Corrigendum: Risk perceptions and changes in tobacco use in relation to Coronavirus Disease 2019 pandemic: A qualitative study on adolescent tobacco users in Hong Kong.

[This corrects the article DOI: 10.18332/tid/167479.].

The significance of an immunohistochemical marker-based panel in assisting the diagnosis of parathyroid carcinoma.

PURPOSE: Parathyroid carcinoma (PC) is an endocrine malignancy with a poor prognosis. However, the diagnosis of PC is still a difficult problem. A model with immunohistochemical (IHC) staining of 5 biomarkers has been reported from limited samples for the differential diagnosis of PC. In the present study, a series of IHC markers was applied in relatively large samples to optimize the diagnostic model for PC. METHODS: In this study, 44 patients with PC, 6 patients with atypical parathyroid tumors and 57 patients with parathyroid adenomas were included. IHC staining for parafibromin, Ki-67, galectin-3, protein-encoding gene product 9.5 (PGP9.5), E-cadherin, and enhancer of zeste homolog 2 (EZH2) was performed on formalin-fixed, paraffin-embedded tissue samples. The effects of clinical characteristics, surgical procedure, and IHC staining results of tumor tissues on the diagnosis and prognosis of PC were evaluated retrospectively. RESULTS: A logistic regression model with IHC results of parafibromin, Ki-67, and E-cadherin was created to differentiate PC with an area under the curve of 0.843. Cox proportional hazards analysis showed that negative parafibromin staining (hazard ratio: 3.26, 95% confidence interval: 1.28-8.34, P = 0.013) was related to the recurrence of PC. CONCLUSION: An IHC panel of parafibromin, Ki-67 and E-cadherin may help to distinguish PC from parathyroid neoplasms. Among the 6 IHC markers and clinical features examined, the risk factor related to PC recurrence was parafibromin staining loss.

Blockade of the lncRNA-DOT1L-LAMP5 axis enhances autophagy and promotes degradation of MLL fusion proteins.

BACKGROUND: Mixed-lineage leukemia (MLL) fusion gene caused by chromosomal rearrangement is a dominant oncogenic driver in leukemia. Due to having diverse MLL rearrangements and complex characteristics, MLL leukemia treated by currently available strategies is frequently associated with a poor outcome. Therefore, there is an urgent need to identify novel therapeutic targets for hematological malignancies with MLL rearrangements. METHODS: qRT-PCR, western blot, and spearman correction analysis were used to validate the regulation of LAMP5-AS1 on LAMP5 expression. In vitro and in vivo experiments were conducted to assess the functional relevance of LAMP5-AS1 in MLL leukemia cell survival. We utilized chromatin isolation by RNA purification (ChIRP) assay, RNA pull-down assay, chromatin immunoprecipitation (ChIP), RNA fluorescence in situ hybridization (FISH), and immunofluorescence to elucidate the relationship among LAMP5-AS1, DOT1L, and the LAMP5 locus. Autophagy regulation by LAMP5-AS1 was evaluated through LC3B puncta, autolysosome observation via transmission electron microscopy (TEM), and mRFP-GFP-LC3 puncta in autophagic flux. RESULTS: The study shows the crucial role of LAMP5-AS1 in promoting MLL leukemia cell survival. LAMP5-AS1 acts as a novel autophagic suppressor, safeguarding MLL fusion proteins from autophagic degradation. Knocking down LAMP5-AS1 significantly induced apoptosis in MLL leukemia cell lines and primary cells and extended the survival of mice in vivo. Mechanistically, LAMP5-AS1 recruits the H3K79 histone methyltransferase DOT1L to LAMP5 locus, directly activating LAMP5 expression. Importantly, blockade of LAMP5-AS1-LAMP5 axis can represses MLL fusion proteins by enhancing their degradation. CONCLUSIONS: The findings underscore the significance of LAMP5-AS1 in MLL leukemia progression through the regulation of the autophagy pathway. Additionally, this study unveils the novel lncRNA-DOT1L-LAMP5 axis as promising therapeutic targets for degrading MLL fusion proteins.

Design and Fabrication of a 3D-Printed Microfluidic Immunoarray for Ultrasensitive Multiplexed Protein Detection.

Microfluidic technology has revolutionized device fabrication by merging principles of fluid dynamics with technologies from chemistry, physics, biology, material science, and microelectronics. Microfluidic systems manipulate small volumes of fluids to perform automated tasks with applications ranging from chemical syntheses to biomedical diagnostics. The advent of low-cost 3D printers has revolutionized the development of microfluidic systems. For measuring molecules, 3D printing offers cost-effective, time, and ease-of-designing benefits. In this paper, we present a comprehensive tutorial for design, optimization, and validation for creating a 3D-printed microfluidic immunoarray for ultrasensitive detection of multiple protein biomarkers. The target is the development of a point of care array to determine five protein biomarkers for aggressive cancers. The design phase involves defining dimensions of microchannels, reagent chambers, detection wells, and optimizing parameters and detection methods. In this study, the physical design of the array underwent multiple iterations to optimize key features, such as developing open detection wells for uniform signal distribution and a flap for covering wells during the assay. Then, full signal optimization for sensitivity and limit of detection (LOD) was performed, and calibration plots were generated to assess linear dynamic ranges and LODs. Varying characteristics among biomarkers highlighted the need for tailored assay conditions. Spike-recovery studies confirmed the assay's accuracy. Overall, this paper showcases the methodology, rigor, and innovation involved in designing a 3D-printed microfluidic immunoarray. Optimized parameters, calibration equations, and sensitivity and accuracy data contribute valuable metrics for future applications in biomarker analyses.

A COM-B analysis of facilitators of and barriers to smoking cessation among Chinese smokers: A qualitative study.

INTRODUCTION: Smoking prevalence remains high in China with a low cessation motivation level, despite the government's tobacco control efforts. There is a lack of research specifically examining perceptions, attitudes, and behaviors related to smoking cessation in this region, particularly from a theory-based deductive perspective. Utilizing the COM-B (Capability, Opportunity, Motivation-Behavior) model as a theoretical framework, this study aimed to identify facilitators and barriers to smoking cessation among Chinese smokers. METHODS: The study employed semi-structured individual interviews with 40 participants. Each interview spanned approximately 30 minutes. The participants, constituting both current and former smokers, were all aged ≥18 years (n=40). Interview data were then examined using a directed content analysis approach. RESULTS: Analysis revealed three interrelated themes. Capability: Smokers face challenges when resisting peer pressure and dealing with life after quitting. They also lack knowledge about smoking, quitting techniques, and withdrawal symptoms. Opportunity: Changing societal attitudes towards smoking create opportunities for quitting, but these are hindered by inadequate cessation services and a lack of family support. Motivation: Smokers' motivation to quit is mainly driven by health concerns. Resistance to quitting often stems from the belief that smoking is a personal choice or just a habit. Excessive emphasis on willpower may hinder motivation to quit. CONCLUSIONS: To enhance smoking cessation efforts in China, three key aspects should be considered: capability, opportunity, and motivation. Publicity and educational campaigns should target common misconceptions about smoking as a personal freedom, correct the overemphasis on willpower, and widely promote available cessation services. A crucial aspect is shifting societal norms to foster anti-smoking sentiments. Effective strategies may involve using real-life stories to illustrate smoking's health consequences, disseminating information about cessation services in maternity centers, enhancing services through mobile health initiatives, and empowering families to support smokers in their quit attempts.

Cyst fluid glycoproteins accurately distinguishing malignancies of pancreatic cystic neoplasm.

Pancreatic cystic neoplasms (PCNs) are recognized as precursor lesions of pancreatic cancer, with a marked increase in prevalence. Early detection of malignant PCNs is crucial for improving prognosis; however, current diagnostic methods are insufficient for accurately identifying malignant PCNs. Here, we utilized mass spectrometry (MS)-based glycosite- and glycoform-specific glycoproteomics, combined with proteomics, to explore potential cyst fluid diagnostic biomarkers for PCN. The glycoproteomic and proteomic landscape of pancreatic cyst fluid samples from PCN patients was comprehensively investigated, and its characteristics during the malignant transformation of PCN were analyzed. Under the criteria of screening specific cyst fluid biomarkers for the diagnosis of PCN, a group of cyst fluid glycoprotein biomarkers was identified. Through parallel reaction monitoring (PRM)-based targeted glycoproteomic analysis, we validated these chosen glycoprotein biomarkers in a second cohort, ultimately confirming N-glycosylated PHKB (Asn-935, H5N2F0S0; Asn-935, H4N4F0S0; Asn-935, H5N4F0S0), CEACAM5 (Asn-197, H5N4F0S0) and ATP6V0A4 (Asn-367, H6N4F0S0) as promising diagnostic biomarkers for distinguishing malignant PCNs. These glycoprotein biomarkers exhibited robust performance, with an area under the curve ranging from 0.771 to 0.948. In conclusion, we successfully established and conducted MS-based glycoproteomic analysis to identify novel cyst fluid glycoprotein biomarkers for PCN. These findings hold significant clinical implications, providing valuable insights for PCN decision-making, and potentially offering therapeutic targets for PCN treatment.

Follicular lymphoma evolves with a surmountable dependency on acquired glycosylation motifs in the B-cell receptor.

ABSTRACT: An early event in the genesis of follicular lymphoma (FL) is the acquisition of new glycosylation motifs in the B-cell receptor (BCR) due to gene rearrangement and/or somatic hypermutation. These N-linked glycosylation motifs (N-motifs) contain mannose-terminated glycans and can interact with lectins in the tumor microenvironment, activating the tumor BCR pathway. N-motifs are stable during FL evolution, suggesting that FL tumor cells are dependent on them for their survival. Here, we investigated the dynamics and potential impact of N-motif prevalence in FL at the single-cell level across distinct tumor sites and over time in 17 patients. Although most patients had acquired at least 1 N-motif as an early event, we also found (1) cases without N-motifs in the heavy or light chains at any tumor site or time point and (2) cases with discordant N-motif patterns across different tumor sites. Inferring phylogenetic trees of the patients with discordant patterns, we observed that both N-motif-positive and N-motif-negative tumor subclones could be selected and expanded during tumor evolution. Comparing N-motif-positive with N-motif-negative tumor cells within a patient revealed higher expression of genes involved in the BCR pathway and inflammatory response, whereas tumor cells without N-motifs had higher activity of pathways involved in energy metabolism. In conclusion, although acquired N-motifs likely support FL pathogenesis through antigen-independent BCR signaling in most patients with FL, N-motif-negative tumor cells can also be selected and expanded and may depend more heavily on altered metabolism for competitive survival.

Direct measurement of engineered cancer mutations and their transcriptional phenotypes in single cells.

Genome sequencing studies have identified numerous cancer mutations across a wide spectrum of tumor types, but determining the phenotypic consequence of these mutations remains a challenge. Here, we developed a high-throughput, multiplexed single-cell technology called TISCC-seq to engineer predesignated mutations in cells using CRISPR base editors, directly delineate their genotype among individual cells and determine each mutation's transcriptional phenotype. Long-read sequencing of the target gene's transcript identifies the engineered mutations, and the transcriptome profile from the same set of cells is simultaneously analyzed by short-read sequencing. Through integration, we determine the mutations' genotype and expression phenotype at single-cell resolution. Using cell lines, we engineer and evaluate the impact of >100 TP53 mutations on gene expression. Based on the single-cell gene expression, we classify the mutations as having a functionally significant phenotype.

Risk perceptions and changes in tobacco use in relation to Coronavirus Disease 2019 pandemic: A qualitative study on adolescent tobacco users in Hong Kong.

INTRODUCTION: Tobacco use is associated with an increased risk of Coronavirus Disease 2019 (COVID-19) infection, severe COVID-19 outcomes requiring intensive care, and mortality. We investigated the perceived risk of and changes in cigarette, e-cigarette (EC) and heated tobacco product (HTP) use in relation to COVID-19 in Hong Kong adolescent tobacco users. METHODS: We conducted semi-structured telephone interviews from January to April 2021 and in February 2022 on 40 adolescents (65% boys, Secondary school grades 2-6) who participated in our previous smoking surveys and were using cigarettes, ECs or HTPs before the first wave of the COVID-19 pandemic in January 2020. RESULTS: Adolescents generally perceived higher risks of contracting and having more severe COVID-19 from using cigarettes than ECs/HTPs, but they had limited knowledge of COVID-19 risks from EC/HTP use, particularly. Both increased and reduced consumption were found in tobacco, with EC use being the less affected product. Changes also included switching to ECs for convenience and lower cost and shifting from smoking cigarettes outside to mainly at home or in hidden areas. COVID-related policies, fear of infection, non-COVID-related health concerns, less social opportunities and pocket money, and limited access to tobacco products were barriers to tobacco use. In contrast, greater freedom at home versus school and negative emotions due to social distancing were facilitators. Family/peer influence had mixed impacts. CONCLUSIONS: Adolescent tobacco users perceived lower COVID risks associated with HTPs and ECs than cigarettes, and various changes in tobacco use were found amid the pandemic in Hong Kong. COVID-19 and related social changes may both facilitate or deter adolescent tobacco use.

Six polyacetylenes from Atractylodes macrocephala Koidz and their anti-colon cancer activity.

Six undescribed polyacetylenes Atracetylenes A-F (1-6) and three known ones (7-9) were isolated from the rhizomes of Atractylodes macrocephala Koidz.. The comprehensive interpretation of NMR, HR-ESI-MS, DP4+ calculations, and electronic circular dichroism (ECD) calculations resulted in the elucidation of their structures and absolute configurations. The anti-colon cancer activities of (1-9) were evaluated by assaying the cytotoxicity and apoptosis on CT-26 cell lines. Notably, 5 (IC50 17.51 ± 1.41 µM) and 7 (IC50 18.58 ± 1.37 µM) exhibited significant cytotoxicity, and polyacetylenes 3-6 showed excellent abilities to promote apoptosis of CT-26 cell lines by Annexin V-FITC/PI assay. The results demonstrated that the polyacetylenes in A. macrocephala may be prospective for the treatment of colorectal cancer.

METTL3 protects METTL14 from STUB1-mediated degradation to maintain m6 A homeostasis.

N6 -Methyladenosine (m6 A) is an important RNA modification catalyzed by methyltransferase-like 3 (METTL3) and METTL14. m6 A homeostasis mediated by the methyltransferase (MTase) complex plays key roles in various biological processes. However, the mechanism underlying METTL14 protein stability and its role in m6 A homeostasis remain elusive. Here, we show that METTL14 stability is regulated by the competitive interaction of METTL3 with the E3 ligase STUB1. STUB1 directly interacts with METTL14 to mediate its ubiquitination at lysine residues K148, K156, and K162 for subsequent degradation, resulting in a significant decrease in total m6 A levels. The amino acid regions 450-454 and 464-480 of METTL3 are essential to promote METTL14 stabilization. Changes in STUB1 expression affect METTL14 protein levels, m6 A modification and tumorigenesis. Collectively, our findings uncover an ubiquitination mechanism controlling METTL14 protein levels to fine-tune m6 A homeostasis. Finally, we present evidence that modulating STUB1 expression to degrade METTL14 could represent a promising therapeutic strategy against cancer.

The snoRNA-like lncRNA LNC-SNO49AB drives leukemia by activating the RNA-editing enzyme ADAR1.

Long noncoding RNAs (lncRNAs) are usually 5' capped and 3' polyadenylated, similar to most typical mRNAs. However, recent studies revealed a type of snoRNA-related lncRNA with unique structures, leading to questions on how they are processed and how they work. Here, we identify a novel snoRNA-related lncRNA named LNC-SNO49AB containing two C/D box snoRNA sequences, SNORD49A and SNORD49B; and show that LNC-SNO49AB represents an unreported type of lncRNA with a 5'-end m7G and a 3'-end snoRNA structure. LNC-SNO49AB was found highly expressed in leukemia patient samples, and silencing LNC-SNO49AB dramatically suppressed leukemia progression in vitro and in vivo. Subcellular location indicated that the LNC-SNO49AB is mainly located in nucleolus and interacted with the nucleolar protein fibrillarin. However, we found that LNC-SNO49AB does not play a role in 2'-O-methylation regulation, a classical function of snoRNA; instead, its snoRNA structure affected the lncRNA stability. We further demonstrated that LNC-SNO49AB could directly bind to the adenosine deaminase acting on RNA 1(ADAR1) and promoted its homodimerization followed by a high RNA A-to-I editing activity. Transcriptome profiling shows that LNC-SNO49AB and ADAR1 knockdown respectively share very similar patterns of RNA modification change in downstream signaling pathways, especially in cell cycle pathways. These findings suggest a previously unknown class of snoRNA-related lncRNAs, which function via a manner in nucleolus independently on snoRNA-guide rRNA modification. This is the first report that a lncRNA regulates genome-wide RNA A-to-I editing by enhancing ADAR1 dimerization to facilitate hematopoietic malignancy, suggesting that LNC-SNO49AB may be a novel target in therapy directed to leukemia.

Patterns and Predictors of Cervical Lymph Node Metastasis in Parathyroid Carcinoma.

BACKGROUND: Parathyroid carcinoma (PC) is a rare endocrine malignancy with poor outcomes. Over 60% of PC patients experience repeated disease recurrence or metastasis. The significance of cervical lymph node dissection (LND) for PC remains inconclusive. METHODS: PC patients diagnosed at Peking Union Medical College Hospital between 1992 and 2021 were reviewed retrospectively. Clinical data, initial tumor histological staging, parafibromin histochemical staining results, Ki67 index, CDC73 gene mutation status and outcome information were collected systemically. The risk factors for recurrence and lymph node or distant metastasis were explored. RESULTS: Sixty-eight PC patients receiving LND were enrolled. Cervical lymph node metastasis was identified in 19.4% of patients at initial surgery and 25.0% of patients including reoperations for recurrences. The independent risk factor for PC recurrence was a Ki67 index ≥ 5% (HR4.41, 95% confidence interval (CI)1.30-14.95, p = 0.017). Distant metastasis was an independent prognostic factor for PC patient overall survival (HR 5.44, 95% CI 1.66-17.82, p = 0.005). High-risk Schulte staging (p = 0.021) and CDC73 abnormalities (p = 0.012) were risk factors for cervical lymph node metastasis. CONCLUSION: Most PCs were slow-growing, but lymph node metastasis was not rare. For patients planning to undergo remedial surgery after previous local resection of PC, central LND is suggested for tumors with high-risk Schulte staging or CDC73 abnormalities.

Kinsenoside alleviates inflammation and fibrosis in experimental NASH mice by suppressing the NF-κB/NLRP3 signaling pathway.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) has replaced viral hepatitis as the main driver of the rising morbidity and mortality associated with cirrhosis and liver cancer worldwide, while no FDA-approved therapies are currently known. Kinsenoside (KD), naturally isolated from Anoectochilus roxburghii, possesses multiple biological activities, including lipolysis, anti-inflammation, and hepatoprotection. However, the effects of KD on NASH remain unclear. PURPOSE: This study aimed to explore the roles of KD in NASH and its engaged mechanisms. METHODS: Two typical animal models of NASH, mice fed a methionine-choline-deficient (MCD) diet (representing non-obese NASH) and mice fed a high-fat and -fructose diet (HFFD) (representing obese NASH), were used to investigate the effect of KD on NASH in vivo. Transcriptome sequencing was performed to elucidate the underlying mechanisms of KD. Lipopolysaccharide (LPS)-stimulated THP-1 cells and transforming growth factor ß1 (TGF-ß1)-activated LX-2 cells were applied to further explore the effects and mechanisms of KD in vitro. RESULTS: The intragastric administration of KD remarkably alleviated MCD/HFFD-induced murine NASH almost in a dose-dependent manner. Specifically, KD reduced lipid accumulation, inflammation, and fibrosis in the liver of NASH mice. KD ameliorated alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), and malondialdehyde (MDA) abnormalities. In addition, it decreased the level of serum proinflammatory factors (IL-12p70, IL-6, TNF-α, MCP-1, IFN-γ) and the hepatic expression of typical fibrosis-related molecules (α-SMA, Col-I, TIMP-1). Mechanically, KD attenuated the MCD/HFFD-induced NASH through the inhibition of the NF-κB/NLRP3 signaling pathway. Consistently, KD reduced inflammation stimulated by LPS in THP-1 cells via suppressing the NF-κB/NLRP3 pathway. Furthermore, it prevented the activation of LX-2 cells directly, by inhibiting the proliferation stimulated by TGF-ß1, and indirectly, by inactivating the NLRP3 inflammasome in macrophages. CONCLUSION: For the first time, the practical improvement of NASH by KD was revealed. Our study found that KD exerted its alleviative effects on NASH through the inhibition of the NF-κB/NLRP3 signaling pathway. Given its hepatoprotective and nontoxic properties, KD has the potential to be a novel and effective drug to treat NASH.

Methyl 6-O-cinnamoyl-α-d-glucopyranoside Ameliorates Acute Liver Injury by Inhibiting Oxidative Stress Through the Activation of Nrf2 Signaling Pathway.

Excessive stimulation of hepatotoxins and drugs often lead to acute liver injury, while treatment strategies for acute liver injury have been limited. Methyl 6-O-cinnamoyl-α-d-glucopyranoside (MCGP) is a structure modified compound from cinnamic acid, a key chemical found in plants with significant antioxidant, anti-inflammatory, and antidiabetic effects. In this study, we investigated the effects and underlying mechanisms of MCGP on acetaminophen (APAP)- or carbon tetrachloride (CCl4)-induced acute liver injury. As a result, MCGP inhibited cell death and apoptosis induced by APAP or CCl4, and suppressed the reactive oxygen species (ROS) generation stimulated by H2O2 in liver AML12 cells. In vivo, MCGP alleviated APAP/CCl4-induced hepatic necrosis and resumed abnormal aminotransferase activities and liver antioxidase activities. In addition, MCGP depressed APAP- or CCl4-induced oxidative stress through the suppression of CYP2E1 and activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. MCGP also enhanced the number of PCNA-positive hepatocytes, increased hepatic PCNA and Bcl-XL, and decreased BAX expression in APAP-/CCl4-intoxicated mice. Furthermore, MCGP activated the GSDMD-N/cleaved caspase 1 pathway. In summary, MCGP might act as a potential therapeutic drug against drug-induced and chemical-induced acute liver injuries, and its underlying mechanisms might engage on the pressing of oxidative stress, refraining of hepatocyte apoptosis, and facilitating of liver regeneration.

Chromatin-associated orphan snoRNA regulates DNA damage-mediated differentiation via a non-canonical complex.

Small nucleolar RNAs (snoRNAs) are commonly acknowledged as a class of homogeneous non-coding RNAs that guide ribosomal RNA modifications. However, snoRNAs referred to as orphans have largely unknown functions. Here, we systematically profile chromatin-associated snoRNAs (casnoRNAs) in mammalian cells and identify a subgroup of orphan casnoRNAs responding to DNA damage stress, among which SNORA73 shows the most marked reduction in chromatin enrichment. Downregulated SNORA73 maintains cancer genome stability and differentiation block in hematopoietic malignancy. Mechanistically, casnoRNA the 5' end non-canonical structure of SNORA73 is critical for its function and binding to poly (ADP-ribose) polymerase 1 (PARP1). SNORA73 inhibits PARP1 auto-PARylation to affect cancer genome stability by forming a small nucleolar ribonucleoprotein (snoRNP) with PARP1 and canonical H/ACA proteins DKC1/NHP2. Our findings reveal the role of an orphan snoRNA serving as casnoRNA and highlights a link between non-canonical structure of snoRNA and their functional diversity.

The Current Application and Future Prospects of Astragalus Polysaccharide Combined With Cancer Immunotherapy: A Review.

So far, immunotherapy has been shown to have impressive effects on different cancers in clinical trials. All those immunotherapies are generally derived from three main therapeutic approaches: immune checkpoint inhibitors, immune cell vaccination, and adoptive cellular immunotherapy. Our research systematically reviewed a wide range of clinical trials and laboratory studies of astragalus polysaccharide (APS) and elucidated the potential feasibility of using APS in activating adoptive immunotherapy. Apart from being effective in adaptive "passive" immunotherapy such as lymphokine-activated killer treatment and dendritic cell (DC)-cytokine-induced killer treatment, APS could also regulate the anti-programmed cell death protein 1 (PD-1)/PD-L1 on the surface of the immune cells, as a part in the immune checkpoint inhibitory signaling pathway by activating the immune-suppressed microenvironment by regulating cytokines, toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) pathways, and immune cells, such as DCs, macrophages, NK cells, and so on. In view of the multiple functions of APS in immunotherapy and tumor microenvironment, a combination of APS and immunotherapy in cancer treatment has a promising prospect.

The Landscape of the Tumor Microenvironment in Skin Cutaneous Melanoma Reveals a Prognostic and Immunotherapeutically Relevant Gene Signature.

The tumorigenesis of skin cutaneous melanoma (SKCM) remains unclear. The tumor microenvironment (TME) is well known to play a vital role in the onset and progression of SKCM. However, the dynamic mechanisms of immune regulation are insufficient. We conducted a comprehensive analysis of immune cell infiltration in the TME. Based on the differentially expressed genes (DEGs) in clusters grouped by immune infiltration status, a set of hub genes related to the clinical prognosis of SKCM and tumor immune infiltration was explored. Methods: We analyzed immune cell infiltration in two independent cohorts and assessed the relationship between the internal pattern of immune cell infiltration and SKCM characteristics, including clinicopathological features, potential biological pathways, and gene mutations. Genes related to the infiltration pattern of TME immune cells were determined. Furthermore, the unsupervised clustering method (k-means) was used to divide samples into three different categories according to TME, which were defined as TME cluster-A, -B, and -C. DEGs among three groups of samples were analyzed as signature genes. We further distinguished common DEGs between three groups of samples according to whether differences were significant and divided DEGs into the Signature gene-A group with significant differences and the Signature gene-B group with insignificant differences. The Signature gene-A gene set mainly had exon skipping in SKCM, while the Signature gene-B gene set had no obvious alternative splicing form. Subsequently, we analyzed genetic variations of the two signatures and constructed a competing endogenous RNA (ceRNA) regulatory network. LASSO Cox regression was used to determine the immune infiltration signature and risk score of SKCM. Finally, we obtained 13 hub genes and calculated the risk score based on the coefficient of each gene to explore the impact of the high- and low-risk scores on biologically related functions and prognosis of SKCM patients further. The correlation between the risk score and clinicopathological characteristics of SKCM patients indicated that a low-risk score was associated with TME cluster-A classification (p < 0.001) and metastatic SKCM (p < 0.001). Thirteen hub genes also showed different prognostic effects in pan-cancer. The results of univariate and multivariate Cox analyses revealed that risk score could be used as an independent risk factor for predicting the prognosis of SKCM patients. The nomogram that integrated clinicopathological characteristics and immune characteristics to predict survival probability was based on multivariate Cox regression. Finally, 13 hub genes that showed different prognostic effects in pan-cancers were obtained. According to immunohistochemistry staining results, Ube2L6, SRPX2, and IFIT2 were expressed at higher levels, while CLEC4E, END3, and KIR2DL4 were expressed at lower levels in 25 melanoma specimens. Conclusion: We performed a comprehensive assessment of the immune-associated TME. To elucidate the potential development of immune-genomic features in SKCM, we constructed an unprecedented set of immune characteristic genes (EDN3, CLEC4E, SRPX2, KIR2DL4, UBE2L6, and IFIT2) related to the immune landscape of TME. These genes are related to different prognoses and drug responses of SKCM. The immune gene signature constructed can be used as a robust prognostic biomarker of SKCM and a predictor of an immunotherapy effect.