mitochondrial markers in mucosal and cutaneous head and neck melanomas.

Mitochondrial dysfunction is caused by an imbalance in the fission and fusion processes, and it has been implicated in the pathogenesis of several human cancers. However, the role of mitochondrial markers in melanomas still remains poorly understood. In this study, the authors assessed the expression of 3 mitochondrial markers (antimitochondrial, fission protein 1 [FIS1], and mitofusin 2 [MFN2]) in a series of head and neck mucosal and cutaneous melanomas. Patients with cutaneous (n = 56) and mucosal (oral, n = 30, sinonasal, n = 26) melanomas of the head and neck region were enrolled in this study. Clinical and follow-up data were retrieved from medical records. The expression of 3 mitochondrial markers was assessed by the immunohistochemistry, and then digitally quantified and correlated with clinicopathological data and outcome information. In the multivariate model, high mitochondrial content was identified as an independent prognostic value for disease-free survival (DFS) in cutaneous melanomas and overall survival in oral melanomas. FIS1 expression was significantly associated with lower overall survival rates in patients with oral melanomas and strictly correlated with vascular invasion in mucosal melanomas. MFN2 was associated with high risk of distant metastasis in patients with cutaneous melanomas. In summary, the authors demonstrated that mitochondrial content, along with FIS1 and MFN2 expressions, is correlated with important clinicopathological characteristics in patients with cutaneous and mucosal head and neck melanomas.

Phosphorylated Akt1 expression is associated with poor prognosis in cutaneous, oral and sinonasal melanomas.

Melanomas are highly aggressive tumours derived from melanocytes, which occur most commonly in the skin. Occasionally, these tumours may appear in oral and sinonasal mucous membranes. In this study, we performed a comparative analysis of the Phosphorylated Akt1 (p-Akt1) expression in 144 patients affected by cutaneous (CM), 34 oral cavity (OM), and 31 sinonasal melanomas (SNM). Similar to the metastatic cutaneous melanomas, p-Akt1 was overexpressed in 17/34 of the oral cavity and 20/31 of the sinonasal melanomas. In addition, the p-Akt1-nuclear expression was associated with poorer cancer-specific survival in cutaneous (P < .0001), oral (P < .0001), and sinonasal (P = .001) melanomas. Multivariate analysis showed p-Akt1 to be an independent prognostic marker in oral (P = .041) and sinonasal (P < .0001) melanomas patients. In conclusion, p-Akt1 overexpression is an independent prognostic marker in mucosal melanomas and is significantly up-regulated in sinonasal melanomas. As both mucosal and metastatic cutaneous melanomas showed high frequency of p-Akt1 expression, these findings suggest that mucosal melanomas have a biological behaviour, similar to the aggressive cutaneous melanomas.

The clinical significance of c-Kit mutations in metastatic oral mucosal melanoma in China.

c-Kit mutations are frequently detected in mucosal melanomas, but their clinical significance in metastatic oral mucosal melanomas (OMM) remains unclear. The main purpose of this study was to investigate the clinical and pathological features of metastatic OMMs with c-Kit mutations and the efficiency of the tyrosine kinase inhibitor imatinib in treating metastatic OMMs. We found thatresidual primary lesion and neck lymph nodes could act as independent prognostic factors in metastatic OMM patients. c-Kit mutations were detected in 22 out of 139 (15.8%) metastatic OMM patients. Under chemotherapy, the overall survival (OS) of c-Kit mutant patients was significantly shorter than that of wild-type patients. The Ki67 expression was significantly higher in c-Kit mutant patients than in wild-type patients. In distant metastatic OMM patients with c-Kit mutations, the treatment with c-Kit inhibitor resulted in a better OS. In conclusion, residual primary lesion, cervical lymph nodes and c-Kit mutations act as adverse prognostic factors of metastatic OMMs. The Kit inhibitor imatinib could benefit metastatic OMM patients with c-Kit mutations.

Ki67 Proliferation Index as a Histopathological Predictive and Prognostic Parameter of Oral Mucosal Melanoma in Patients without Distant Metastases.

Background: To investigate the relationship between clinical and histopathological characteristics and overall survival of patients with oral mucosal melanoma (OMM) without distal metastasis in order to provide predictive prognostic information of OMM. Methods: Ki67 expression was assessed by immunohistochemistry in 123 patients with OMM without distant metastases. The associations between Ki67 expression and clinical features and overall survival (OS) of patients were statistically analyzed. The Ki67 levels of the primary and recurrent lesions from 14 OMM patients were compared. Results: Univariate analysis showed that tumor type and cervical lymph node (CLN) status, as well as Ki67 expression, were all correlated with survival. Cox proportional hazards regression analysis identified Ki67 expression and CLN status as independent prognostic factors in OMM patients. Further, we found that Ki67 expression was associated with clinical tumor type of OMM. Moreover, with a cut-off point of 20%, patients with lower Ki67 scores showed a survival advantage over those with higher Ki67 scores. Conclusions: Ki67 expression may be a useful pathological predictor of survival of OMM patients without distant metastases.

Mucosal melanomas: Site-specific information, comparisons with cutaneous tumors, and differential diagnosis.

Melanoma of the skin is the fifth leading new cancer diagnosis, having accounted for almost 77,000 cases and more than 9000 deaths in the United States in 2013. Although cutaneous neoplasms of this type are relatively common, their mucosal counterparts are not. Mucosal melanomas comprise approximately 1.3% of all melanocytic malignancies. Although they are rare, these lesions present at an advanced stage with more adverse prognoses. In addition, at a molecular level, they have proven to be distinct entities because they possess genetic mutations not usually seen in their cutaneous counterparts. Conversely, a sizable proportion of mucosal melanomas lack the gene aberrations seen in cutaneous melanomas. Such findings indicate different pathways in tumorigenesis for the two subtypes. Because melanomas arising from the mucosae are not often encountered, very little has been published on staging guidelines and prognostic factors. This causes dilemmas for both patients and physicians. Further work is necessary to define staging systems for all mucosal locations, so that accurate prognoses can be assigned to such lesions.

Comparison of International Guidelines on Mucosal Melanoma of the Head and Neck: A Comprehensive Review of the Role of Radiation Therapy.

Mucosal melanomas of the head and neck are rare pathological entities that correlate with poor prognosis due to their high propensity for local failure and distant metastases. The exact role of radiation therapy in the management of mucosal melanoma patients has not yet been fully proven, even though in everyday clinical practice these patients are referred for radiotherapy, in an effort to improve locoregional control. The guidelines of various societies on the role of radiation therapy for the treatment of mucosal melanoma of the head and neck region are very limited. We reviewed and analyzed the guidelines developed in the U.S.A. (National Comprehensive Cancer Network), Canada (Cancer Care Ontario and Canadian Medical Association), Europe (European Society for Medical Oncology and European Society for Radiotherapy and Oncology) and Australia and New Zealand (Cancer Council Australia) and isolated evidence for the management of mucosal melanomas of the head and neck region with radiation therapy worldwide.

Primary amelanotic mucosal melanoma of the oronasal region: report of two new cases and literature review.

Primary amelanotic mucosal melanoma is a rare entity with challenging histopathological features. Because these tumors are thought to be biologically more aggressive, they have a poorer prognosis than that of pigmented melanomas. In this work, we present a literature review about the clinical, histopathological, and immunohistochemical features of primary amelanotic mucosal melanoma of the oronasal region and report two new cases. Amelanotic mucosal melanoma commonly affects men in the seventh decade of life and tend to have a poor prognosis, as seen by the high incidence of metastasis, recurrences, and, ultimately, death. There is a similar pattern in the clinic-pathological predilections (such as age, gender, primary site, and metastatic potential) of amelanotic mucosal melanoma when comparing with data reported for pigmented lesions. This work reinforces knowledge about amelanotic mucosal melanomas and epidemiologic predilections. The optimal management of this lesion remains controversial.

Estudo retrospectivo de melanomas cutâneos e mucosos na população do estado do Rio Grande do Norte, Brasil / Retrospective study of cutaneous and mucosal melanomas in the population of the state of Rio Grande do Norte, Brazil

INTRODUÇÃO E OBJETIVOS: O melanoma é uma neoplasia maligna que pode acometer a pele e as mucosas. A taxa de incidência do melanoma cutâneo de forma geral tem aumentado nos últimos anos. Esta pesquisa realizou uma análise retrospectiva de melanomas cutâneos e mucosos no período de 30 anos em um serviço do Rio Grande do Norte, enfatizando características clínicas, conduta e evolução dos melanomas mucosos orais. MATERIAL E MÉTODO: Nos prontuários clínicos, foram coletadas informações sobre idade, raça, sexo, localização o tipo de comprometimento da lesão, se primária ou metastática. RESULTADOS: Um total de 397 casos de melanoma foi diagnosticado, sendo 332 cutâneos (83,6 por cento) e 46 mucosos (11,6 por cento). O tronco foi mais acometido (27,7 por cento), seguido por membros inferiores (24,7 por cento) e cabeça e pescoço (18,9 por cento). Entre os melanomas mucosos, a maioria era de cabeça e pescoço (76,1 por cento). De forma geral, 52,4 por cento ocorreram em indivíduos do sexo masculino e 47,6 por cento, no feminino. A idade média foi de 54,9 anos. Nos homens esteve mais presente nas sétima e oitava décadas de vida, e nas mulheres, nas quinta e sexta décadas. Seis casos (1,5 por cento) acometeram a mucosa oral e estes ocorreram mais em indivíduos do sexo feminino, acima de 55 anos. CONCLUSÃO: O perfil epidemiológico dos casos de melanomas avaliados se assemelha aos poucos estudos epidemiológicos publicados, apesar de alguns dados, no tocante a sexo e idade em determinadas localizações anatômicas, terem variado e a incidência de melanomas orais ter sido bem mais baixa do que a relatada na maioria das pesquisas.

INTRODUCTION AND OBJECTIVES: Melanoma is a malignant neoplasm that occurs in the skin and mucosae. Overall, the incidence rate of cutaneous melanoma has increased in the last years. It was performed a retrospective analysis of cutaneous and mucosal melanomas diagnosed within a period of 30 years in one health care institution of Rio Grande do Norte, highlighting clinical features, approach and evolution of oral mucosal melanomas. METHODS: Data as to age, race, gender, tumor topography and lesion involvement (metastatic or primary) were collected from clinical records. RESULTS: A total of 397 cases of melanoma were diagnosed, 332 of them were cutaneous (83.6 percent) and 46 of them were mucosal (11.6 percent). The trunk was the most affected area (27.7 percent), followed by lower limbs (24.7 percent) and head/neck (18.9 percent). Within the mucosal melanomas, most cases were on head and neck (76.1 percent). By and large, 52.4 percent affected male individuals and 47.6 percent affected female patients. The mean age was 54.9. Among men it was more prevalent in the 7th and 8th decades of life, whereas among women it was more prevalent in the 5th and 6th decades of life. A total of 6 cases (1.5 percent) occurred in the oral mucosa, with more prevalence among female individuals over 55 years of age. CONCLUSIONS: The epidemiological profile of the analyzed cases of melanoma was similar to the few published epidemiological studies, although some data on gender and age in certain anatomical sites varied and the incidence of oral melanoma was much lower than that reported in most investigations.