Melanoma Tissues Research Articles

Exploiting BRAF Mutation for Treatment of Malignant Melanoma: A Literature Review

Malignant melanoma, a deadly type of skin cancer, presents considerable challenges in clinical management. However, the advent of targeted therapies capitalizing on BRAF mutations has ushered in a new era of optimism for treating melanomas. This investigation delves into the utilization of BRAF mutations as a focused approach in addressing malignant melanoma. Anomalously activated RAS-RAF-MEK-ERK signaling, particularly through the prevalent V600E mutation, instigates uncontrolled cell proliferation and survival in melanocytes. Melanoma arises de novo from melanocytes carrying genetic alterations, with RAF mutations being the most common. Mutated RAF genes, notably the V600E mutation, induce dysregulated cell cycle progression, fueling unchecked proliferation. Given that RAF is a pivotal component of the RAS-MAPK pathway, manipulating this pathway emerges as a promising strategy to impede cancer growth. However, not all melanoma cases exhibit RAS mutations, necessitating molecular testing to identify BRAF mutations before initiating targeted therapy. Detecting typically somatic BRAF mutations mandates melanoma tissue as a specimen. The presence of V600E BRAF mutations becomes a crucial factor for employing BRAF inhibitor therapy. This review article underscores the imperative nature of RAF mutation testing in melanoma patients, serving as a predictive tool to determine the cancer's responsiveness to RAF inhibitor treatment. The identification of BRAF mutations offers a targeted therapeutic avenue, instilling fresh hope for personalized and effective interventions in the management of malignant melanoma. Keywords: skin cancer; melanoma; braf mutation

Identification of biomarkers associated with melanoma based on bioinformatics analysis

Background: Melanoma is an aggressive tumor caused by melanocytes characterized by a poor prognosis at the stage of metastasis. Therefore, it is crucial to identify biomarkers for progression and prognosis for the treatment of melanoma. Objectives: The study aimed to identify the specific genes related to the prognosis of melanoma utilizing bioinformatics analyses. Methods: Differentially expressed genes (DEGs) between melanoma tissues and normal tissues were screened from the GSE 3,189 gene expression profile of Gene Expression Omnibus (GEO). A co-expression network was constructed by weighted correlation network analysis (WGCNA). Functional enrichment analysis for DEGs was performed. The risk prognostic model and nomogram predictive model were established utilizing the least absolute shrinkage and selection operator (LASSO) and Cox regression analysis. Results: Using the differential expression analysis and WGCNA, 1,408 DEGs were screened between melanoma tissues and normal tissues. Functional enrichment analysis proved that these genes primarily participated in the cell cycle and mitotic phase regulation in cancer. In addition, 17 optimal DEGs were identified for constructing the risk score prognostic model. Cox regression analysis further revealed that ectonucleotide pyrophosphatase/phosphodiesterase 4 (ENPP4) and FGR proto-oncogene, Src family tyrosine kinase (FGR) were the key genes significantly associated with survival. A nomogram prediction model was established for individual survival probability by integrating pathological T/N/M stage, age, ENPP4, and FGR. High FGR or ENPP4 expression indicated a better prognosis in melanoma patients. Conclusion: This study identified FGR and ENPP4 as potentially useful prognostic biomarkers for melanoma. The corresponding risk score prognostic model and nomogram may be a reliable tool for predicting the prognosis of melanoma patients.

Abstract LB_C21: Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1

Abstract In experiments in melanoma, prostate, colon, and mammary cells and tissues, the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B: ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S), acts as a tumor suppressor. Following treatment with recombinant human (rh) ARSB in previous experiments and in recent experiments in the B16F10 mouse model of malignant melanoma, specific molecular pathways have been identified that retard tumor progression. These pathways involve more or less binding with chondroitin 4-sulfate when ARSB activity is modified. Galectin-3 binding with C4S increases following exogenous ARSB, leading to lower nuclear galectin-3 and reduced expression of chondroitin sulfate proteoglycan (CSPG)4 and of carbohydrate sulfotransferase (CHST)15 in melanoma cells and tissues. Nuclear Sp1 also declines, inhibiting transcription of both CSPG4, also known as the melanoma-associated chondroitin sulfate proteoglycan (MCSP), and of CHST15, which is required for 6-sulfation of N-acetylgalactosamine 4-sulfate. These proteins are associated with more aggressive tumors, and decline in their expression is consistent with the tumor suppressive function of ARSB. Inverse to the effect on galectin-3, SHP2, the ubiquitous non-receptor tyrosine phosphatase (PTPN11), binds less to C4S following treatment with exogenous ARSB. The resulting activation of SHP2 impacts on MAPK signaling, reducing phospho-ERK1/2 and phospho(T180/Y182)-p38-MAPK. These effects lead to decline in expression of the matrix metalloproteinases MMP2 and MMP9 and thereby restrain invasiveness and metastasis. Treatment of cultured melanoma cells with siRNA for ARSB leads to opposite effects to those of exogenous ARSB, including epigenetic effects modifying Programmed Death-Ligand 1 (PD-L1) expression. Treatment with rhARSB significantly reduces PD-L1 protein and gene expression, in contrast to increases when ARSB is silenced. The effects of rhARSB on PD-L1 expression are attributable to declines in phospho-JNK and nuclear c-Jun when free galectin-3 is less and to associated increases in histone deacetylase (HDAC) 3 expression and activity. Consistent with these effects, the ARSB-silencing induced increase in PD-L1 is inhibited by peptide inhibitors of the JNK-c-Jun interaction and by HDAC3 inhibitor. ARSB silencing reduces PD-L1 promoter acetylation, as shown by chromatin immunoprecipitation with anti-acetylhistone H3 antibody. The impact of rhARSB on signaling pathways, transcriptional events, and epigenetic mechanisms demonstrates how a critical sulfation and the resulting modification of chondroitin 4-sulfate can influence vital cell processes and can suppress tumor development. Effects on subcutaneous and pulmonary melanomas demonstrate the impact of exogenous ARSB in melanoma and suggest future prospects as a therapeutic agent. Recombinant human ARSB is an approved agent for the treatment of Mucopolysaccharidosis VI and is used safely and effectively for this congenital deficiency of ARSB. Citation Format: Joanne K. Tobacman, Insug O-Sullivan, Sumit Bhattacharyya. Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C21.

IDO2-siRNA Carried by Salmonella Combined with Nifuroxazide Attenuates Melanoma Growth.

Melanoma, a highly malignant skin cancer, is a hot topic in oncology treatment research. Nowadays, tumor immunotherapy, especially immunotherapy combined with other therapies, has attracted more and more attention. Indoleamine 2,3-dioxygenase 2 (IDO2), a ratelimiting enzyme of the tryptophan metabolism pathway in the urine of dogs with immunosuppression, is highly expressed in melanoma tissue. Additionally, IDO2 significantly inhibits the anti-tumor immunity of the body and has become a novel target of melanoma treatment. Nifuroxazide, as an intestinal antibacterial agent, was found to be able to inhibit Stat3 expression and exert an anti-tumor effect. Therefore, the present study aimed to examine the therapeutic effect of a self-designed IDO2-small interfering RNA (siRNA) delivered by attenuated Salmonella combined with nifuroxazide on melanoma- bearing mice, as well as determine its underlying mechanism. The effect of nifuroxazide on melanoma was detected by flow cytometry, CCK-8 and colony- forming ability assays, respectively, in vitro. The plasmid of siRNA-IDO2 was constructed, and the mice-bearing melanoma model was established. After the treatment, the tumor growth and survival rate were monitored, and the morphological changes of tumor tissue were detected by HE staining. The expression of related proteins was detected by Western blotting, and the expression of CD4 and CD8 positive T cells in tumor tissue was detected by IHC and IF, and the proportion of CD4 and CD8 positive T cells in spleen was detected by flow cytometry. The results demonstrated that the combination therapy effectively inhibited the phosphorylation of Stat3 and the expression level of IDO2 in melanoma cells, which effectively inhibited tumor growth and prolonged the survival time of tumor-bearing mice. The mechanistic study revealed that, compared with control groups and monotherapy groups, the combination treatment group reduced the atypia of tumor cells, increased the apoptotic rate, enhanced the infiltration of T lymphocytes in tumor tissue and increased the CD4+ and CD8+ T lymphocytes in the spleen, suggesting that the mechanism may be associated with the inhibition of tumor cell proliferation, the increase of apoptosis and the enhancement of the cellular immunity. In conclusion, IDO2-siRNA combined with nifuroxazide therapy could serve a significant role in the treatment of melanoma-bearing mice, enhance the tumor immunity and provide an experimental basis for identifying a novel combination method for the treatment of melanoma clinically.

CircFAT1 Promotes the Proliferation and Invasion of Malignant Melanoma through miR375-SLC7A11 Signal Axis.

Circular RNA, as a member of noncoding RNA, plays an important role in the occurrence, development and metastasis of tumor cells. So far, the correlation between circular RNA and malignant melanoma remains obscure. RNA expression of circFAT1 and miR-375 in malignant melanoma (MM) tissues and cell lines was detected by RT-PCR. The proliferation, cloning, migration and invasion of SK-Mel-28 and A375 cells were assessed using CCK-8 test, clone formation and Transwell assay, respectively. CircRNA immunoprecipitation was used to validate the relationship between circFAT1 and miR-375. The binding between circFAT1 and miR-375, as well as SLC7A11 and miR-375 were verified by luciferase assay. In our study，the circFAT1 was significantly overexpressed in the MM tissue than melanocytic nevi. Conversely, the expression of miR-375 in MM tissue was lower than in melanocytic nevi tissue. The underexpression of circFAT1 with siRNA plasmids significantly suppressed the proliferation, invasion and clone formation of MM cell line. Mechanistically, circFAT1 positively regulates the expression level of SLC7A11 by sponging miR-375. The promotive effects of circFAT1 on the proliferation and invasion ability of MM cells were reversed by the upregulation of miR-375. circFAT1 promotes the proliferation, invasion and clone formation of malignant melanoma cells by improving the expression level of SLC7A11 via sponging miR-375.

Radio-Guided Occult Lesion Localization With 99m Tc for the Localization of Nonpalpable Melanoma and Soft Tissue Sarcoma Lesions: A Feasibility Study.

Localization techniques are needed to facilitate resection of nonpalpable lesions. In this study, the feasibility of radio-guided occult lesion localization (ROLL) with 99m Tc is investigated for the localization of nonpalpable, small, suspicious, or proven melanoma or soft tissue sarcoma lesions at various locations throughout the body. Patients with nonpalpable, suspicious, or proven melanoma or soft tissue sarcoma lesions were selected for this study. Within 24 hours before surgery, a median dose of 33.92 MBq 99m Tc-labeled human albumin particles ( 99m Tc-NA or 99m Tc-MAA) was injected in the lesion under ultrasound guidance. A hand-held gamma probe was used to detect the radioactive signal and guidance during surgery. In this study, 20 patients with a total of 25 lesions were included and analyzed. The median size of the lesions was 1.8 cm (interquartile range [IQR], 1.8-4.0 cm), of which 44% were intramuscular located and 36% were subcutaneous, and 20% consisted of suspicious lymph nodes, mostly in the lower extremity. At median 4 hours (IQR, 3-6 hours) postinjection, 99m Tc ROLL showed a 100% intraoperative identification rate with proper signal identification with the gamma probe in all patients. With a median surgery time of 76 minutes (IQR, 45-157 minutes), all targeted lesions could be resected without 99m Tc-related complications, resulting in 88% microscopically margin-negative resection. No reoperations were needed for the same lesion. The 99m Tc ROLL procedure is feasible for the localization and excision of small, nonpalpable melanoma and soft tissue sarcoma lesions at various locations in the body.

The role of diabetes in metastatic melanoma patients treated with nivolumab plus relatlimab

BackgroundThe combination of nivolumab + relatlimab is superior to nivolumab alone in the treatment of naive patients and has activity in PD-1 refractory melanoma. We had previously observed a reduced expression of LAG3 in melanoma tissue from patients with type 2 diabetes.MethodTo evaluate the impact of diabetes on oncological outcomes of patients with advanced melanoma treated with nivolumab plus the LAG3 inhibitor relatlimab we performed a retrospective multicenter study.ResultsOverall, 129 patients were included: 88 without diabetes before the treatment, 37 who were diagnosed with type 2 diabetes before the start of treatment, and 4 without diabetes before treatment who developed immune checkpoint inhibitor-induced diabetes (ICI-DM). PFS was 21.71 months (95% CI: 15.61–27.81) in patients without diabetes, 10.23 months (95% CI: 5.81–14.66) in patients with type 2 diabetes, and 50.85 months (95% CI: 23.04–78.65) in patients who developed ICI-DM. OS was 37.94 months (95% CI: 31.02–44.85) in patients without diabetes, 22.12 months (95% CI: 14.41–29.85) in those with type 2 diabetes and 57.64 months (95% CI: 42.29–72.99) in those who developed ICI-DM. Multivariate analysis showed that the presence of diabetes and LDH was correlated with OS and PFS. The mean OS was 64.63 months in subjects with low levels of glucose (< 137 mg/dl) and 36.27 months in those with high levels (hazard ratio 0.16, 95% CI: 0.04–0.58; p = 0.005). The patients whose glucose blood level increased after 3 months of treatment with nivolumab + relatinib compared to baseline (ratio of blood level at baseline/after 3 months > 1.5) had a worse prognosis than those whose glucose level had not increased. This result was observed also in subgroups treated either in first line or further lines. Patients who developed ICI-DM during the study period had better outcomes than the overall population and patients without diabetes.ConclusionsLAG3 inhibition for treating metastatic or unresectable melanoma has a reduced efficacy in patients with type 2 diabetes, possibly due to a low expression of LAG3 in tumor tissue. Higher level evidence should be obtained.

DNA methylation-regulated LINC02587 inhibits ferroptosis and promotes the progression of glioma cells through the CoQ-FSP1 pathway

BackgroundLong noncoding RNAs (lncRNAs) are considered key players in the formation and development of tumors. Herein, Gene Expression Profiling Interactive Analysis (GEPIA) was employed as a bioinformatics technology. LINC02587 is differentially expressed in bladder urothelial cancer, glioblastoma, lung adenocarcinoma, lung SCC, melanoma, and other tumor tissue and cells. However, its impact on the emergence of glioma and its mechanism is remaining elusive.MethodsSome of the in vitro assays employed in this study were the CCK-8 / Annexin-V / Transwell assays, colony formation, and wound healing, together with Western blot (WB) evaluation. MSP / BSP assays were employed for assessing the CpG island’s methylation status in the LINC02587 promoter. Through transcriptome, ferroptosis-related experiments, and WB evaluation, it was confirmed that LINC02587 is correlated with the regulation of ferroptosis in tumor cells, and CoQ-Fsp1 is one of its regulatory pathways. Moreover, the underlined in-vitro results were further validated by in-vivo studies.ResultsThe current study shows that the promoter sequence of LINC02587 is regulated by methylation. The silencing of LINC02587 can inhibit cellular proliferative, migrative, and invasive properties, and induce ferroptosis within gliomas through the CoQ-FSP1 pathway.ConclusionsLINC02587 is likely to be a novel drug target in treating glioma.

Lnc-PKNOX1-1 inhibits tumor progression in cutaneous malignant melanoma by regulating NF-κB/IL-8 axis.

Cutaneous malignant melanoma is one of the most lethal cutaneous malignancies. Accumulating evidence has demonstrated the potential influence of long non-coding RNAs (lncRNAs) in biological behaviors of melanoma. Herein, we reported a novel lncRNA, lnc-PKNOX1-1 and systematically studied its functions and possible molecular mechanisms in melanoma. Reverse transcription-quantitative PCR assay showed that lnc-PKNOX1-1 was significantly decreased in melanoma cells and tissues. Low lnc-PKNOX1-1 expression was significantly correlated with invasive pathological type and Breslow thickness of melanoma. In vitro and in vivo experiments showed lnc-PKNOX1-1 dramatically inhibited melanoma cell proliferation, migration and invasion. Mechanically, protein microarray analysis suggested that interleukin-8 (IL-8) was negatively regulated by lnc-PKNOX1-1 in melanoma, which was confirmed by western blot and ELISA. Western blot analysis also showed that lnc-PKNOX1-1 could promote p65 phosphorylation at Ser536 in melanoma. Subsequent rescue assays proved IL-8 overexpression could partly reverse the tumor-suppressing function of lnc-PKNOX1-1 overexpression in melanoma cells, indicating that lnc-PKNOX1-1 suppressed the development of melanoma by regulating IL-8. Taken together, our study demonstrated the tumor-suppressing ability of lnc-PKNOX1-1 in melanoma, suggesting its potential as a novel diagnostic biomarker and therapeutic target for melanoma.

DSE inhibits melanoma progression by regulating tumor immune cell infiltration and VCAN

Dermatan sulfate epimerase (DSE) is a C5 epiminase that plays a key role in converting chondroitin sulfate into dermal sulfate. DSE is often upregulated during carcinogenesis of some types of cancer and can regulate growth factor signaling in cancer cells. However, the expression and function of DSE in human melanoma have not been reported. In this study, we investigated the influence of tumor-derived DSE in melanoma progression and the potential mechanism of their action. First, proteomic analysis of collected melanoma tissues revealed that DSE was significantly down-regulated in melanoma tissues. DSE silenced or overexpressed melanoma cells were constructed to detect the effect of DSE on melanoma cells, and it was found that the up-regulation of DSE significantly inhibited the proliferation, migration and invasion of melanoma cells. Data analysis and flow cytometry were used to evaluate the immune subpopulations in tumors, and it was found that the high expression of DSE was closely related to the invasion of killer immune cells. Mechanistically, DSE promoted the expression of VCAN, which inhibited the biological activity of melanoma cells. Together, these results suggest that DSE is downregulated in melanoma tissues, and that high expression of DSE can promote melanoma progression by inducing immune cell infiltration and VCAN expression.

Upregulation of exosome secretion from tumor-associated macrophages plays a key role in the suppression of anti-tumor immunity

Macrophages play a pivotal role in tumor immunity. We report that reprogramming of macrophages to tumor-associated macrophages (TAMs) promotes the secretion of exosomes. Mechanistically, increased exosome secretion is driven by MADD, which is phosphorylated by Akt upon TAM induction and activates Rab27a. TAM exosomes carry high levels of programmed death-ligand 1 (PD-L1) and potently suppress the proliferation and function of CD8+ Tcells. Analysis of patient melanoma tissues indicates that TAM exosomes contribute significantly to CD8+ Tcell suppression. Single-cell RNA sequencing analysis showed that exosome-related genes are highly expressed in macrophages in melanoma; TAM-specific RAB27A expression inversely correlates with CD8+ Tcell infiltration. In a murine melanoma model, lipid nanoparticle delivery of small interfering RNAs (siRNAs) targeting macrophage RAB27A led to better Tcell activation and sensitized tumors to anti-programmed cell death protein 1 (PD-1) treatment. Our study demonstrates tumors use TAM exosomes to combat CD8 Tcells and suggests targeting TAM exosomes as a potential strategy to improve immunotherapies.

RaT: Raman Transformer for highly accurate melanoma detection with critical features visualization

Melanoma is an important cause of death from skin cancer. Early and accurate diagnosis can effectively reduce mortality. But the current diagnosis relies on the experience of pathologists, increasing the rate of misdiagnosis. In this paper, Raman Transformer (RaT) model is proposed by combining Raman spectroscopy and a Transformer encoder to distinguish the Raman spectra of melanoma and normal tissue. To make the spectral data more suitable for the Transformer encoder, we split the Raman spectrum into segments and map them into block vectors, which are then input into the Transformer encoder and classified using the multi-head self-attention mechanism and the Multilayer Perceptron (MLP). The RaT model achieves 99.69% accuracy, 99.61% sensitivity, and 99.82% specificity, which is higher than the classical principal component analysis with the neural network (PCA + NNET) method. In addition, we visualize and explain the fingerprint peaks found by the RaT model and their corresponding biological information. Our proposed RaT model provides a novel and reliable method for processing Raman spectral data, which is expected to help distinguish melanoma from normal cells, diagnose other diseases, and save human lives.

Phosphatase and Tensin Homolog (PTEN) Expression as a Surrogate Biomarker Correlated With the Depth of Invasion in Cutaneous Malignant Melanoma.

Objective The aim of this study is to evaluate the expression of the phosphatase and tensin homolog (PTEN), which is a tumor suppressor gene that is implicated in the pathogenesis of cutaneous malignant melanoma, in normal skin and melanoma tissue samples. The study also aimed to correlate PTEN expression levels with various clinicopathological parameters of melanoma lesions, thus highlighting the utility of PTEN expression as a prognostic biomarker for melanoma. Study design Immunohistochemistry (IHC) staining was performed on tissue microarray samples representing normal skin and melanoma biopsies of different clinicopathological parameters. Tissue photomicrographs were evaluated with Aperio ImageScope, which has a positive-pixel-counting algorithm built in. Subsequently, a histochemical score (H-score) was derived from the percentage of positive cells (%-staining) and their staining intensity. The H-scores were averaged in groups of tissue samples representing the different melanomas' tumor (T), node (N), and distant metastasis (M), also known as TNM parameters, as set forth by the American Joint Committee on Cancer (AJCC) classification. The mean H-scores were statistically compared using a two-tailed unpaired t-test. Results The PTEN protein expression was measured by IHC and found to be correlated with tumor thickness (T), which is a reliable indicator for survival rates. Specifically, PTEN was significantly downregulated in tumors with a thickness over 2 mm (T3+T4) compared to tumors with a thickness at or below 2 mm (T1+T2). Conclusions The PTEN protein expression, as measured by immunohistochemistry, helped differentiate between tumors with a thickness over 2 mm and tumors with a thickness at or below 2 mm, suggesting PTEN as a potential surrogate marker for the melanoma's invasion depth along with possible prognostic implications. Longitudinal studies evaluating risk stratification based on the expression of PTEN are needed to establish the utility of this promising biomarker in the clinic as an adjunct for pathological examination.

Putative Cancer Stem Cell Markers are Frequently Expressed by Melanoma Cells in Vitro and in Situ but are also Present in Benign Differentiated Cells.

Currently, there remains an incomplete view of cancer stem cells (CSCs) in solid tumours. We studied a panel of putative CSC surface markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) in 40 established melanoma cell lines and four early-passage melanoma strains by flow cytometry. We additionally examined 40 formalin-fixed paraffin-embedded melanoma tissues using immunofluorescence microscopy. This was compared with their expression in healthy skin, normal differentiated melanocytes and fibroblasts. Most of the putative CSC markers were expressed by both melanoma cell lines and tissues. When present, these proteins were expressed by the majority of cells in the population. However, the expression of these markers by cells in healthy skin sections, normal differentiated melanocytes, and fibroblasts revealed that differentiated non-malignant cells also expressed CSC markers indicating that they lack of specificity for CSCs. Culturing cell lines under conditions more characteristic of the tumour microenvironment upregulated CSC marker expressions in a proportion of cell lines, which correlated with improved cell growth and viability. The testing of melanoma cell lines (n = 40), early-passage cell strains (n = 4), and melanoma tissues (n = 40) showed that several putative CSC markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) are commonly present in a large proportion of melanoma cells in vitro and in situ. Further, we showed that these putative markers lack specificity for CSCs because they are also expressed in differentiated non-malignant cell types (melanocytes, fibroblasts, and skin), which could limit their use as therapeutic targets. These data are consistent with the emerging notion of CSC plasticity and phenotype switching within cancer cell populations.

Bioinformatics-based analysis reveals elevated CYTL1 as a potential therapeutic target for BRAF-mutated melanoma.

Introduction: Despite many recent emerging therapeutic modalities that have prolonged the survival of melanoma patients, the prognosis of melanoma remains discouraging, and further understanding of the mechanisms underlying melanoma progression is needed. Melanoma patients often have multiple genetic mutations, with BRAF mutations being the most common. In this study, public databases were exploited to explore a potential therapeutic target for BRAF-mutated melanoma. Methods: In this study, we analyzed differentially expressed genes (DEGs) in normal tissues and melanomas, Braf wild-type and Braf mutant melanomas using information from TCGA databases and the GEO database. Subsequently, we analyzed the differential expression of CYTL1 in various tumor tissues and its effect on melanoma prognosis, and resolved the mutation status of CYTL1 and its related signalling pathways. By knocking down CYTL1 in melanoma cells, the effects of CYTL1 on melanoma cell proliferation, migration and invasion were further examined by CCK8 assay, Transwell assay and cell migration assay. Results: 24 overlapping genes were identified by analyzing DEGs common to melanoma and normal tissue, BRAF-mutated and BRAF wild-type melanoma. Among them, CYTL1 was highly expressed in melanoma, especially in BRAF-mutated melanoma, and the high expression of CYTL1 was associated with epithelial-mesenchymal transition (EMT), cell cycle, and cellular response to UV. In melanoma patients, especially BRAF-mutated melanoma patients, clinical studies showed a positive correlation between increased CYTL1 expression and shorter overall survival (OS) and disease-free survival (DFS). In vitro experiments further confirmed that the knockdown of CYTL1 significantly inhibited the migration and invasive ability of melanoma cells. Conclusion: CYTL1 is a valuable prognostic biomarker and a potentially effective therapeutic target in melanoma, especially BRAF-mutated melanoma.

Plasma Methylated DNA Markers for Melanoma Surveillance.

Surveillance after primary melanoma treatment aims to detect early signs of low-volume systemic disease. The current standard of care, surveillance imaging, is costly and difficult to access. We therefore sought to develop methylated DNA markers (MDMs) as promising alternatives for disease surveillance. We used reduced representation bisulfite sequencing (RRBS) to identify MDMs in DNA samples obtained from metastatic melanoma, benign nevi, and normal skin tissues. The identified MDMs underwent validation in an independent cohort of tissue and buffy coat DNA samples. Subsequently, we tested the validated MDMs in the plasma DNA of patients with metastatic melanoma undergoing surveillance with total body imaging and compared them with cancer-free controls. To estimate the overall predictive accuracy of the MDMs, we used random forest modeling with bootstrap cross-validation. Forty MDMs demonstrated discrimination between melanoma cases and controls consisting of benign nevi and normal skin. Nine MDMs passing biological validation in tissue were run on 77 plasma samples from individuals with a history of metastatic melanoma, 49 of whom had evidence of disease detected by imaging at the time of blood draw, and 100 cancer-free controls. The cross-validated sensitivity of the panel for imaging-positive disease was 80% with a specificity of 100% in cancer-free controls, resulting in an overall AUC of 0.88 (95% CI, 0.81 to 0.96). The survival estimates for patients with melanoma who tested positive for the panel at 6 months and 1 year were 67% and 56%, respectively, while those who tested negative had survival rates of 100% and 92%. MDMs identified by RRBS demonstrate a high degree of concordance with imaging results in the plasma of patients with metastatic melanoma. Further prospective studies in larger intended use cohorts are needed to confirm these findings.

Learning how to detect: A deep reinforcement learning method for whole-slide melanoma histopathology images.

Cutaneous melanoma represents one of the most life-threatening malignancies. Histopathological image analysis serves as a vital tool for early melanoma detection. Deep neural network (DNN) models are frequently employed to aid pathologists in enhancing the efficiency and accuracy of diagnoses. However, due to the paucity of well-annotated, high-resolution, whole-slide histopathology image (WSI) datasets, WSIs are typically fragmented into numerous patches during the model training and testing stages. This process disregards the inherent interconnectedness among patches, potentially impeding the models' performance. Additionally, the presence of excess, non-contributing patches extends processing times and introduces substantial computational burdens. To mitigate these issues, we draw inspiration from the clinical decision-making processes of dermatopathologists to propose an innovative, weakly supervised deep reinforcement learning framework, titled Fast medical decision-making in melanoma histopathology images (FastMDP-RL). This framework expedites model inference by reducing the number of irrelevant patches identified within WSIs. FastMDP-RL integrates two DNN-based agents: the search agent (SeAgent) and the decision agent (DeAgent). The SeAgent initiates actions, steered by the image features observed in the current viewing field at various magnifications. Simultaneously, the DeAgent provides labeling probabilities for each patch. We utilize multi-instance learning (MIL) to construct a teacher-guided model (MILTG), serving a dual purpose: rewarding the SeAgent and guiding the DeAgent. Our evaluations were conducted using two melanoma datasets: the publicly accessible TCIA-CM dataset and the proprietary MELSC dataset. Our experimental findings affirm FastMDP-RL's ability to expedite inference and accurately predict WSIs, even in the absence of pixel-level annotations. Moreover, our research investigates the WSI-based interactive environment, encompassing the design of agents, state and reward functions, and feature extractors suitable for melanoma tissue images. This investigation offers valuable insights and references for researchers engaged in related studies. The code is available at: https://github.com/titizheng/FastMDP-RL.

Invasion depth estimation of carcinoma cells using adaptive stain normalization to improve epidermis segmentation accuracy.

Submucosal invasion depth is a significant prognostic factor when assessing lymph node metastasis and cancer itself to plan proper treatment for the patient. Conventionally, oncologists measure the invasion depth by hand which is a laborious, subjective, and time-consuming process. The manual pathological examination by measuring accurate carcinoma cell invasion with considerable inter-observer and intra-observer variations is still challenging. The increasing use of medical imaging and artificial intelligence reveals a significant role in clinical medicine and pathology. In this paper, we propose an approach to study invasive behavior and measure the invasion depth of carcinoma from stained histopathology images. Specifically, our model includes adaptive stain normalization, color decomposition, and morphological reconstruction with adaptive thresholding to separate the epithelium with blue ratio image. Our method splits the image into multiple non-overlapping meaningful segments and successfully finds the homogeneous segments to measure accurate invasion depth. The invasion depths are measured from the inner epithelium edge to outermost pixels of the deepest part of particles in image. We conduct our experiments on skin melanoma tissue samples as well as on organotypic invasion model utilizing myoma tissue and oral squamous cell carcinoma. The performance is experimentally compared to three closely related reference methods and our method provides a superior result in measuring invasion depth. This computational technique will be beneficial for the segmentation of epithelium and other particles for the development of novel computer-aided diagnostic tools in biobank applications.

Clinical-mediated discovery of pyroptosis in CD8+ T cell and NK cell reveals melanoma heterogeneity by single-cell and bulk sequence

Histologically, melanoma tissues had fewer positive cells percentage of pyroptosis-related genes (PRGs), GZMA, GSDMB, NLRP1, IL18, and CHMP4A in epidermal than in normal skin. Pyroptosis, a new frontier in cancer, affects the tumor microenvironment and tumor immunotherapy. Nevertheless, the role of pyroptosis remains controversial, which reason is partly due to the heterogeneity of the cellular composition in melanoma. In this study, we present a comprehensive analysis of the single-cell transcriptome landscape of pyroptosis in melanoma specimens. Our findings reveal dysregulation in the expression of PRGs, particularly in immune cells, such as CD8+ cells (representing CD8+ T cells) and CD57+ cells (representing NK cells). Additionally, the immunohistochemical and multiplex immunofluorescence staining experiments results further confirmed GZMA+ cells and GSDMB+ cells were predominantly expressed in immune cells, especially in CD8 + T cells and NK cells. Melanoma specimens secreted a minimal presence of GZMA+ merged CD8+ T cells (0.11%) and GSDMB+ merged CD57+ cells (0.08%), compared to the control groups exhibiting proportions of 4.02% and 0.62%, respectively. The aforementioned findings indicate that a reduced presence of immune cells within tumors may play a role in diminishing the ability of pyroptosis, consequently posing a potential risk to the anti-melanoma properties. To quantify clinical relevance, we constructed a prognostic risk model and an individualized nomogram (C-index=0.58, P = 0.002), suggesting a potential role of PRGs in malignant melanoma prevention. In conclusion, our integrated single-cell and bulk RNA-seq analysis identified immune cell clusters and immune gene modules with experiment validation, contributing to our better understanding of pyroptosis in melanoma.

Poly(A)-specific ribonuclease protein promotes the proliferation, invasion and migration of esophageal cancer cells.

Bioinformatics analysis showed that the expression of the poly(A)-specific ribonuclease (PARN) gene in gastric cancer, head and neck squamous cell carcinoma, melanoma, cervical cancer and lung squamous cell carcinoma tissues was significantly higher than that in normal tissues and was associated with high stage and poor prognosis. The expression of the PARN gene in esophageal cancer (EC) tissue is also significantly higher than that in normal tissues, but the effect of PARN on the proliferation, migration and invasion of EC cells remains unclear. To investigate the relationship between PARN and the proliferation, migration and invasion of EC cells. The EC tissues of 91 patients after EC surgery and 63 paired precancerous healthy tissues were collected. PARN mRNA levels were measured using a tissue microarray, and the PARN expression level was evaluated using immunohistochemistry to analyze the relationship between PARN expression and clinicopathologic features as well as the survival and prognosis of patients. In addition, the effects of PARN gene knockout on tumor cell proliferation, invasion and migration were studied by using shRNA during the in vitro culture of EC cell lines Eca-109 and TE-1, and the effects of the PARN gene on tumor growth in vivo were verified by a xenotransplantation nude mice model. The expression of PARN in EC tissues was higher than that in adjacent normal tissues, and the level of PARN expression was significantly positively correlated with lymphatic metastasis. Patients with high PARN levels had poor overall survival. BIM, IGFBP-5 and p21 levels were significantly increased in the PARN knockout group, while the expression levels of the antiapoptotic proteins Survivin and sTNF-R1 were significantly decreased in the apoptotic antibody array data. In addition, the expression levels of Akt, p-Akt, PIK3CA and CCND1 in the downstream signaling pathway regulating EC progression were significantly decreased. The culture of EC cell lines confirmed that the apoptosis rate of EC cells was significantly increased, the growth and proliferation of tumor cells were significantly inhibited, and the invasion and migration ability of tumor cells were significantly decreased after PARN gene knockout. In vivo experiments of BALB/c nude mice transfected with Eca-109 cells expressing control shRNA (sh-NC) and PARN shRNA (sh-PARN) showed that the tumor volume and weight of nude mice treated with sh-PARN were significantly decreased compared with those of nude mice treated with sh-NC, indicating that PARN knockdown significantly inhibited tumor growth in vivo. PARN has antiapoptotic effects on EC cells and promotes their proliferation, invasion and migration, which is associated with the development of EC and poor patient prognosis. PARN may become a potential target for the diagnosis, prognosis prediction and treatment of EC.