Stemness and hybrid epithelial-mesenchymal profiles guide peritoneal dissemination of malignant mesothelioma and pseudomyxoma peritonei.

Intrabdominal dissemination of malignant mesothelioma (MM) and pseudomyxoma peritonei (PMP) is poorly characterized with respect to the stemness window which malignant cells activate during their reshaping on the epithelial-mesenchymal (E/M) axis. To gain insights into stemness properties and their prognostic significance in these rarer forms of peritoneal metastases (PM), primary tumor cultures from 55 patients selected for cytoreductive surgery with hyperthermic intraperitoneal chemotherapy were analyzed for cancer stem cells (CSC) by aldehyde dehydrogenase 1 (ALDH1) and spheroid formation assays, and for expression of a set of plasticity-related genes to measure E/M transition (EMT) score. Intratumor heterogeneity was also analyzed. Samples from PM of colorectal cancer were included for comparison. Molecular data were confirmed using principal component and cluster analyses. Associations with survival were evaluated using Kaplan-Meier and Cox regression models. The activity of acetylsalicylic acid (ASA), a stemness modifier, was tested in five cultures. Significantly increased amounts of ALDH1bright-cells identified high-grade PMP, and discriminated solid masses from ascitic/mucin-embedded tumor cells in both forms of PM. Epithelial/early hybrid EMT scores and an early hybrid expression pattern correlated with pluripotency factors were significantly associated with early peritoneal progression (p = .0343 and p = .0339, respectively, log-rank test) in multivariable models. ASA impaired spheroid formation and increased cisplatin sensitivity in all five cultures. These data suggest that CSC subpopulations and hybrid E/M states may guide peritoneal spread of MM and PMP. Stemness could be exploited as targetable vulnerability to increase chemosensitivity and improve patient outcomes. Additional research is needed to confirm these preliminary data.

Sentinel Lymph Node Biopsy in Atypical Spitz Tumor: A Systematic Review.

BACKGROUND: Atypical Spitz tumor (AST) is an intermediate category among Spitz melanocytic neoplasms. Sentinel node biopsy (SNB) has been proposed in the clinical management of AST patients, but this approach remains the subject of debate. This systematic review aims to summarize the available evidence on SNB procedures in AST patients. METHODS: A comprehensive search was conducted, including MEDLINE/Pubmed, EMBASE, and SCOPUS, through April 2023. Case series, cohort studies, and case-control studies of AST patients were eligible for inclusion. PRISMA guidelines were followed. RESULTS: Twenty-two studies with a total of 756 AST patients were included. The pooled SNB prevalence was 54% (95% CI 32 to 75%), with substantial heterogeneity (I2 90%). The pooled SNB+ prevalence was 35% (95% CI 25 to 46%) with moderate heterogeneity (I2 39%). Lymphadenectomy was performed in 0-100% of SNB+ patients. Overall survival rates ranged from 93% to 100%, and disease-free survival ranged from 87% to 100% in AST patients. Overall and disease-free survival rates were 100% in SNB patients. Pooled survival estimates were not calculated due to the heterogeneous timing of the survival assessment and/or the small size of the subgroups. All studies clearly reported inclusion criteria and measured the condition in a standard way for all participants, but only 50% indicated valid methods for the identification of the condition. CONCLUSIONS: The oncologic behavior of AST is related to an almost always favorable outcome. SNB does not seem to be relevant as a staging or prognostic procedure, and its indication remains debatable and controversial.

SlideTiler: A dataset creator software for boosting deep learning on histological whole slide images.

The introduction of deep learning caused a significant breakthrough in digital pathology. Thanks to its capability of mining hidden data patterns in digitised histological slides to resolve diagnostic tasks and extract prognostic and predictive information. However, the high performance achieved in classification tasks depends on the availability of large datasets, whose collection and preprocessing are still time-consuming processes. Therefore, strategies to make these steps more efficient are worth investigation. This work introduces SlideTiler, an open-source software with a user-friendly graphical interface. SlideTiler can manage several image preprocessing phases through an intuitive workflow that does not require specific coding skills. The software was designed to provide direct access to virtual slides, allowing custom tiling of specific regions of interest drawn by the user, tile labelling, quality assessment, and direct export to dataset directories. To illustrate the functions and the scalability of SlideTiler, a deep learning-based classifier was implemented to classify 4 different tumour histotypes available in the TCGA repository. The results demonstrate the effectiveness of SlideTiler in facilitating data preprocessing and promoting accessibility to digitised pathology images for research purposes. Considering the increasing interest in deep learning applications of digital pathology, SlideTiler has a positive impact on this field. Moreover, SlideTiler has been conceived as a dynamic tool in constant evolution, and more updated and efficient versions will be released in the future.

Clinical, histopathological and molecular features of dedifferentiated melanomas: An EORTC Melanoma Group Retrospective Analysis.

PURPOSE: Dedifferentiated melanoma (DedM) poses significant diagnostic challenges. We aimed to investigate the clinical, histopathological and molecular features of DedM. Methylation signature (MS) and copy number profiling (CNP) were carried out in a subgroup of cases. PATIENTS AND METHODS: A retrospective series of 78 DedM tissue samples from 61 patients retrieved from EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group centres were centrally reviewed. Clinical and histopathological features were retrieved. In a subgroup of patients, genotyping through Infinium Methylation microarray and CNP analysis was carried out. RESULTS: Most patients (60/61) had a metastatic DedM showing most frequently an unclassified pleomorphic, spindle cell, or small round cell morphology akin to undifferentiated soft tissue sarcoma, rarely associated with heterologous elements. Overall, among 20 successfully analysed tissue samples from 16 patients, we found retained melanoma-like MS in only 7 tissue samples while a non-melanoma-like MS was observed in 13 tissue samples. In two patients from whom multiple specimens were analysed, some of the samples had a preserved cutaneous melanoma MS while other specimens exhibited an epigenetic shift towards a mesenchymal/sarcoma-like profile, matching the histological features. In these two patients, CNP was largely identical across all analysed specimens, in line with their common clonal origin, despite significant modification of their epigenome. CONCLUSIONS: Our study further highlights that DedM represents a real diagnostic challenge. While MS and genomic CNP may help pathologists to diagnose DedM, we provide proof-of-concept that dedifferentiation in melanoma is frequently associated with epigenetic modifications.

TRK Protein Expression in Merkel Cell Carcinoma Is Not Caused by NTRK Fusions.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignant tumor with neuroendocrine differentiation, with a rapidly growing incidence rate, high risk of recurrence, and aggressive behavior. The available therapeutic options for advanced disease are limited and there is a pressing need for new treatments. Tumors harboring fusions involving one of the neurotrophin receptor tyrosine kinase (NTRK) genes are now actionable with targeted inhibitors. NTRK-fused genes have been identified in neuroendocrine tumors of other sites; thus, a series of 76 MCCs were firstly analyzed with pan-TRK immunohistochemistry and the positive ones with real-time RT-PCR, RNA-based NGS, and FISH to detect the eventual underlying gene fusion. Despite 34 MCCs showing pan-TRK expression, NTRK fusions were not found in any cases. As in other tumors with neural differentiation, TRK expression seems to be physiological and not caused by gene fusions.

Per- and polyfluoroalkyl substances (PFAS) exposure in melanoma patients: a retrospective study on prognosis and histological features.

Per- and polyfluoroalkyl substances (PFAS) are endocrine disrupting chemicals which could be associated with cancer development, such as kidney and testicular cancers, pancreatic and hepatocellular carcinoma and thyroid tumor. Available scientific literature offers no information on the role of PFAS in melanoma development/progression. Since 1965, a massive environmental contamination by PFAS has occurred in northeastern Italy. This study compared histopathology and prognosis between melanoma patients exposed (n = 194) and unexposed (n = 488) to PFAS. All patients were diagnosed and/or treated for melanoma at the Veneto Oncological Institute and the University Hospital of Padua (Italy) in 1998-2014. Patients were categorized in exposed or unexposed groups according to their home address and the geographical classification of municipalities affected by PFAS contamination as provided by Veneto Government in 2018. Presence of mitoses was found in 70.5% of exposed patients and 58.7% of unexposed patients (p = 0.005). Median follow-up was 90 months (IQR 59-136). 5-year overall survival was 83.7% in exposed patients and 88.0% in unexposed patients (p = 0.20); 5-year disease-specific survival was 88.0% in exposed patients and 90.9% in unexposed patients (p = 0.50); 5-year disease-free survival was 83.8% in exposed patients and 87.3% in unexposed patients (p = 0.20). Adjusting for imbalanced characteristics at baseline (presence of mitoses), survival was not statistically different between exposed and unexposed patients (overall survival: HR 1.10, 95% CI 0.77 to 1.58, p = 0.57; disease-specific survival: HR 0.99, 95% CI 0.62 to 1.59, p = 0.99; disease-free survival: HR 1.10, 95% CI 0.74 to 1.64, p = 0.62). Although the magnitude of PFAS exposure was not quantifiable, our findings suggested that exposure to PFAS was associated with higher level of mitosis in melanoma patients, but this did not translate into a survival difference. Further studies are required to investigate this relationship and all effects of PFAS on prognosis.

Altitude Effect on Cutaneous Melanoma Epidemiology in the Veneto Region (Northern Italy): A Pilot Study.

The incidence of cutaneous melanoma has been increasing in the last decades among the fair-skinned population. Despite its complex and multifactorial etiology, the exposure to ultraviolet radiation (UVR) is the most consistent modifiable risk factor for melanoma. Several factors influence the amount of UVR reaching the Earth's surface. Our study aimed to explore the relationship between melanoma and altitude in an area with mixed geographic morphology, such as the Veneto region (Italy). We included 2752 melanoma patients who were referred to our centers between 1998 and 2014. Demographics, histological and clinical data, and survival information were extracted from a prospectively maintained local database. Head/neck and acral melanoma were more common in patients from the hills and the mountains, while limb and trunk melanoma were more common in patients living in plain and coastal areas. Breslow thickness, ulceration and mitotic rate impaired with increased altitude. However, the geographical area of origin was not associated with overall or disease-free survival. The geographical area of origin of melanoma patients and the "coast-plain-hill gradient" could help to estimate the influence of different sun exposure and to explain the importance of vitamin D levels in skin-cancer control.

The Morpho-Molecular Landscape of Spitz Neoplasms.

Spitz neoplasms are a heterogeneous group of melanocytic proliferations with a great variability in the histological characteristics and in the biological behavior. Thanks to recent discoveries, the morpho-molecular landscape of Spitz lineage is becoming clearer, with the identification of subtypes with recurrent features thus providing the basis for a more solid and precise tumor classification. Indeed, specific mutually exclusive driver molecular events, namely HRAS or MAP2K1 mutations, copy number gains of 11p, and fusions involving ALK, ROS, NTRK1, NTRK2, NTRK3, MET, RET, MAP3K8, and BRAF genes, correlate with distinctive histological features. The accumulation of further molecular aberrations, instead, promotes the increasing malignant transformation of Spitz neoplasms. Thus, the detection of a driver genetic alteration can be achieved using the appropriate diagnostic tests chosen according to the histological characteristics of the lesion. This allows the recognition of subtypes with aggressive behavior requiring further molecular investigations. This review provides an update on the morpho-molecular correlations in Spitz neoplasms.

Gastric metastases of breast cancer: Histopathological and molecular characterization of a single Institution case series.

The metastatic spread of breast carcinoma to the stomach is a rare event and often represents a diagnostic challenge. In the present study, 23 cases of gastric metastases from breast cancer were retrospectively identified dating back until 2007. Primitive histotype, localization, gross appearance, microscopic architecture were analyzed. Cytokeratins 7 and 20, sex hormones, HER2 and Ki67 expression was evaluated. According to the results, the series was characterized by an enrichment of lobular primitive histotype (43.7%). In most cases gastric metastases were described as parietal nodules, polypoid masses or ulcerated lesions, mainly involving the antro-angular region. In a relatively high rate (10.5%) of cases, endoscopic examinations resulted negative for macroscopic lesions. More than half of the cases (52.2%) microscopically resembled primitive poorly cohesive gastric cancer. Because gross and histological findings can be deceiving, immunohistochemistry may be essential for the diagnosis of gastric metastases from breast cancer. Accordingly with the results of our analysis and literature review, an immunohistochemical panel composed of cytokeratins 7 and 20, Estrogen and Progesteron Receptors would drastically improve diagnostic accuracy. Interaction among the clinician, endoscopist and the pathologist is also essential to provide the patient the best therapeutic option.

Necroptosis-driving genes RIPK1, RIPK3 and MLKL-p are associated with intratumoral CD3+ and CD8+ T cell density and predict prognosis in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly lethal cancer and the second leading cause of cancer-related deaths worldwide. As demonstrated in other solid neoplasms and HCC, infiltrating CD8+ T cells seem to be related to a better prognosis, but the mechanisms affecting the immune landscape in HCC are still mostly unknown. Necroptosis is a programmed, caspase-independent cell death that, unlike apoptosis, evokes immune response by releasing damage-associated molecular factors. However, in HCC, the relationship between the necroptotic machinery and the tumor-infiltrating lymphocytes has not been fully investigated so far. METHODS: We investigated the association between the main necroptosis-related genes, that is, RIPK1, RIPK3, MLKL-p, and CD3+/CD8+ tumor-infiltrating T cell by RNA-seq data analysis in 371 patients with primary HCC from The Cancer Genome Atlas and then by immunohistochemistry in two independent cohorts of HCC patients from Italy (82) and Japan (86). RESULTS: Our findings highlighted the immunogenetic role of necroptosis and its potential prognostic role in HCC: RIPK1, RIPK3 and MLKL-p were found significantly associated with intratumoral CD3+ and CD8+ T cells. In addition, multivariate survival analysis showed that the expression of RIPK1, RIPK3 and MLKL-p was associated with better overall survival in the two independent cohorts. CONCLUSIONS: Our results confirmed the immunogenetic properties of necroptosis (NCP) in human HCC, showing that tumor-infiltrating lymphocytes (TILs) and, specifically, CD8+ T cells accumulate in tumors with higher expression of the necroptosis-related genes. These results suggest the importance of further studies to better assess the specific composition, as well as the functional features of the immune environment associated with a necroptotic signature in order to explore new possible diagnostic and immunotherapeutic scenarios.

Corrigendum: Melanoma in Adolescents and Young Adults (AYA): Evaluation of the Characteristics, Treatment Strategies, and Prognostic Factors in a Monocentric Retrospective Study.

[This corrects the article DOI: 10.3389/fonc.2021.725523.].

RIPK3 and AXL Expression Study in Primary Cutaneous Melanoma Unmasks AXL as Predictor of Sentinel Node Metastasis: A Pilot Study.

Malignant melanoma (MM) is the most lethal skin cancer. AXL is a tyrosine kinase receptor involved in several oncogenic processes and might play a role in blocking necroptosis (a regulated cell death mechanism) in MM through the downregulation of the necroptotic-related driver RIPK3. The aim of this study was to evaluate the clinical impact of the expression of AXL and RIPK3 in 108 primary cutaneous MMs. Association between AXL and RIPK3 immunoreactivity and clinical-pathological variables, sentinel lymph node status, and tumor-infiltrating lymphocytes (TILs) was assessed. Immunoreaction in tumor cells was detected in 30 cases (28%; range, 5%-80%) and in 17 cases (16%; range, 5%-50%) for AXL and RIPK3, respectively. Metastases in the sentinel lymph nodes were detected in 14 out of 61 patients, and these were associated with AXL-positive immunoreactivity in the primary tumor (p < 0.0001). No association between AXL and TILs was found. RIPK3 immunoreactivity was not associated with any variables. A final logistic regression analysis showed Breslow and AXL-positive immunoreactivity as the stronger predictor for positive sentinel node status [area under the receiver operating characteristic curve (AUC) of 0.96]. AXL could be a potential new biomarker for MM risk assessment, and it deserves to be further investigated in larger studies.

NTRK Gene Fusion Detection in Atypical Spitz Tumors.

Atypical Spitz tumors (AST) deviate from stereotypical Spitz nevi for one or more atypical features and are now regarded as an intermediate category of melanocytic tumors with uncertain malignant potential. Activating NTRK1/NTRK3 fusions elicit oncogenic events in Spitz lesions and are targetable with kinase inhibitors. However, their prevalence among ASTs and the optimal approach for their detection is yet to be determined. A series of 180 ASTs were screened with pan-TRK immunohistochemistry and the presence of NTRK fusions was confirmed using FISH, two different RNA-based NGS panels for solid tumors, and a specific real time RT-PCR panel. Overall, 26 ASTs showed pan-TRK immunostaining. NTRK1 fusions were detected in 15 of these cases showing cytoplasmic immunoreaction, whereas NTRK3 was detected in one case showing nuclear immunoreaction. Molecular tests resulted all positive in only two ASTs (included the NTRK3 translocated), RNA-based NGS and real time RT-PCR were both positive in three cases, and FISH and real time RT-PCR in another two cases. In seven ASTs NTRK1 fusions were detected only by FISH and in two cases only by real time RT-PCR. The frequency of NTRK fusions in ASTs is 9%, with a clear prevalence of NTRK1 compared to NTRK3 alterations. Pan-TRK immunohistochemistry is an excellent screening test. Confirmation of NTRK fusions may require the use of different molecular techniques.

Melanoma in Adolescents and Young Adults: Evaluation of the Characteristics, Treatment Strategies, and Prognostic Factors in a Monocentric Retrospective Study.

The "Veneto Cancer Registry" records melanoma as the most common cancer diagnosed in males and the third common cancer in females under 50 years of age in the Veneto Region (Italy). While melanoma is rare in children, it has greater incidence in adolescents and young adults (AYA), but literature offers only few studies specifically focused on AYA melanoma. The aim of this study was to describe the characteristics, surgical treatment, and prognosis of a cohort of AYA melanoma in order to contribute to the investigation of this malignancy and provide better patient care. This retrospective cohort study included 2,752 Caucasian patients (702 AYA and 2,050 non-AYA patients) from the Veneto Region who were over 15 years of age at diagnosis, and who received diagnosis and/or treatment from our institutions between 1998 and 2014. Patients were divided in adolescents and youth (15-25 years), young adults (26-39 years) and adults (more than 39 years) for the analysis. We found statistically significant differences in gender, primary site, Breslow thickness, ulceration, pathologic TNM classification (pTNM) stage and tumor subtype among the age groups. Disease-specific survival and disease-free survival were also different among the age groups. Our findings suggest that the biological behavior of melanoma in young people is different to that in adults, but not such as to represent a distinct pathological entity. Additional and larger prospective studies should be performed to better evaluate potential biological and cancer-specific differences between AYAs and the adult melanoma population.

Epithelial-to-Mesenchymal Transition and Neoangiogenesis in Laryngeal Squamous Cell Carcinoma.

The mechanism of epithelial-mesenchymal transition (EMT) is fundamental for carcinogenesis, tumor progression, cancer cell invasion, metastasis, recurrence, and therapy resistance, comprising important events, such as cellular junction degradation, downregulation of epithelial phenotype markers, overexpression of mesenchymal markers, and increase in cellular motility. The same factors that drive epithelial cells toward a mesenchymal phenotype may also drive endothelial cells toward a proangiogenic phenotype. The aim of this exploratory study was to investigate a potential interplay between EMT and angiogenesis (quantified through CD105 expression) in laryngeal carcinoma (LSCC). CD105-assessed microvessel density (MVD) and EMT markers (E-cadherin, N-cadherin, Snail, Slug, Zeb1, and Zeb2) were assessed on 37 consecutive LSCC cases. The univariate Cox regression model identified pN+ status (p = 0.0343) and Slug expression (p = 0.0268) as predictive of disease-free survival (DFS). A trend toward significance emerged for CD105-assessed MVD (p = 0.0869) and N-cadherin expression (p = 0.0911). In the multivariate Cox model, pN-status, Slug, and N-cadherin expressions retained their significant values in predicting DFS (p = 0.0346, p = 0.0430, and p = 0.0214, respectively). Our data support the hypothesis of a mutual concurrence of EMT and angiogenesis in driving LSCC cells toward an aggressive phenotype. To better characterize the predictive performance of prognostic models based on EMT and angiogenesis, further large-scale prospective studies are required.

Corrigendum: Melanoma of Unknown Primary: Evaluation of the Characteristics, Treatment Strategies, Prognostic Factors in a Monocentric Retrospective Study.

[This corrects the article DOI: 10.3389/fonc.2021.627527.].

The immune cell landscape of metastatic uveal melanoma correlates with overall survival.

BACKGROUND: Uveal melanoma (UM) represents the most common primary intra-ocular malignancy in adults. Up to 50% of the patients develop distant metastases within 10 years from diagnosis, with the liver as the most common site. Upon metastatization, life expectancy strongly reduces and immune checkpoint inhibitors that prove effective in cutaneous melanoma do not modify clinical outcome. To date, few studies have focused on deciphering the immunomodulatory features of metastatic UM microenvironment, and there are no prognostic models for clinical use. This highlights the urgent need to understand the delicate interplay between tumor and immune cells acting at the site of metastasis. METHODS: We collected a patient cohort comprising 21 metastatic UM patients. Hepatic and extra-hepatic UM metastasis samples were studied by multiplex immunofluorescence to assess the tumor immune cell composition. Quantitative analyses were performed to correlate immune cell densities with treatment response, metastasis site and patient survival. RESULTS: Compared to patients with progressive disease, those with controlled disease had a higher intra-tumoral/peritumoral ratio of CD8 + Granzyme B+ cells, higher density of intra-tumoral CD8+ cytotoxic T lymphocytes (CTL) and an increased percentage of UM cells in close proximity to T lymphocytes, reflecting a role of tumor-killing T cells in the disease. In liver metastases (LM), the intra-tumoral densities of CD163+ tumor-associated macrophages (TAM) and of total CD8+ T cells were higher than in extra-hepatic UM metastases, but the percentage of Granzyme B+ CTL was lower. Moreover, LM displayed more UM cells adjacent to both CTL and TAM, and also more T cells in proximity to TAM, all signs of an impaired immune response. The percentage of activated CTL within the tumor represented a prognostic indicator, as patients with a higher intra-tumoral percentage of CD8 + Granzyme B+ cells had the better outcome. A temptative Immunoscore was generated and proved capable to stratify patients with improved survival. Finally, CD4 + FoxP3+ T cells appeared a crucial population for response to immunotherapy. CONCLUSION: The results of this study underly the clinical relevance and functional importance of composition and localization of antitumor effector cells for the progression of UM metastasis.

Intraosseous lipoma of the patella: a case report and review of the literature.

An intraosseous lipoma is considered one of the rarest primary bone tumors. The etiology of this lesion remains unclear; many lesions are asymptomatic and appear only as incidental findings during routine radiographic evaluations. Magnetic resonance imaging of intraosseous lipomas can help to establish a diagnosis and to stage the neoplasm. This is a case report of a 53-year old man with a rare intraosseous lipoma of the patella.

The Molecular Landscape of Primary Acral Melanoma: A Multicenter Study of the Italian Melanoma Intergroup (IMI).

Acral melanoma (AM) is a rare and aggressive subtype of melanoma affecting the palms, soles, and nail apparatus with similar incidence among different ethnicities. AM is unrelated to ultraviolet radiation and has a low mutation burden but frequent chromosomal rearrangements and gene amplifications. Next generation sequencing of 33 genes and somatic copy number variation (CNV) analysis with genome-wide single nucleotide polymorphism arrays were performed in order to molecularly characterize 48 primary AMs of Italian patients in association with clinicopathological and prognostic features. BRAF was the most commonly mutated gene, followed by NRAS and TP53, whereas TERT promoter, KIT, and ARID1A were less frequently mutated. Gains and losses were recurrently found in the 1q, 6p, 7, 8q, 20 and 22 chromosomes involving PREX2, RAC1, KMT2C, BRAF, CCND1, TERT, and AKT3 genes, and in the 6q, 9, 10, 11q and 16q chromosomes including CDKN2A, PTEN, and ADAMTS18 genes, respectively. This study confirmed the variety of gene mutations and the high load of CNV in primary AM. Some genomic alterations were associated with histologic prognostic features. BRAF mutations, found with a higher rate than previously reported, correlated with a low Breslow thickness, low mitotic count, low CNV of the AMs, and with early-stage of disease.

Melanoma of Unknown Primary: Evaluation of the Characteristics, Treatment Strategies, Prognostic Factors in a Monocentric Retrospective Study.

BACKGROUND: Melanoma of unknown primary (MUP), accounts for up to 3% of all melanomas and consists of a histologically confirmed melanoma metastasis to either lymph nodes, (sub)cutaneous tissue, or visceral sites without any evidence of a primary cutaneous, ocular, or mucosal melanoma. This study aimed to investigate the characteristics, treatment strategies, and prognostic factors of MUP patients, in order to shed some light on the clinical behavior of this malignancy. METHODS: All the consecutive patients with a diagnosis of MUP referring to our institutions between 1985 and 2018 were considered in this retrospective cohort study. The records of 173 patients with a suspected diagnosis of MUP were retrospectively evaluated for inclusion in the study. Patient selection was performed according to the Das Gupta criteria, and a total of 127 MUP patients were finally included in the study, representing 2.7% of the patients diagnosed with melanoma skin cancer at our institutions during the same study period. A second cohort of all consecutive 417 MKP patients with AJCC stages IIIB-IV, referring tions in the period considered (1985-2018), was included in the study to compare survival between MUP and MKP patients. All the diagnoses were based on histopathologic, cytologic and immunohistochemical examination of the metastases. All tumors were re-staged according to the 2018 American Joint Committee on Cancer (AJCC) 8th Edition. RESULTS: Median follow-up was 32 months (IQR: 15-84). 3-year progression-free survival (PFS) was 54%, while 3-year overall survival (OS) was 62%. Worse OS and PFS were associated with older age (P = 0.0001 for OS; P = 0.008 for PFS), stage IV (P < 0.0001 for OS; P = 0.0001 for PFS) and higher Charlson Comorbidity Index (P < 0.0001 for OS and P = 0.01 for PFS). Patients with lymph node disease showed longer PFS (P = 0.001) and OS (P = 0.0008) than those with (sub)cutis disease. Complete lymph node dissection (CLND) was the most common surgical treatment; a worse OS in these patients was associated with the number of positive lymph nodes (P = 0.01), without significant association with the number of retrieved lymph nodes (P = 0.79). Survival rates were lower in patients undergoing chemotherapy (CT) and target therapy (TT), and higher in those receiving immunotherapy (IT). 417 patients with AJCC stages IIIB-IV of Melanoma Known Primary (MKP) were included for the survival comparison with MUP. 3-year PFS rates were 54 and 58% in MUP and MKP, respectively (P = 0.30); 3-year OS rates were 62 and 70% in MUP and MKP, respectively (P = 0.40). CONCLUSIONS: The most common clinical scenario of our series was a male patient around 59 years with lymph node disease. We report that CLND associated with IT was the best treatment in terms of survival outcome. In the current era of IT and TT for melanoma, new studies have to clarify the impact of novel drugs on MUP.