Melanoma Markers Research Articles

Evaluation of 2-methoxyestradiol serum levels as a potential prognostic marker in malignant melanoma.

Experimental findings indicated that 2-methoxyestradiol (2-ME), an endogenous metabolite of 17β-estradiol, may exhibit anti-tumorigenic properties in various types of tumour, such as melanoma and endometrial carcinoma. In patients with endometrial cancer, the serum levels of 2-ME are decreased compared with those in healthy controls, and this finding has been associated with a poor outcome. The aim of the present study was to examine whether the serum levels of 2-ME are decreased in patients with melanoma, and whether this decrease may be correlated with disease stage and, therefore, serve as a prognostic indicator. ELISA was used to detect serum levels of 2-ME in patients with stage I-IV malignant melanoma (MM). A cohort of 78 patients with MM was analysed, along with 25 healthy controls, among whom 15 were women in the second trimester of pregnancy (positive control). As expected, significantly elevated levels of serum 2-ME were observed in pregnant control patients compared with those in patients with MM and healthy controls. There was no observed correlation between 2-ME serum levels in patients with MM and disease stage, tumour thickness, lactate dehydrogenase or S100 calcium-binding protein B levels. In addition, the 2-ME levels of patients with MM did not differ significantly from those of normal healthy controls. Overall, the findings of the present study indicated that the 2-ME serum levels in patients with MM were not decreased, and there was no correlation with early- or advanced-stage disease. Therefore, in contrast to published results on endometrial cancer, endogenous serum 2-ME levels in MM were not found to be correlated with tumour stage and did not appear to be a suitable prognostic factor in MM.

Plasma thymidine kinase activity (TKa) as a novel prognostic biomarker in metastatic melanoma.

9556 Background: In the recent decade, new effective immunotherapies and targeted therapies have emerged for the treatment of disseminated melanoma. However, a considerable fraction of patients does not respond or get lasting effects and the treatments also have significant side effects. Biomarkers can contribute with more knowledge on prognosis and the efficacy of these therapies in different patients. In other cancer types, the plasma activity of the enzyme thymidine kinase (TKa), has been demonstrated as a marker of tumor stage and prognosis. The TK enzyme is part of a reaction chain to introduce thymidine into the DNA strand. TK thereby has a key function in DNA-synthesis, -repair and cell division. Dividing cells release TK during mitotic exit and TK can thus be detected in the blood. This study is the first to investigate plasma TKa as a potential biomarker in melanoma patients. Methods: Plasma samples were collected within five days prior to treatment start in patients with unresectable metastatic cutaneous melanoma, treated with immunotherapy (anti-CTLA-4 and/or anti-PD-1) or targeted therapy (BRAF±MEK inhibitors). Plasma TKa levels were determined using the DiviTum TKa ELISA assay (Biovica, Sweden). TKa levels were correlated with the patients’ baseline criteria, response rate (RR), progression free survival (PFS) and overall survival (OS). Results: Among the 124 study patients, the median TKa was 50 Du/L (range < 20-3491 Du/L). Significantly higher plasma TKa levels were found in patients with ECOG performance status ≥1 vs. 0-1 ( P< 0.001), M1c-d vs. M1a-b disease ( P< 0.001), ≥3 vs. 1-2 affected organs ( P= 0.002) or elevated vs. non-elevated LDH ( P< 0.001). In the patients treated with immunotherapy (n = 86) the RR was 63.2% vs. 37.9% in those with low ( < 60 Du/L) vs. high TKa ( P= 0.024). The median PFS and OS was 19.9 and > 60 months in those with low TKa vs. 12.6 and 18.5 months in those with high TKa (HR for PFS: 1.73 (95% CI, 1.01-2.97), P= 0.044 and HR for OS: 2.16 (95% CI, 1.17-3.98), P= 0.011). In the patients treated with BRAF±MEK inhibitor (n = 38) a similar trend was observed, with shorter PFS and OS in those with high TKa, but the differences were not statistically significant. Conclusions: In this first study on plasma TKa in melanoma patients, high pretreatment TKa was significantly associated with poor baseline factors and poor response and survival in immunotherapy treated patients. Currently, plasma LDH is the only non-clinical factor that is routinely used as a prognostic marker in melanoma. Several other candidate markers have been described, such as PD-L1 tumor immunohistochemistry, tumor mutational burden, gut microbiome and circulating tumor DNA. Compared to these assays, TKa measured with DiviTum is a simpler, ELISA based test for a single plasma marker. TKa is hence a novel and interesting marker in melanoma and should be further studied to define its role as a prognostic and predictive marker in this disease.

Circulating tumor DNA mutation as a prognostic marker in melanoma with brain metastasis.

e21560 Background: Prognosis in melanoma with brain metastasis is poor with a median survival of four months and a one-year survival rate of 10–20%. There is an unmet need for surveillance methods that can supplement imaging at regular intervals. Serial analysis of circulating tumor DNA (ctDNA) may aid surveillance and prognostication. A PCR-based, “specimen in/result out” testing device was employed to detect BRAF variants in plasma-derived ctDNA to evaluate the utility of rapid biomarker detection in the management of melanoma with brain metastasis. Methods: Serial blood samples from patients diagnosed with BRAF mutation-positive metastatic melanoma were collected at regular intervals. We employed a real-time PCR-based automated mutation detection system (Idylla; Biocartis, Belgium) to interrogate the plasma samples. The ctDNA mutation detection trend was analyzed relative to disease progression. Results: 39 patients with BRAF mutation positive melanoma were enrolled. 29 patients were treated in the metastatic setting, 10 in the adjuvant setting. 18 of the 29 patients with metastatic disease (62%) had brain metastases. Circulating BRAF mutation was detected in 17 of the 29 (59%) patients with metastatic disease, and was not detected in any patients treated adjuvantly. In the group with metastatic disease, this circulating biomarker changed from undetectable to detectable in eight (28%) and detectable to undetectable in three (10%). No change in circulating mutation status occurred in 18 (62%). In the eight patients who had an initial negative test that later became positive, seven (87%) had brain metastases. In three patients, ctDNA mutation detection occurred before the diagnosis of brain metastases on imaging, with a median lead time of five weeks (range, 3-12 weeks). In one patient with de novo metastatic disease admitted to the ICU, tissue was unavailable for BRAF testing but plasma was found to be positive for ctDNA BRAF detection. BRAF/MEK targeted therapy resulted in a sustained objective response. Five of six (83%) patients that had persistent ctDNA positivity had brain metastases. Among patients with brain metastases, median overall survival (mOS) of patients demonstrating >50% test positivity was numerically longer than those with <50% positivity (mOS 12.3 vs 53.5 months; p = 0.133). Conclusions: Plasma-based, rapid ctDNA testing may be useful as an aid in detecting progression and gauging prognosis in patients with melanoma treated in the metastatic setting. The dynamics of ctDNA test positivity may indicate a need for more urgent imaging, particularly of the brain. Blood-based, semi-automated ctDNA detection may serve as an attractive adjunct to scheduled imaging surveillance in melanoma.

Single-cell characterization of macrophages in glioblastoma reveals MARCO as a mesenchymal pro-tumor marker

BackgroundMacrophages are the most common infiltrating immune cells in gliomas and play a wide variety of pro-tumor and anti-tumor roles. However, the different subpopulations of macrophages and their effects on the tumor microenvironment remain poorly understood.MethodsWe combined new and previously published single-cell RNA-seq data from 98,015 single cells from a total of 66 gliomas to profile 19,331 individual macrophages.ResultsUnsupervised clustering revealed a pro-tumor subpopulation of bone marrow-derived macrophages characterized by the scavenger receptor MARCO, which is almost exclusively found in IDH1-wild-type glioblastomas. Previous studies have implicated MARCO as an unfavorable marker in melanoma and non-small cell lung cancer; here, we find that bulk MARCO expression is associated with worse prognosis and mesenchymal subtype. Furthermore, MARCO expression is significantly altered over the course of treatment with anti-PD1 checkpoint inhibitors in a response-dependent manner, which we validate with immunofluorescence imaging.ConclusionsThese findings illustrate a novel macrophage subpopulation that drives tumor progression in glioblastomas and suggest potential therapeutic targets to prevent their recruitment.

CRISPR/Cas9 uPAR Gene Knockout Results in Tumor Growth Inhibition, EGFR Downregulation and Induction of Stemness Markers in Melanoma and Colon Carcinoma Cell Lines.

uPAR is a globular protein, tethered to the cell membrane by a GPI-anchor involved in several cancer-related properties and its overexpression commonly correlates with poor prognosis and metastasis. We investigated the consequences of uPAR irreversible loss in human melanoma and colon cancer cell lines, knocking out its expression by CRISPR/Cas9. We analyzed through flow cytometry, western blotting and qPCR, the modulation of the most known cancer stem cells-associated genes and the EGFR while we observed the proliferation rate exploiting 2D and 3D cellular models. We also generated uPAR “rescue” expression cell lines as well as we promoted the expression of only its 3’UTR to demonstrate the involvement of uPAR mRNA in tumor progression. Knocking out PLAUR, uPAR-encoding gene, we observed an inhibited growth ratio unexpectedly coupled with a significant percentage of cells acquiring a stem-like phenotype. In vivo experiments demonstrated that uPAR loss completely abrogates tumorigenesis despite the gained stem-like profile. Nonetheless, we proved that the reintroduction of the 3’UTR of PLAUR gene was sufficient to restore the wild-type status validating the hypothesis that such a region may act as a “molecular sponge”. In particular miR146a, by binding PLAUR 3’ UTR region might be responsible for uPAR-dependent inhibition of EGFR expression.

Prognostic Value of ctDNA Mutation in Melanoma: A Meta-Analysis.

Purpose Melanoma is the most aggressive form of skin cancer. Circulating tumor DNA (ctDNA) is a diagnostic and prognostic marker of melanoma. However, whether ctDNA mutations can independently predict survival remains controversial. This meta-analysis assessed the prognostic value of the presence or change in ctDNA mutations in melanoma patients. Methods We identified studies from the PubMed, EMBASE, Web of Science, and Cochrane databases. We estimated the combined hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS) using either fixed-effect or random-effect models based on heterogeneity. Results Sixteen studies including 1,781 patients were included. Both baseline and posttreatment detectable ctDNA were associated with poor OS (baseline detectable vs. undetectable, pooled HR = 1.97, 95% CI = 1.64–2.36, P < 0.00001; baseline undetectable vs. detectable, pooled HR = 0.19, 95% CI = 0.11–0.36, P < 0.00001; posttreatment detectable vs. undetectable, pooled HR = 2.36, 95% CI = 1.30–4.28, P=0.005). For PFS, baseline detectable ctDNA may be associated with adverse PFS (baseline detectable vs. undetectable, pooled HR = 1.41, 95% CI = 0.84–2.37, P=0.19; baseline undetectable vs. detectable, pooled HR = 0.43, 95% CI = 0.19–0.95, P=0.04) and baseline high ctDNA and increased ctDNA were significantly associated with adverse PFS (baseline high vs. low/undetectable, pooled HR = 3.29, 95% CI = 1.73–6.25, P=0.0003; increase vs. decrease, pooled HR = 4.48, 95% CI = 2.45–8.17, P < 0.00001). The baseline BRAFV600 ctDNA mutation-positive group was significantly associated with adverse OS compared with the baseline ctDNA-negative group (pooled HR = 1.90, 95% CI = 1.58–2.29, P < 0.00001). There were no significant differences in PFS between the baseline BRAFV600 ctDNA mutation-detectable group and the undetectable group (pooled HR = 1.02, 95% CI = 0.72–1.44, P=0.92). Conclusion The presence or elevation of ctDNA mutation or BRAFV600 ctDNA mutation was significantly associated with worse prognosis in melanoma patients.

Suitability of CD133 as a Marker for Cancer Stem Cells in Melanoma.

Objectives:CD133 is considered a cancer stem cell (CSC) marker in various malignancies; however, its role as a biomarker of malignant melanoma remains controversial. The present study was conducted to evaluate the suitability of CD133 surface antigen as a CSC marker in melanoma. Methods:Human melanoma cells were fractionally separated by magnetic cell separation depending on the CD133 phenotype and transplanted into immunodeficient mice to evaluate their tumorigenic capacity. Furthermore, the time until the development of a palpable tumor and the growth rate were measured, and the final tumor volume was assessed after 8 weeks. The immunohistochemical expression of CD133 in the induced neoplasia was then compared using histomorphometry. Results:Notably, neoplasms were induced in all the groups (n = 48), including in the CD133-negative group. Tumors induced by unsorted cells had the largest volume (p = 0.014) but were detected significantly later in this group (p ≤ 0.001). Interestingly, all explanted tumors expressed CD133, with no significant differences among groups. Conclusions:In contrast to the results obtained in prior studies, the suitability of CD133 as a CSC marker could not be demonstrated. The current encouraging progress in targeted therapy for malignant melanoma highlights the need to identify more effective targets.

Time-related changes in cell morphology and biomarker immunoreactivity for cells stored in a buffer-based cell medium.

Buffer-based cell media (BBCM) are a valuable tool in the post-collection processing of cytology samples, though with poorly defined effects on cell properties. In this study, time-related changes in cell morphology and biomarker immunoreactivity were evaluated for cells stored at room temperature in a BBCM prepared with bovine serum albumin (BSA) and ethylene diamine tetraacetic acid (EDTA). Cytospins were prepared at five consecutive 24-hour intervals (0, 24, 48, 72, 96) from three human cell lines (MCF7, SK-MEL-28, FaDu) suspended and stored in BBCM. Preservation of cell morphology was evaluated on Papanicolaou-stained cytospins from the percentages of apoptotic cells. Preservation of immunoreactivity was evaluated for cytokeratins, oestrogen receptors, Ki67, and melanoma markers from the percentages of cells positive for the corresponding immunocytochemical reactions. Cell morphology was well preserved for the majority of cells of the three lines stored for 24 and 48hours (93%, 97%, 98% and 62%, 81%, 88%, respectively), while the majority of cells were apoptotic after 72 and 96hours (70%, 47%, 39% and 77%, 70%, 59%, respectively). The immunoreactivity of cytokeratins remained unchanged during the entire 96hours, while that of melanoma markers (S100, HMB45, Melan-A) decreased by 27%, 2%, and 3%, respectively. The immunoreactivity of oestrogen receptors and Ki67 decreased by 29% and 17% after the first 24hours, and was completely lost after 96hours. A BBCM with the addition of BSA and EDTA facilitates good preservation of cell morphology and immunoreactivity of biomarkers for up to 48hours at room temperature.

PLA1A expression as a diagnostic marker of BRAF-mutant metastasis in melanoma cancer

BRAF and NRAS are the most reported mutations associated to melanomagenesis. The lack of accurate diagnostic markers in response to therapeutic treatment in BRAF/NRAS-driven melanomagenesis is one of the main challenges in melanoma personalized therapy. In order to assess the diagnostic value of phosphatidylserine-specific phospholipase A1-alpha (PLA1A), a potent lysophospholipid mediating the production of lysophosphatidylserine, PLA1A mRNA and serum levels were compared in subjects with malignant melanoma (n = 18), primary melanoma (n = 13), and healthy subjects (n = 10). Additionally, the correlation between histopathological subtypes of BRAF/NRAS-mutated melanoma and PLA1A was analyzed. PLA1A expression was significantly increased during melanogenesis and positively correlated to disease severity and histopathological markers of metastatic melanoma. PLA1A mRNA and serum levels were significantly higher in patients with BRAF-mutated melanoma compared to the patients with NRAS-mutated melanoma. Notably, PLA1A can be used as a diagnostic marker for an efficient discrimination between naïve melanoma samples and advanced melanoma samples (sensitivity 91%, specificity 57%, and AUC 0.99), as well as BRAF-mutated melanoma samples (sensitivity 62%, specificity 61%, and AUC 0.75). Our findings suggest that PLA1A can be considered as a potential diagnostic marker for advanced and BRAF-mutated melanoma.

The value of melanoma inhibitory activity and LDH with melanoma patients in a Chinese population.

Malignant melanoma is a highly malignant tumor originating from the melanocytes of the neural crest, which is prone to metastasis and has a poor prognosis. Previous research demonstrated that melanoma inhibitory activity (MIA) and lactate dehydrogenase (LDH) could serve as serum markers in malignant melanoma and indicate prognosis in the Caucasian race. Researchers suspected that both MIA and LDH could prompt the prognosis of malignant melanoma in the Chinese population. This study aimed to investigate the value of MIA and LDH in the prognosis of acral malignant melanoma.From January 1, 2014, to December 31, 2017, in Jiangsu Province, 44 acral malignant melanoma patients with complete data were chosen from the clinic. The LDH levels were extracted from their clinical data, and MIA levels were measured by enzyme-linked immunosorbent assay method. 8 paired advancing samples before and after metastasis were examined. 22 health donors were matched to the patient group. Receiver operating characteristic (ROC) curves of MIA and LDH were drawn to determine acral malignant melanoma tumorigenesis and metastasis and finally got the cut-off value. Cumulative survival was illustrated with the Kaplan-Meier plot, and factors were compared using the Log-rank test.Compared with age-matched healthy donors, MIA was significantly high in patients (P < .001). Moreover, serum MIA was significantly higher in III-IV stage patients than I-II stage patients (P < .001). However, there was no such association between LDH and melanoma stage and risk. Further study indicated that the MIA cut-off > 914.7pg/mL predicted disease progression with 86.4% specificity and 95.5% sensitivity. In the Kaplan-Meier analysis, MIA levels were independent risk factors for long-term mortality of acral malignant melanoma patients.It concluded that the quantification of MIA in the serum should be performed as a general standard of care in patients at risk of developing metastatic melanoma.

Immunohistochemistry analysis reveals lysyl oxidase-like 3 as a novel prognostic marker for primary melanoma.

Lysyl oxidase-like 3 (LOXL3) is an extracellular enzyme involved in the synthesis of collagen and elastin, and it has been reported to promote melanoma cell proliferation and invasion in vitro. However, the expression level of LOXL3 at different stages of melanocytic lesions and the role of LOXL3 in melanoma pathogenesis remain unknown. Immunohistochemical staining of LOXL3 in a tissue microarray of 373 biopsies at different melanocytic stages was conducted. The correlation between LOXL3 expression and patient survival was examined using Kaplan-Meier survival analysis. Univariate and multivariate Cox regression analyses were conducted to study the hazard ratios. The tissue microarray study revealed that stronger expression of LOXL3 protein was found at more advanced melanocytic stages (P < 0.0001; χ2 test). Increased LOXL3 expression was associated with enhanced tumor thickness and mitosis. Survival analysis showed significantly worsened prognosis in primary melanoma patients when the LOXL3 expression level was higher (P = 0.043; log-rank test). Multivariate Cox regression analysis further showed that LOXL3 expression is a prognostic factor for primary melanoma patient survival (P = 0.04). LOXL3 expression is positively correlated with tumor progression and invasion, and its overexpression is associated with worse prognosis of primary melanoma patients. LOXL3 can serve as a prognostic marker to help identify primary melanoma patients at higher risks of death.

Primary malignant melanoma of small intestine – A rare case report

Primary malignant melanoma of the intestine is an extremely rare lesion that may arise from large bowel. Hereby, we report a case of a 35-year-old female who presented with abdominal pain of 1 month duration. On positron emission tomography-computed tomography, a hypermetabolic mass observed in the pelvic ileal loop with no lesions elsewhere in the body. The surgically excised specimen was diagnosed as primary small intestinal melanoma by histopathological examination, which was further confirmed by the presence of melanoma markers, that is, Melan-A, HMB45, and S100 protein.

Decreased Expression of CPEB3 Predicts a Poor Prognosis in Patients with Melanoma: A Study Based on TCGA Data.

Aim Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) has been acknowledged as a tumor-suppressive gene in several cancers; however, there are few reports on the clinical significance of CPEB3 in melanoma. The aim of this study was to investigate the role of CPEB3 in predicting the prognosis of melanoma patients. Methods The association of CPEB3 expression and clinical pathologic features was performed using The Cancer Genome Atlas (TCGA) data set. The role of CPEB3 expression in prognosis was also analyzed. In addition, CPEB3 expression-related pathways were enriched by gene set enrichment analysis (GSEA). Association analysis of CPEB3 gene expression and immune infiltration was performed by ssGSEA. Results The mRNA was significantly less in melanoma than in normal tissues (p < 0.001). The decrease in CPEB3 expression in melanoma was significantly correlated with T staging (p < 0.001), clinical staging (p = 0.029), melanoma Clark level (p = 0.014), and melanoma ulceration (p = 0.003), while it was marginally significant in N staging (p = 0.089). Melanoma with low CPEB3 expression was associated with worse OS (overall survival), progression-free survival (PFS), and disease-specific survival (DSS) than in that with high expression. In the univariate analysis, expression of CPEB3, melanoma ulceration, Clark level of melanoma, age, clinical stage, T stage, and N stage were correlated with OS (p < 0.05). Further analysis by multivariate Cox regression showed that N stage (p = 0.029), melanoma ulceration (p = 0.004), and CPEB3 expression (p < 0.001) were independent prognostic factors of OS in melanoma. Moreover, GSEA showed that several pathways were enriched in CPEB3, such as PD1 signaling, CTLA4 pathway, CTCF pathway, CHEMOKIN signaling, VEGF signaling, and JAK-STAT pathway. CPEB3 was significantly correlated with the infiltration level of B cells (p < 0.001), T cells (p < 0.001), T helper cells (p < 0.001), and central memory T (Tcm) cells (p < 0.001). Conclusion CPEB3 may be a potential prognostic marker in melanoma with poor survival. Moreover, PD1 signaling, CTLA4 pathway, CTCF pathway, CHEMOKIN signaling, VEGF signaling, and JAK-STAT pathway may be the key pathway regulated by CPEB3. Moreover, the expression of CPEB3 in melanoma is related to the level of immune infiltration.

Dedifferentiated and Undifferentiated Melanomas: Report of 35 New Cases With Literature Review and Proposal of Diagnostic Criteria.

Dedifferentiated melanoma (DM) and undifferentiated melanoma (UM) is defined as a primary or metastatic melanoma showing transition between conventional and undifferentiated components (DM) or lacking histologic and immunophenotypic features of melanoma altogether (UM). The latter is impossible to verify as melanoma by conventional diagnostic tools alone. We herein describe our experience with 35 unpublished cases to expand on their morphologic, phenotypic, and genotypic spectrum, along with a review of 50 previously reported cases (total: 85) to establish the diagnostic criteria. By definition, the dedifferentiated/undifferentiated component lacked expression of 5 routinely used melanoma markers (S100, SOX10, Melan-A, HMB45, Pan-melanoma). Initial diagnoses (known in 66 cases) were undifferentiated/unclassified pleomorphic sarcoma (n=30), unclassified epithelioid malignancy (n=7), pleomorphic rhabdomyosarcoma (n=5), other specific sarcoma types (n=6), poorly differentiated carcinoma (n=2), collision tumor (n=2), atypical fibroxanthoma (n=2), and reactive osteochondromatous lesion (n=1). In only 11 cases (16.6%) was a diagnosis of melanoma considered. Three main categories were identified: The largest group (n=56) comprised patients with a history of verified previous melanoma who presented with metastatic DM or UM. Axillary or inguinal lymph nodes, soft tissue, bone, and lung were mainly affected. A melanoma-compatible mutation was detected in 35 of 48 (73%) evaluable cases: BRAF (n=20; 40.8%), and NRAS (n=15; 30.6%). The second group (n=15) had clinicopathologic features similar to group 1, but a melanoma history was lacking. Axillary lymph nodes (n=6) was the major site in this group followed by the lung, soft tissue, and multiple site involvement. For this group, NRAS mutation was much more frequent (n=9; 60%) than BRAF (n=3; 20%) and NF1 (n=1; 6.6%). The third category (n=14) comprised primary DM (12) or UM (2). A melanoma-compatible mutation was detected in only 7 cases: BRAF (n=2), NF1 (n=2), NRAS (n=2), and KIT exon 11 (n=1). This extended follow-up study highlights the high phenotypic plasticity of DM/UM and indicates significant underrecognition of this aggressive disease among general surgical pathologists. The major clues to the diagnosis of DM and UM are: (1) presence of minimal differentiated clone in DM, (2) earlier history of melanoma, (3) undifferentiated histology that does not fit any defined entity, (4) locations at sites that are unusual for undifferentiated/unclassified pleomorphic sarcoma (axilla, inguinal, neck, digestive system, etc.), (5) unusual multifocal disease typical of melanoma spread, (6) detection of a melanoma-compatible gene mutation, and (7) absence of another genuine primary (eg, anaplastic carcinoma) in other organs.

Investigating Serum and Tissue Expression Identified a Cytokine/Chemokine Signature as a Highly Effective Melanoma Marker.

Simple SummaryIn this study, we investigated the expression of 27 cytokines/chemokines in the serum of 232 individuals (136 melanoma patients vs. 96 controls). It identified several cytokines/chemokines differently expressed in melanoma patients as compared to the healthy controls, as a function of the presence of the melanoma, age, tumor thickness, and gender, indicating different systemic responses to the melanoma presence. We also analyzed the gene expression of the same 27 molecules at the tissue level in 511 individuals (melanoma patients vs. controls). From the gene expression analysis, we identified several cytokines/chemokines showing strongly different expression in melanoma as compared to the controls, and the 4-gene signature “IL-1Ra, IL-7, MIP-1a, and MIP-1b” as the best combination to discriminate melanoma samples from the controls, with an extremely high accuracy (AUC = 0.98). These data indicate the molecular mechanisms underlying melanoma setup and the relevant markers potentially useful to help the diagnosis of biopsy samples.The identification of reliable and quantitative melanoma biomarkers may help an early diagnosis and may directly affect melanoma mortality and morbidity. The aim of the present study was to identify effective biomarkers by investigating the expression of 27 cytokines/chemokines in melanoma compared to healthy controls, both in serum and in tissue samples. Serum samples were from 232 patients recruited at the IDI-IRCCS hospital. Expression was quantified by xMAP technology, on 27 cytokines/chemokines, compared to the control sera. RNA expression data of the same 27 molecules were obtained from 511 melanoma- and healthy-tissue samples, from the GENT2 database. Statistical analysis involved a 3-step approach: analysis of the single-molecules by Mann–Whitney analysis; analysis of paired-molecules by Pearson correlation; and profile analysis by the machine learning algorithm Support Vector Machine (SVM). Single-molecule analysis of serum expression identified IL-1b, IL-6, IP-10, PDGF-BB, and RANTES differently expressed in melanoma (p < 0.05). Expression of IL-8, GM-CSF, MCP-1, and TNF-α was found to be significantly correlated with Breslow thickness. Eotaxin and MCP-1 were found differentially expressed in male vs. female patients. Tissue expression analysis identified very effective marker/predictor genes, namely, IL-1Ra, IL-7, MIP-1a, and MIP-1b, with individual AUC values of 0.88, 0.86, 0.93, 0.87, respectively. SVM analysis of the tissue expression data identified the combination of these four molecules as the most effective signature to discriminate melanoma patients (AUC = 0.98). Validation, using the GEPIA2 database on an additional 1019 independent samples, fully confirmed these observations. The present study demonstrates, for the first time, that the IL-1Ra, IL-7, MIP-1a, and MIP-1b gene signature discriminates melanoma from control tissues with extremely high efficacy. We therefore propose this 4-molecule combination as an effective melanoma marker.

Correlation between the Expression of Angiogenic Factors and Stem Cell Markers in Human Uveal Melanoma.

Uveal melanoma (UM) is the most common malignant tumor of the eye with extremely high metastatic potential. UM tumor cells can disseminate only hematogenously, thus, angiogenic signals have a particular role in the prognosis of the disease. Although the presence of cancer stem cells (CSCs) in densely vascularized UMs has been reported previously, their role in the process of hematogenous spread of UM has not been studied. In this study, we investigated the regulation of angiogenesis in UM in correlation with the presence of CSCs. Seventy UM samples were collected to analyze the expression of CSC markers and angiogenic factors. The expression of CSC markers was studied by RT-PCR, Western blotting techniques and IHC-TMA technique. RT-PCR showed high expression of CSC markers, particularly nestin, FZD6 and SOX10 and somewhat lower expression of NGFR. The protein expression of FZD6, HIF-1α and VEGFA was further evaluated in 52 UM samples by the IHC-TMA technique. We report here for the first time a significant correlation between FZD6 and VEGFA expression in UM samples. The observed correlation between FZD6 and VEGFA suggests the presence of CSCs in UM that are associated with the vascularization process.

Ran GTPase is an independent prognostic marker in malignant melanoma which promotes tumour cell migration and invasion

AimsRan GTPase is involved in nucleocytoplasmic shuttling of proteins and is overexpressed in several cancers. The expression of Ran in malignant melanoma (MM) and its functional activity have not been...

Review and prospect of CD146 research

CD146 was initially identified as a marker of melanoma in 1987. The research history of more than 30 years revealed its physiological functions in early development, immune response, metabolism, and pathological functions in inflammation, autoimmune diseases, tumor and pregnancy diseases. The understanding of the functional mechanisms of CD146 has also changed from mediating cell migration as an adhesion molecule to receiving extracellular signals and activating various intracellular signaling pathways as a cell membrane receptor. The correlation of CD146 to various diseases, especially its roles in tumors, inspired the application explorations of CD146 in diagnosis, prognosis and targeted therapy of those diseases, some of which has entered the clinical stage. In this review, to understand CD146 in an all-round way and develop its application potential in biomedicine, we comprehensively reviewed and summarized the research history of CD146 and addressed the prospect of the fundamental questions in its research and development.

Identification of key genes and pathways for melanoma in the TRIM family.

Certain members of the TRIM family have been shown to have abnormal expression and prognostic value in cancer. However, in the development and progression of melanoma, the role of different TRIM family members remains unknown. To address this issue, this study used the Oncomine, UCSC, Human Protein Atlas, DAVID, and GEPIA databases to study the role of TRIMs in the prognosis of melanoma. Differential expression of TRIM2, TRIM7, TRIM8, TRIM18 (MID1), TRIM19 (PML), TRIM27, and TRIM29 may play an important role in the development of melanoma. The expression TRIM7 and TRIM29 appeared to be helpful in the identification of primary tumors and metastases. Survival analysis suggested that the expression of TRIM27 significantly affected the overall survival and disease‐free survival of melanoma, and its expression was confirmed by qRT‐PCR. Our results indicated that the expression level of TRIM27 might be a prognostic marker of melanoma.

Gene expression analysis in formalin fixed paraffin embedded melanomas is associated with density of corresponding immune cells in those tissues

Immune cell infiltrates in melanoma have important prognostic value. Gene expression analysis may simultaneously quantify numbers and function of multiple immune cell subtypes in formalin-fixed paraffin-embedded (FFPE) tissues. Prior studies report single gene expression can represent individual immune cell subtypes, but this has not been shown in FFPE melanomas. We hypothesized that gene expression profiling of human melanomas using a new RNA expression technology in FFPE tissue would correlate with the same immune cells identified by immunohistochemistry (IHC). This retrospective study included melanoma specimens analyzed by IHC on tumor tissue microarray (TMA) cores and by gene expression profiling with EdgeSeq Immuno-Oncology Assay using qNPA technology on the corresponding tumors. Standardized gene expression levels were analyzed relative to enumerated cells by IHC using Spearman rank test to calculate r-values. Multivariate analysis was performed by Kruskal–Wallis test. 119 melanoma specimens had both IHC and gene expression information available. There were significant associations between the level of gene expression and its quantified IHC cell marker for CD45+, CD3+, CD8+, CD4+, and CD20+ cells (all p < 0.001). There were also significant associations with exhaustion markers FoxP3+, PD-1+, and PD-L1+ (all p ≤ 0.0001). This new qNPA technology is useful to quantify intratumoral immune cells on FFPE specimens through RNA gene expression in metastatic melanoma. As previous studies have shown on other solid human tumors, we also confirm that the expression level of a single gene may be used to represent a single IHC immune cell marker in melanoma.