Integrated bioinformatics analysis and experimental validation reveal ISG20 as a novel prognostic indicator expressed on M2 macrophage in glioma.

BACKGROUND: Glioma is the most common malignant primary brain tumor and is characterized by a poor prognosis and limited therapeutic options. ISG20 expression is induced by interferons or double-stranded RNA and is associated with poor prognosis in several malignant tumors. Nevertheless, the expression of ISG20 in gliomas, its impact on patient prognosis, and its role in the tumor immune microenvironment have not been fully elucidated. METHODS: Using bioinformatics, we comprehensively illustrated the potential function of ISG20, its predictive value in stratifying clinical prognosis, and its association with immunological characteristics in gliomas. We also confirmed the expression pattern of ISG20 in glioma patient samples by immunohistochemistry and immunofluorescence staining. RESULTS: ISG20 mRNA expression was higher in glioma tissues than in normal tissues. Data-driven results showed that a high level of ISG20 expression predicted an unfavorable clinical outcome in glioma patients, and revealed that ISG20 was possibly expressed on tumor-associated macrophages and was significantly associated with immune regulatory processes, as evidenced by its positive correlation with the infiltration of regulatory immune cells (e.g., M2 macrophages and regulatory T cells), expression of immune checkpoint molecules, and effectiveness of immune checkpoint blockade therapy. Furthermore, immunohistochemistry staining confirmed the enhanced expression of ISG20 in glioma tissues with a higher WHO grade, and immunofluorescence assay verified its cellular localization on M2 macrophages. CONCLUSIONS: ISG20 is expressed on M2 macrophages, and can serve as a novel indicator for predicting the malignant phenotype and clinical prognosis in glioma patients.

LncRNA CRNDE promotes cell proliferation, migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis.

Ovarian cancer seriously threatens the health of women. LncRNA CRNDE is known to be upregulated in ovarian cancer. However, the mechanism by which CRNDE regulates the progress of ovarian cancer is largely unknown. MTT assay was applied to measure the cell viability. Colony formation assay was used to measure the cell proliferation. Cell migration was tested by wound healing, and Transwell assay was performed to detect cell invasion. In addition, the expression of miR-423-5p, CRNDE and FSCN1 were detected by RT-qPCR and western blotting, respectively. Meanwhile, dual-luciferase reporter assay and RIP assay were performed to explore the correlation between miR-423-5p and CRNDE (or FSCN1). CRNDE and FSCN1 were upregulated in ovarian cancer cells (SKOV3, CAOV-3, IGROV1, A2780 and C13K), while miR-423-5p was downregulated. Moreover, silencing of FSCN1/CRNDE significantly decreased proliferation, migration and invasion of ovarian cancer cells (SKOV3 and CI3K) via suppressing MMP-2 and MMP-9. In addition, CRNDE could sponge miR-423-5p, and FSCN1 was confirmed to be the direct target of miR-423-5p. Furthermore, CRNDE knockdown-induced inhibition of FSCN1 was notably reversed by miR-423-5p downregulation. Knockdown of CRNDE inhibited cell proliferation, migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis. Thus, CRNDE may serve a new target for ovarian cancer.

MMP-9-cleaved osteopontin isoform mediates tumor immune escape by inducing expansion of myeloid-derived suppressor cells.

As an extracellular matrix protein, osteopontin (OPN) has been shown to play an important role in regulation of the immune response to tumors. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid progenitors, are major components of the immune suppressive tumor microenvironment and contribute to tumor evasion of the immune response. However, the specific regulating mechanisms underlying MDSCs expansion remain unclear. Here, we found that MDSCs accumulated in the spleen and tumors of 3LL tumor-bearing mice. Supernatant collected from 3LL cells was able to induce the expansion of MDSCs in peripheral blood mononuclear cell (PBMC) in vitro. Results of enzyme linked immunosorbent assay showed high levels of OPN and matrix metalloproteinase-9 (MMP-9) in this supernatant. Silencing OPN can effectively reduce MDSCs frequency in vivo and in vitro. Furthermore, a specific fragment of OPN, OPN-32 kDa cleaved by MMP-9 was detected in the supernatant from 3LL cells. Overexpression of OPN-32 kDa in 3LL cells induced MDSCs expansion. Inhibition of MMP-9 by monoclonal antibody and inhibitor (TIMP-1) reduced MDSCs expansion in vitro and in vivo. These findings suggest that the MMP-9-cleaved OPN fragment, OPN-32kDa, was responsible for MDSCs expansion, which may contribute to tumor's evasion of the immune response.

Expression and significance of ARID1A mRNA in endometriosis- associated ovarian cancer.

PURPOSE: To investigate the expression and relevant clinical and pathological significance of AT-rich interactive domaincontaining protein 1A (ARID1A) mRNA in endometriosis-associated ovarian cancer. METHODS: The clinical and pathological data of 63 patients with ovarian clear cell carcinoma (OCCC) and of 43 patients with ovarian endometrioid adenocarcinoma (OEAC) were collected. The expression of ARID1A-encoded protein, baf250a, in ovarian cancer tissues was detected using immunohistochemistry. The ARID1A mRNA expression was detected via RNAscope hybridization in situ, and its correlation with the clinical and pathological features of patients was analyzed. RESULTS: The age at the onset of OEAC patients accompanied with endometriosis (CM-EAC) was lower than that of those not accompanied with endometriosis (NCM-EAC) (p<0.001). For patients with OCCC, the lymph node metastasis (LNM) rate of CM-CCC patients was significantly lower compared to NCM-CCC (p=0.02) and FIGO stage was earlier (stage I and II) (p=0.013). The expression of baf250a in OCCC group was significantly lower than that in the EAC group (p=0.033). In the OCCC group, baf250a was significantly related to early FIGO staging (stage I and II) (p=0.026), while its expression was not significantly associated with FIGO staging of EAC, age, tumor size, occurrence site and LND. The mRNA expression of ARID1A was positively correlated with the expression of baf250a (in OCCC group, rp=0.936, p<0.01; in OEAC group, rp=0.325, p=0.035). Analysis of prognosis showed that baf250a was an important prognostic factor rather than an independent prognostic factor, affecting the overall survival (OS) of patients with OCCC, while patients with low ARID1A mRNA expression had a longer-term OS. CONCLUSION: The decreased gene and protein expression levels of ARID1A are related to the occurrence and development of endometriosis-associated ovarian cancer, especially OCCC. The detection of ARID1A mRNA expression may be used to predict the OS of OCCC.

Whole-exome sequencing of endometriosis identifies frequent alterations in genes involved in cell adhesion and chromatin-remodeling complexes.

Endometriosis is a complex and enigmatic disease that arises from the interplay among multiple genetic and environmental factors. The defining feature of endometriosis is the deposition and growth of endometrial tissues at sites outside of the uterine cavity. Studies to date have established that endometriosis is heritable but have not addressed the causal genetic variants for this disease. Here, we conducted whole-exome sequencing to comprehensively search for somatic mutations in both eutopic and ectopic endometrium from 16 endometriosis patients and five normal control patients using laser capture microdissection. We compared the mutational landscape of ectopic endometrium with the corresponding eutopic sample from endometriosis patients compared with endometrium from normal women and identified previously unreported mutated genes and pathway alternations. Statistical analysis of exome data identified that most genes were specifically mutated in both eutopic and ectopic endometrium cells. In particular, genes that are involved in biological adhesion, cell-cell junctions, and chromatin-remodeling complex(es) were identified, which partially supports the retrograde menstruation theory that proposes that endometrial cells are refluxed through the fallopian tubes during menstruation and implanted onto the peritoneum or pelvic organs. Conspicuously, when we compared exomic mutation data for paired eutopic and ectopic endometrium, we identified a mutational signature in both endometrial types for which no overlap in somatic single nucleotide variants were observed. These mutations occurred in a mutually exclusive manner, likely because of the discrepancy in endometriosis pathology and physiology, as eutopic endometrium rapidly regrows, and ectopic endometrial growth is inert. Our findings provide, to our knowledge, an unbiased view of the landscape of genetic alterations in endometriosis and vital information for indicating that genetic alterations in cytoskeletal and chromatin-remodeling proteins could be involved in the pathogenesis of endometriosis, thus implicating a novel therapeutic possibility for endometriosis.

A hypoxia- and lactate metabolism-related gene signature to predict prognosis of sepsis: discovery and validation in independent cohorts.

BACKGROUND: High throughput gene expression profiling is a valuable tool in providing insight into the molecular mechanism of human diseases. Hypoxia- and lactate metabolism-related genes (HLMRGs) are fundamentally dysregulated in sepsis and have great predictive potential. Therefore, we attempted to build an HLMRG signature to predict the prognosis of patients with sepsis. METHODS: Three publicly available transcriptomic profiles of peripheral blood mononuclear cells from patients with sepsis (GSE65682, E-MTAB-4421 and E-MTAB-4451, total n = 850) were included in this study. An HLMRG signature was created by employing Cox regression and least absolute shrinkage and selection operator estimation. The CIBERSORT method was used to analyze the abundances of 22 immune cell subtypes based on transcriptomic data. Metascape was used to investigate pathways related to the HLMRG signature. RESULTS: We developed a prognostic signature based on five HLMRGs (ERO1L, SIAH2, TGFA, TGFBI, and THBS1). This classifier successfully discriminated patients with disparate 28-day mortality in the discovery cohort (GSE65682, n = 479), and consistent results were observed in the validation cohort (E-MTAB-4421 plus E-MTAB-4451, n = 371). Estimation of immune infiltration revealed significant associations between the risk score and a subset of immune cells. Enrichment analysis revealed that pathways related to antimicrobial immune responses, leukocyte activation, and cell adhesion and migration were significantly associated with the HLMRG signature. CONCLUSIONS: Identification of a prognostic signature suggests the critical role of hypoxia and lactate metabolism in the pathophysiology of sepsis. The HLMRG signature can be used as an efficient tool for the risk stratification of patients with sepsis.

An immune-related gene signature predicts the 28-day mortality in patients with sepsis.

Introduction: Sepsis is the leading cause of death in intensive care units and is characterized by multiple organ failure, including dysfunction of the immune system. In the present study, we performed an integrative analysis on publicly available datasets to identify immune-related genes (IRGs) that may play vital role in the pathological process of sepsis, based on which a prognostic IRG signature for 28-day mortality prediction in patients with sepsis was developed and validated. Methods: Weighted gene co-expression network analysis (WGCNA), Cox regression analysis and least absolute shrinkage and selection operator (LASSO) estimation were used to identify functional IRGs and construct a model for predicting the 28-day mortality. The prognostic value of the model was validated in internal and external sepsis datasets. The correlations of the IRG signature with immunological characteristics, including immune cell infiltration and cytokine expression, were explored. We finally validated the expression of the three IRG signature genes in blood samples from 12 sepsis patients and 12 healthy controls using qPCR. Results: We established a prognostic IRG signature comprising three gene members (LTB4R, HLA-DMB and IL4R). The IRG signature demonstrated good predictive performance for 28-day mortality on the internal and external validation datasets. The immune infiltration and cytokine analyses revealed that the IRG signature was significantly associated with multiple immune cells and cytokines. The molecular pathway analysis uncovered ontology enrichment in myeloid cell differentiation and iron ion homeostasis, providing clues regarding the underlying biological mechanisms of the IRG signature. Finally, qPCR detection verified the differential expression of the three IRG signature genes in blood samples from 12 sepsis patients and 12 healthy controls. Discussion: This study presents an innovative IRG signature for 28-day mortality prediction in sepsis patients, which may be used to facilitate stratification of risky sepsis patients and evaluate patients' immune state.

The level of macrophage migration inhibitory factor is negatively correlated with the efficacy of PD-1 blockade immunotherapy combined with chemotherapy as a neoadjuvant therapy for esophageal squamous cell carcinoma.

PURPOSE: This study aimed to screen biomarkers to predict the efficacy of programmed cell death 1 (PD-1) blockade immunotherapy combined with chemotherapy as neoadjuvant therapy for esophageal squamous cell carcinoma (ESCC). METHODS: In the first stage of the study, the baseline concentrations of 40 tumor-related chemokines in the serum samples of 50 patients were measured to screen for possible biomarkers. We investigated whether the baseline concentration of the selected chemokine was related to the therapeutic outcomes and tumor microenvironment states of patients treated with the therapy. In the second stage, the reliability of the selected biomarkers was retested in 34 patients. RESULTS: The baseline concentration of macrophage migration inhibitory factor (MIF) was negatively correlated with disease-free survival (DFS) and overall survival (OS) in patients treated with the therapy. In addition, a low baseline expression level of MIF is related to a better tumor microenvironment for the treatment of ESCC. A secondary finding was that effective treatment decreased the serum concentration of MIF. CONCLUSION: Baseline MIF levels were negatively correlated with neoadjuvant therapy efficacy. Thus, MIF may serve as a predictive biomarker for this therapy. The accuracy of the prediction could be improved if the serum concentration of MIF is measured again after the patient received several weeks of treatment.

The genomic mutational landscape and its correlation with TMB, PD-L1 expression and CD8+ T cell infiltration in Chinese lung large cell neuroendocrine carcinoma.

OBJECTIVES: The aim of this study was to illustrate the genomic mutational landscape, tumor mutation burden (TMB), PD-L1 expression, CD8+ T cell infiltration and their prognostic value in resectable Lung large cell neuroendocrine carcinoma (LCNEC). MATERIALS AND METHODS: The tumor tissues and corresponding normal tissues of 37 LCNEC patients undergoing surgical resection were collected and determined by whole exome sequencing (WES). Subsequently, the tumor samples were stained with the antibodies of PD-L1 and CD8 via multiplex immunohistochemistry (Multi IHC) to evaluate PD-L1 expression and CD8+ T cells infiltration in stroma and tumor regions. Univariate and multivariate analysis were applied to assess the association among genomic features, immune profiles, clinical data and prognosis of LCNEC patients. RESULTS: The median TMB was 5.42 mutations per megabase. Mutations in Wnt (p = 0.049) and Hippo (p = 0.005) pathways were markedly associated with higher TMB value, mutations in p53 pathway were related with higher stromal PD-L1 expression (p = 0.041). LCNEC patients with KEAP1 mutation (p = 0.044) or without adjuvant radiochemotherapy (p = 0.023) had significantly shorter OS. Multivariate analysis showed that high stromal CD8 + T cells infiltration was an independent favorable factor for disease free survival (p = 0.030). The patient stratification of KEAP1 mutation status and stroma PD-L1 expression was independent prognostic factors for overall survival (p = 0.049). CONCLUSION: The investigation of prognostic factor in resectable LCNEC may provide guidance for timely intervention and new therapy strategy for LCENC patients.

Identification of Immune-Related Genes Concurrently Involved in Critical Illnesses Across Different Etiologies: A Data-Driven Analysis.

Severe trauma and sepsis can lead to multiple organ dysfunction syndrome, which is a leading cause of death in intensive care units with mortality rates in excess of 50%. In addition to infection, the degree of immuno-inflammatory response also influences the outcome. The genomic changes observed after a variety of pathophysiological insults, such as trauma, sepsis, burns are similar, and consist of innate immune activation and adaptive immunity suppression. However, the characteristics of the shared mechanisms of aforementioned critical illnesses and the clinical relevance remain less explored. In the present study, we performed a data analysis to identify functional genes concurrently involved in critical illnesses across differing etiologies (trauma and sepsis derived from community-acquired pneumonia/abdominal source) and explored the shared signaling pathways these common genes involved in to gain insight into the underlying molecular mechanisms. A number of immune-related biological functions were found to be dysregulated in both trauma and sepsis in the present study, so we continued to identify immune-related common genes, profiled the immune cell proportion, and explored the relationships between them. The diagnostic and prognostic value of the immune-related common genes was also evaluated to address their potential clinical utilization as novel biomarkers. Notably, we identified a list of 14 immune-related genes concurrently dysregulated in trauma and sepsis showing favorable diagnostic value, among which S100P can predict prognosis of sepsis patients. Moreover, a spectrum of immune cell subsets including naïve B cells, CD8+ T cells, CD4+ memory resting T cells, activated NK cells, resting dendritic cells, plasma cells, Tregs, macrophages M0 and macrophages M1 was found to be concurrently dysregulated in both trauma and sepsis, and a close relation between above identified immune-related genes and immune cell subsets was observed. Our data-driven findings lay a foundation for future research to elucidate the pathophysiology regarding the aspect of inflammatory and immune response in critical illnesses, and suggest future studies focus on interpreting the function roles of the identified immune-related genes, as well as the reactive immune cell subsets.

Prognostic and predictive values of pERK1/2 and pAkt-1 expression in non-small cell lung cancer patients treated with adjuvant chemotherapy.

Ras/ERK and PI3K/Akt pathways are reported to play a prognostic role and contribute to drug resistance in many cancers. The objective of this study was to explore associations between the expression levels of several molecules in Ras/ERK and PI3K/Akt pathways and their clinical significance in predicting the effectiveness of postoperative adjuvant chemotherapy in patients with non-small cell lung cancer (NSCLC). The expressions of K-ras, Raf-1, ERK1/2, phosphorylated ERK1/2 (pERK1/2), Akt-1, phosphorylated Akt-1 (pAkt-1), and Bcl-2 were detected by immunohistochemistry in tumor specimens from 144 NSCLC patients. The correlations between the expression levels of these molecules and the clinicopathological characteristics were analyzed. Patient survival was analyzed by Kaplan-Meier method, log-rank test, and Cox regression. The positive expression rates of K-ras, Raf-1, ERK1/2, pERK1/2, Akt-1, pAkt-1, and Bcl-2 were 21.5%, 41.7%, 59.7%, 27.1%, 50.7%, 36.1%, and 30.6%, respectively. Univariate analysis showed that patients with pERK1/2-positive (P = 0.01), Bcl-2-positive (P = 0.023), or pAkt-1 negative (P = 0.021) had significantly better recurrence-free survival (RFS) than those with pERK1/2-negative, Bcl-2-negative, or pAkt-1-positive. Multivariate analysis showed that earlier stage (P ≤ 0.001), non-adenocarcinoma (P ≤ 0.001), pERK1/2-positive (P ≤ 0.001), and pAkt-1-negative (P = 0.016) were independent prognostic factors for a better RFS in NSCLC. pERK1/2-positive and pAkt-1-negative proved to contribute to a better RFS in postoperative NSCLC patients who received adjuvant chemotherapy after taking the stage and histological subtype into account. pERK1/2 and pAkt-1 could be considered as new independent prognostic biomarkers for predicting RFS and selecting patients who are more likely to benefit from postoperative adjuvant chemotherapy.

A nomogram-based immunoprofile predicts clinical outcomes for stage II and III human colorectal cancer.

An immunoscore for colorectal cancer (CRC) has higher prognostic significance than the TNM staging system. However, the tumor immune microenvironment contains various components that affect clinical prognosis. Therefore, a broader range of immune markers is required to establish an accurate immunoprofile to assess the prognosis of patients with CRC. Using immunohistochemistry combined with multispectral immunohistochemistry and objective assessments, the infiltration of four immune cell types (CD4+/CD8+/forkhead box p3+/CD33+ cells), as well as the expression of six co-signaling molecules [programmed cell death 1 (PD1) ligand 1/PD1/T-cell immunoglobulin mucin family member 3/lymphocyte-activating 3/tumor necrosis factor receptor superfamily, member 4/inducible T-cell costimulator] and indoleamine 2,3-dioxygenase 1 were investigated in two independent cohorts of CRC. The patients' overall survival (OS) was evaluated using the Kaplan-Meier method. Using the Cox proportional hazards model, independent prognostic factors of patients were assessed and a nomogram-based immunoprofile system was developed. The predictive ability of the nomogram was determined using a concordance index (C-index) and calibration curve. To facilitate clinical application, a simplified nomogram-based immunoprofile was constructed. Using receiver operating characteristic (ROC) analysis, the predictive accuracy for OS was compared between the immunoprofile and the TNM staging system for patients with stage II/III CRC. According to multivariate analysis for the primary cohort, independent prognostic factors for OS were CD8+ tumor-infiltrating lymphocytes, CD33+ myeloid-derived suppressor cells and TNM stage, which were included in the nomogram. The C-index of the nomogram for predicting OS was 0.861 (95% CI: 0.796-0.925) for the internal validation and 0.759 (95% CI: 0.714-0.804) for the external validation cohort. The simplified nomogram-based immunoprofile system was able to separate same-stage patients into different risk subgroups, particularly for TNM stage II (P<0.0001) and III (P=0.0002) patients. Pairwise comparison of ROC curves for the immunoprofile and TNM stage systems for patients with stage II/III CRC revealed statistically significant differences (P=0.046) and the Z-statistic value was 1.995. In conclusion, the nomogram-based immunoprofile system provides prognostic accuracy regarding clinical outcomes and is a useful supplement to the TNM staging system for patients with stage II/III CRC.

The Ratio of IP10 to IL-8 in Plasma Reflects and Predicts the Response of Patients With Lung Cancer to Anti-PD-1 Immunotherapy Combined With Chemotherapy.

Antibodies against checkpoint inhibitors such as anti-programmed cell death protein 1 (PD-1) and its ligand anti-programmed death ligand 1 (PD-L1) have shown clinical efficacy in the treatment of multiple cancers. However, there are only a few studies on biomarkers for these targeted immunotherapies, especially in peripheral blood. We first studied the role of interferon-induced protein-10 (IP10) combined with interleukin-8 (IL-8) in peripheral blood as a biomarker of immune-combined chemotherapy for lung cancer and multiple cancers. We used the high-throughput cytokine detection platform and performed bioinformatics analysis of blood samples from 67 patients with lung cancer and 24 with multiple cancers. We selected the ratio of IP-10 to IL-8 (S2/S0, ratio of changes at 10-12 weeks after treatment to baseline) to predict the response to immunotherapy combined with chemotherapy and evaluate the survival of lung cancer patients and mixed cancer patients. In patients treated with the combination therapy, the specificity and sensitivity of IL-8 and IP10 together as predictors were improved compared with those of IL-8 and IP10 alone. Our conclusion was verified in not only lung cancer but also multiple cancer research cohorts. We then further validated the predictive effect of biomarkers in different histologic types of NSCLC and chemotherapy combined with different PD-1 drug groups. Subsequent validation should be conducted with a larger number of patients. The proposed marker IP10 (S2/S0)/IL-8 (S2/S0), as a predictive immunotherapy biomarker, has broad prospects for future clinical applications in treating patients with multiple intractable neoplasms.

Patients with advanced non-small cell lung cancer with EGFR mutations in addition to complex mutations treated with osimertinib have a poor clinical outcome: A real-world data analysis.

The present study aimed to investigate the clinical characteristics and outcomes of patients with advanced non-small cell lung cancer (NSCLC) treated with osimertinib, and focused on the resistance mechanism to osimertinib in a real-world setting. Data from 128 patients with advanced NSCLC who were treated with osimertinib between March 2015 and November 2018 at the Chinese People's Liberation Army General Hospital (Beijing, China) were retrospectively collected, and the associations between mutation types and survival were analysed. In patients treated with osimertinib, the objective response rate reached 60.9% (78/128) and the disease control rate reached 81.3% (104/128), with a median progression-free survival (PFS) time of 12.2 months. A number of complex mutations were identified in the re-analysis after the development of osimertinib resistance, including TP53, KRAS and PIK3CA mutations, epidermal growth factor receptor (EGFR) and MYC amplifications, and mutations associated with SCLC transformation, demonstrating that these mutations may account for osimertinib resistance. The median PFS time for patients with the EGFR T790M mutation (n=41) was significantly longer than that for patients with the T790M mutation and the aforementioned complex mutations (n=13) (16.7 vs. 10.8 months; P=0.001). Patients with a single EGFR mutation (n=87) had a longer median PFS time than those with an EGFR mutation and complex mutations (n=24) (14.63 vs. 6.63 months; P<0.0001). In conclusion, the present study analysed the effects of osimertinib in patients with advanced NSCLC with EGFR mutations, particularly T790M mutations. The results indicated that the efficacy of osimertinib was weakened when patients had complex mutations, suggesting that complex mutations may be responsible for resistance to osimertinib.

A DNA methylation signature to improve survival prediction of gastric cancer.

BACKGROUND: The current Union International Committee on Cancer or the American Joint Committee on Cancer TNM stage system has shown valuable but insufficient estimation for subsets of gastric cancer and prediction for prognosis patients. Thus, there is an urgent need to identify diagnostic, prognostic, and predictive biomarkers to improve patients' outcomes. Our aim was to perform an integrative analysis on publicly available datasets to identify epigenetic changes that may play key role in the initiation and progression of gastric cancer, based on which we set to develop a DNA methylation signature to improve survival prediction of gastric cancer. RESULTS: A total of 340 methylation-related differentially expression genes (mrDEGs) were screened in gastric cancer patients from The Cancer Genome Atlas (TCGA) project. Pathway enrichment analysis revealed that they were involved in the biological process related to initiation and progression of gastric cancer. Based on the mrDEGs identified, we developed a DNA methylation signature consisting of ten gene members (SCNN1B, NFE2L3, CLDN2, RBPMS2, JPH2, GBP6, COL4A5, SMKR1, PPP1R14A, and ARL4D) according to their methylation ß value. This innovative DNA methylation signature was associated with cancer recurrence, while it showed independence of cancer recurrence and TNM stage for survival prediction. Combination of this DNA methylation signature and TNM stage improved overall survival prediction in the receiver operating characteristic analysis. We also verified that two individual genes (PPP1R14A and SCNN1B) of the identified prognostic signature were regulated by promoter region methylation in a panel of gastric cell lines. CONCLUSIONS: This study presents a powerful DNA methylation signature by performing analyses integrating multi-source data including transcriptome, methylome, and clinical outcome of gastric cancer patients from TCGA. The identified DNA methylation signature may be used to refine the current prognostic model and facilitate further stratification of patients in the future clinical trials. Further experimental studies are warranted to unveil the regulatory mechanism and functional role of all the individual genes of the DNA methylation signature. Also, clinical investigations in large GC patient cohorts are greatly needed to validate our findings.

Current state and future of co-inhibitory immune checkpoints for the treatment of glioblastoma.

In the interaction between a tumor and the immune system, immune checkpoints play an important role, and in tumor immune escape, co-inhibitory immune checkpoints are important. Immune checkpoint inhibitors (ICIs) can enhance the immune system's killing effect on tumors. To date, impressive progress has been made in a variety of tumor treatments; PD1/PDL1 and CTLA4 inhibitors have been approved for clinical use in some tumors. However, glioblastoma (GBM) still lacks an effective treatment. Recently, a phase III clinical trial using nivolumab to treat recurrent GBM showed no significant improvement in overall survival compared to bevacizumab. Therefore, the use of immune checkpoints in the treatment of GBM still faces many challenges. First, to clarify the mechanism of action, how different immune checkpoints play roles in tumor escape needs to be determined; which biomarkers predict a benefit from ICIs treatment and the therapeutic implications for GBM based on experiences in other tumors also need to be determined. Second, to optimize combination therapies, how different types of immune checkpoints are selected for combined application and whether combinations with targeted agents or other immunotherapies exhibit increased efficacy need to be addressed. All of these concerns require extensive basic research and clinical trials. In this study, we reviewed existing knowledge with respect to the issues mentioned above and the progress made in treatments, summarized the state of ICIs in preclinical studies and clinical trials involving GBM, and speculated on the therapeutic prospects of ICIs in the treatment of GBM.

Alveolar macrophages in patients with non-small cell lung cancer.

OBJECTIVE: To observe alveolar macrophages (AMs) in the microenvironment of patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: 20 NSCLC patients received bronchoalveolar lavage, and the bronchial alveolar lavage fluid (BALF) was collected. The phenotypes of AMs were detected by the opal multiplex immunofluorescence assay (mIF), flow cytometry, and western blot. RESULTS: AMs could easily be made into paraffin sections after agar pre-embedding. The mIF results showed that AMs highly expressed M1-type marker CD86, and M2-type marker CD163 under PerkinElmer Vectra microscope, while there was a significant difference between the expression of CD86 and CD163 (**P<0.01), consistent with the flow cytometry results. Western blot revealed that the other markers of M1-type (CD16 and iNOS) expression in the AMs were compared with M2-type markers CD206 and ARG (*P<0.05). CONCLUSIONS: Our results showed that AMs simultaneously expressed M1-type markers and M2-type markers, while the M2 markers still dominated. This suggests agar pre-embedding is a very convenient method to embed cells to paraffin tissue, so that cell membrane or nuclear antigens are very easily detected by mIF.

PTK2 promotes cancer stem cell traits in hepatocellular carcinoma by activating Wnt/ß-catenin signaling.

Emerging evidence indicates that cancer stem cells (CSCs) are involved in tumorigenesis, tumor recurrence, and therapeutic resistance in hepatocellular carcinoma (HCC). However, the mechanisms underlying HCC CSC regulation remain largely unknown. Here we report our analysis of 97 paraffin-embedded HCC tumor specimens. We found that protein tyrosine kinase 2 (PTK2) expression correlated with liver CSC marker expression, overall survival, and recurrence-free survival in HCC patients. Our results further showed that PTK2 activated Wnt/ß-catenin signaling by promoting nuclear accumulation of ß-catenin in HCC cells. In this manner, PTK2 activates CSC traits and drives tumorigenicity in HCC cells, leading to HCC recurrence and sorafenib resistance. Moreover, PTK2 expression was negatively correlated with its level of promoter methylation. PTK2 apparently acts as an oncogene by increasing CSC traits and tumorigenicity in HCC. The present data suggest that PTK2 may be a novel prognostic biomarker for HCC recurrence, and a therapeutic target for HCC treatment.

A nomogram-based immunoprofile predicts overall survival for previously untreated patients with esophageal squamous cell carcinoma after esophagectomy.

BACKGROUND: Immunoscore, as a prognostic tool defined to quantify in situ immune cell infiltrates, appears to be superior to the TNM staging system. In esophageal squamous cell carcinoma (ESCC), no immunoscore has been established; however, in situ tumor immunology is recognized as highly important. Our study aimed to construct a comprehensive immunoprofile for ESCC. METHODS: The infiltration of four immune cell types (CD8+/CD4+/Foxp3+/CD33+ cells), the expression of both inhibitory (PD-1/PD-L1/Tim-3/LAG-3) and stimulatory checkpoints (OX-40/ICOS), and IDO1 were evaluated by IHC staining and multi-color immunofluorescence in two independent cohorts (95 patients in the primary cohort and 55 patients in the validation cohort). The association with patients' overall survival was analyzed by the Kaplan-Meier method and the Cox model. Nomogram-based immunoprofile was established using the independent prognostic variables. To determine its predictive accuracy and discriminatory capacity, the C-index and calibration curve were calculated. RESULTS: Significant correlation of PD-L1 expression in tumor cells with PD-1+ T cell infiltration was found (P = 0.035), indicating the activation of the inhibitory PD-1/PD-L1 pathway in ESCC cases. More PD-L1+ ICs, Tim-3+ ICs and LAG-3+ ICs were found in the CD8-rich tumor microenvironment, which is in accordance with the feedback nature of immune system. After adjustment by TNM stage, four immune variables including the infiltration of CD8+/Foxp3+/CD33+ cells and the PD-L1 expression by tumor cells were selected to construct a prognostic nomogram. The calibration curves showed good accuracy of the nomogram for survival prediction. To overcome the complexity of applying a nomogram in a clinical setting, a simple immunoprofile was then established according to the points of each factor from the nomogram. Our immunoprofile model could separate same-stage patients into different risk subgroups, and showed superior accuracy for survival prediction than the TNM staging system based on the C-index calculation and ROC analysis. CONCLUSIONS: Our nomogram-based immunoprofile can provide more accurate prognosis prediction and is an important complement to the TNM staging system for operable ESCC patients.

miR-409 Inhibits Human Non-Small-Cell Lung Cancer Progression by Directly Targeting SPIN1.

Lung cancers, the leading cause of cancer mortality worldwide, are characterized by a high metastatic potential. Growing evidence reveals that Spindlin 1 (SPIN1) is involved in tumor progression and carcinogenesis. However, the role of SPIN1 in non-small-cell lung cancer (NSCLC) and the molecular mechanisms underlying SPIN1 in human NSCLC remain undetermined. Here we examined the function of SPIN1 in human NSCLC and found that the expression of SPIN1 was closely correlated with the overall survival and poor prognosis of NSCLC patients. Aberrant regulation of microRNAs (miRNAs) has an important role in cancer progression. We revealed that miR-409 inhibits the expression of SPIN1 by binding directly to the 3' UTR of SPIN1 using dual-luciferase reporter assays. Overexpression of miR-409 significantly suppressed cell migration, growth, and proliferation by inhibiting SPIN1 in vitro and in vivo. SPIN1 overexpression in miR-409-transfected NSCLC cells effectively rescued the suppression of cell migration, growth, and proliferation regulated by miR-409. miR-409 regulates the PI3K/AKT (protein kinase B) pathway in NSCLC. Moreover, clinical data showed that NSCLC patients with high levels of miR-409 experienced significantly better survival. miR-409 expression was also negatively associated with SPIN1 expression. Taken together, these findings highlight that the miR-409/SPIN1 axis is a useful pleiotropic regulatory network and could predict the metastatic potential in NSCLC patients early, indicating the possibility that miR-409 and SPIN1 might be attractive prognostic markers for treating NSCLC patients.