Reprograming the tumor immunologic microenvironment using neoadjuvant chemotherapy in osteosarcoma.

Tumor-infiltrating immune cells play a crucial role in tumor progression and response to treatment. However, the limited studies on infiltrating immune cells have shown inconsistent and even controversial results for osteosarcoma (OS). In addition, the dynamic changes of infiltrating immune cells after neoadjuvant chemotherapy are largely unknown. We downloaded the RNA expression matrix and clinical information of 80 OS patients from the TARGET database. CIBERSORT was used to evaluate the proportion of 22 immune cell types in patients based on gene expression data. M2 macrophages were found to be the most abundant immune cell type and were associated with improved survival in OS. Another cohort of pretreated OS samples was evaluated by immunohistochemistry to validate the results from CIBERSORT analysis. Matched biopsy and surgical samples from 27 patients were collected to investigate the dynamic change of immune cells and factors before and after neoadjuvant chemotherapy. Neoadjuvant chemotherapy was associated with increased densities of CD3+ T cells, CD8+ T cells, Ki67 + CD8+ T cells and PD-L1+ immune cells. Moreover, HLA-DR-CD33+ myeloid-derived suppressive cells (MDSC) were decreased after treatment. We determined that the application of chemotherapy may activate the local immune status and convert OS into an immune "hot" tumor. These findings provide rationale for investigating the schedule of immunotherapy treatment in OS patients in future clinical trials.

ALYREF promotes the metastasis of nasopharyngeal carcinoma by increasing the stability of NOTCH1 mRNA.

Approximately 70% of treatment failures in nasopharyngeal carcinoma (NPC) patients are attributed to distant metastasis, yet the underlying mechanisms remain unclear. RNA 5-methylcytosine (m5C) is an emerging regulatory modification that controls gene expression and plays a critical role in tumor progression. However, there is little information on the potential roles of RNA m5C modification in NPC metastasis. In this study, we found that the m5C reader Aly/REF export factor (ALYREF) is significantly upregulated in NPC, whereby its high expression is associated with metastasis and poor prognosis. ALYREF overexpression was found to promote tumor metastasis of NPC cells in vitro and in vivo. Mechanistically, m5C-modified NOTCH1 mRNA was identified as a target of ALYREF. Moreover, ALYREF was found to upregulate NOTCH1 expression by enhancing its RNA stability in an m5C modification-dependent manner, thereby promoting the activation of the NOTCH signaling pathway and facilitating NPC metastasis. Overall, our data reveal the crucial role of ALYREF in NPC metastasis and provide a potential therapeutic target for NPC.

Osteosarcoma Cells Secrete CXCL14 That Activates Integrin α11ß1 on Fibroblasts to Form a Lung Metastatic Niche.

Cooperation between primary malignant cells and stromal cells can mediate the establishment of lung metastatic niches. Here, we characterized the landscape of cell populations in the tumor microenvironment in treatment-naïve osteosarcoma using single-cell RNA sequencing and identified a stem cell-like cluster with tumor cell-initiating properties and prometastatic traits. CXCL14 was specifically enriched in the stem cell-like cluster and was also significantly upregulated in lung metastases compared with primary tumors. CXCL14 induced stromal reprogramming and evoked a malignant phenotype in fibroblasts to form a supportive lung metastatic niche. Binding of CXCL14 to heterodimeric integrin α11ß1 on fibroblasts activated actomyosin contractility and matrix remodeling properties. CXCL14-stimulated fibroblasts produced TGFß and increased osteosarcoma invasion and migration. mAbs targeting the CXCL14-integrin α11ß1 axis inhibited fibroblast TGFß production, enhanced CD8+ T cell-mediated antitumor immunity, and suppressed osteosarcoma lung metastasis. Taken together, these findings identify cross-talk between osteosarcoma cells and fibroblasts that promotes metastasis and demonstrate that targeting the CXCL14-integrin α11ß1 axis is a potential strategy to inhibit osteosarcoma lung metastasis. SIGNIFICANCE: Cooperation between stem-like osteosarcoma cells and fibroblasts mediated by a CXCL14-integrin α11ß1 axis creates a tumor-supportive lung metastatic niche and represents a therapeutic target to suppress osteosarcoma metastasis.

Epstein-Barr virus-based prognostic model in nodular sclerosis classic Hodgkin lymphoma.

The role of Epstein-Barr virus (EBV) in lymphoma cells of nodular sclerosis classic Hodgkin lymphoma (NScHL) is controversial. Our aim was to explore this and establish a clinically feasible model for risk stratification. We interrogated data from 542 consecutive subjects with NScHL receiving ABVD therapy and demonstrated EBV-infection in their lymphoma cells with EBV-encoded small RNAs (EBERs) in situ hybridization. Subjects were divided into training and validation datasets. As data from the training dataset suggested EBERs-positivity was the only independent prognostic factor for both progression-free survival (PFS) and overall survival (OS), we developed corresponding prognostic models based on it. Our models showed excellent performance in both training and validation cohort. These data indicate the close association of EBV infection and the outcomes of persons with NScHL receiving ABVD. Additionally, our newly developed models should help physicians estimate prognosis and select individualized therapy.

Breast Cancer Subtypes Based on Hypoxia-Related Gene Sets Identify Potential Therapeutic Agents.

Purpose: The hypoxic tumor microenvironment was reported to be involved in different tumorigenesis mechanisms of breast cancer (BC). We aimed to establish a hypoxia-related gene signature to identify a new BC subtype through the clustering analysis and explore potential compounds targeting the BC subtypes. Methods: Gene expression data and clinical features of BC and adjacent non-tumor tissues were downloaded from the Cancer Genome Atlas-Breast cancer (TCGA-BRCA) database. We comprehensively revealed the activity changes of Gene Ontology (GO) biological processes (BP) gene sets in BC by gene set variation analysis (GSVA) and identified three hypoxia-related BC subtypes. We then matched the differentially expressed gene profile of each subtype with the gene profile in CMap database to identify the potential agents targeting the BC subtypes. Results: 562 of Gene Ontology biological processes gene sets significantly correlated with hypoxia score in breast cancer. 969 BC patients were clustered into three subtypes based on the enrichment score of hypoxia-associated gene sets. Subtype 1 patients displayed better survival than subtype 2 and 3. KEGG pathway enrichment analysis of each subtype was performed based on the unique differential expression genes profile. In subtype 1, the upregulated genes were associated with lipid and amino acid metabolism regulation; in subtype 2, the upregulated genes were associated with metabolic energy regulation, while in subtype 3, the upregulated genes were associated with apoptosis and protein process. Using the CMap database, 55, 111 and 63 compounds were identified, targeting subtype 1, 2, and 3, respectively. Conclusion: In this study, novel hypoxia-related subtypes were developed for patients with BC. In addition, biological processes associated with differential expression genes profile and potential therapeutic target compounds were identified in each subtype. The new classification might provide a better understanding of the role of hypoxia in breast cancer and more individualized treatment for patients.

DAB2IP attenuates chemoresistance of triple-negative breast cancer through sequestration of RAC1 to prevent ß-catenin nuclear accumulation.

BACKGROUND: Although chemotherapy, the most widely used systemic treatment in triple-negative breast cancer (TNBC), markedly improved the patients' outcome, chemoresistance always occurs. This study purposed to explore new therapeutic strategies for the treatment of chemoresistance. METHODS AND RESULTS: The expression and prognostic value of DAB2IP were investigated in TNBC tissues and cell lines. Low DAB2IP expression predicted high mortality risk in TNBC. Inhibition of DAB2IP expression conferred cancer stem cell capacity and chemoresistance in TNBC cell lines. Using murine breast cancer (BC) xenograft models, we evaluated the association with DAB2IP and chemoresistance. DAB2IP inhibited TNBC tumourigenesis and chemoresistance in vivo. Further, we revealed that DAB2IP inhibited ß-catenin nuclear transport through competitive interaction with RAC1 and decreased ß-catenin accumulation in the cell nucleus. Finally, we found that the DNA methylation level was negatively associated with DAB2IP expression in TNBC. Inhibition of DNA methylation restored the DAB2IP expression and attenuated chemoresistance in TNBC. CONCLUSIONS: We revealed that DAB2IP attenuates chemoresistance of TNBC via inhibition of RAC1-mediated ß-catenin nuclear accumulation. Decitabine treatment results in re-expression of DAB2IP by inhibiting DNA methylation and could be a potential therapeutic strategy for chemoresistance in TNBC.

BCL6-SPECC1L: A Novel Fusion Gene in Nasopharyngeal Carcinoma.

Background: Nasopharyngeal carcinomas (NPCs) are malignant tumors originating from the lining epithelium of the nasopharynx. Fusion genes have been confirmed to play important roles in the occurrence and development of various malignant tumors, but the role of fusion genes in NPC is poorly understood. We aimed to explore new fusion genes that promote the occurrence and development of NPC. Methods: RNA-seq was used to search for interchromosomal translocations in 18 NPC tissues. Polymerase chain reaction (PCR) and Sanger sequencing were applied to verify the presence of BCL6-SPECC1L (BS); quantitative PCR (qPCR) and Western blotting were used to measure the expression level of BCL-6 in NPC cells; MTT and in vivo tumorigenesis assays were applied to evaluate the cell proliferation ability; immunofluorescence assays were used to determine the cellular localization of BCL6 and BS; and a luciferase reporter assay was performed to evaluate the ability of BCL6 and BS to inhibit transcription. Results: BS was present in 5.34% (11/206) of primary NPC biopsies and 2.13% (1/47) of head and neck cancer biopsies. The expression of BCL6 was downregulated in NPC, and silencing of endogenous BCL6 promoted NPC cell proliferation in vitro. Overexpression of BCL6 but not BS inhibited the growth of NPC cells in vivo and in vitro. Mechanistically, BCL6 localized in the nucleus can inhibit the G1/S transition to suppress the growth of NPC cells. However, after the fusion of BCL6 and SPECC1L, the product cannot localize to the nucleus, and the transcriptional inhibitory function of BCL6 is abolished, eventually abolishing its tumor suppressor effect and leading to the development of NPC. Conclusion: BS is a novel fusion gene in NPC that may play an important role in the occurrence and development of this cancer. The clinical significance of the BS fusion gene needs further elucidation.

ARNTL2 is a Prognostic Biomarker and Correlates with Immune Cell Infiltration in Triple-Negative Breast Cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is associated with poor prognosis. The aberrant expression of circadian genes contributes to the origin and progression of breast cancer. The present study was designed to explore the potential function and prognosis value of circadian genes in TNBC. METHODS: The transcriptome data of circadian genes were downloaded from The Cancer Genomic Atlas (TCGA), GSE25066 and GSE31448 datasets. The differential expressed circadian genes between non-TNBC and TNBC patients were analysed by Wilcoxon test. Univariate and multivariate Cox regression analyses were employed to identify the prognostic circadian genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed to study the biological functions of ARNTL2. The composition of 22 immune cells in the tumour samples was estimated with CIBERSORT algorithm. The correlations between ARNTL2 expression and tumour-infiltrating immune cells were evaluated by Spearman correlation coefficient. RESULTS: A total of 8 circadian genes were found to be differentially expressed between non-TNBC and TNBC, but only ARNTL2 has prognostic value. Multivariate Cox analysis identified that ARNTL2 was an independent prognosis factor for overall survival and relapse-free survival in TNBC patients. Functionally, ARNTL2 was mainly involved in immune response processes such as positive regulation of cytokine production, regulation of innate immune response, and cellular responses to molecules of bacterial origin. High expression of ARNTL2 was positively correlated with activated CD4 memory T cells, activated mast cells, and neutrophil infiltration and the expression of markers of neutrophils (ITGAM), dendritic cells (HLA-DRA, HLA-DPA1, ITGAM), Th1 (IL1B, STAT1), Th2 (IL13), Th17 (STAT3) and mast cells (TPSB2, TPSAB1). CONCLUSION: ARNTL2 may be linked with the functional modulation of the tumour immune microenvironment and serve as a potential biomarker for predicting the prognosis of TNBC patients.

Circular RNA circKIF4A facilitates the malignant progression and suppresses ferroptosis by sponging miR-1231 and upregulating GPX4 in papillary thyroid cancer.

Circular RNAs (circRNAs) are one type of non-coding RNA. They act as important role in regulating various biological processes in the malignant progression. But we don't clearly know the specific mechanism of the majority circRNAs in papillary thyroid tumor progression. In the current study, we explored circKIF4A and the result showed that it had high expression in papillary thyroid cancer. The functions of circKIF4A were explored by CCK-8, transwell, and mouse xenograft experiments. Knockdown of circKIF4A could suppress papillary thyroid cell growth and migration. In addition, RIP assays and dual luciferase vector reporter assays were further conducted. Our consequence showed circKIF4A facilitated the malignant progress of papillary thyroid tumor by sponging miR-1231 and upregulating GPX4 expression. In conclusion, our study proved that circKIF4A-miR-1231-GPX4 axis played a vital role in cancer proliferation and ferroptosis by competing endogenous RNAs. Therefore, targeting circKIF4A is very likely to be a potential method for treatment of papillary thyroid cancer in the future.

Decreased expression of nuclear factor 90 correlates with worse outcomes in nasopharyngeal carcinoma.

BACKGROUND: Nuclear factor 90 (NF90), one of the double-stranded RNA binding proteins (DSRBP), is involved in many cellular biological processes, includes cell proliferation, differentiation, angiogenesis, cell cycle and immunity. However, its role in nasopharyngeal carcinoma (NPC), and the significance of expression in clinics, remains unknown. The aim of this study was to uncover the relationship between the NF90 expression by immunohistochemistry (IHC) and clinical outcomes in NPC patients. METHODS: We retrospectively detected the NF90 expression in 216 NPC specimens by IHC. The cutoff value of NF90 was evaluated using receiver operating characteristic (ROC) curve, the prognostic value of NF90 in NPC was evaluated by Kaplan-Meier and cox multifactor statistical model. RESULTS: Elevated expression of NF90 was found in 60.2% (130/216). Positive correlation was found between NF90 expression and clinical stage (P=0.018), T stage (P=0.004), and therapy (P=0.020). Over-expression of NF90 predicted favorable progression-free survival (PFS) (P=0.017) and overall survival (OS) (P<0.001) in NPC. Further analysis suggested that the level of NF90 expression was an independent risk factor in NPC prognosis. CONCLUSIONS: These results demonstrate, for the first time, that decreased expression of NF90 is an independent biomarker of worse prognosis for NPC patients, and is a great potential tool for screening the high-risk population for advanced NPC patients.

Gene Expression Classifier Reveals Prognostic Osteosarcoma Microenvironment Molecular Subtypes.

Osteosarcoma (OSA) is the most common bone malignancy and displays high heterogeneity of molecular phenotypes. This study aimed to characterize the molecular features of OSA by developing a classification system based on the gene expression profile of the tumor microenvironment. Integrative analysis was performed using specimens and clinical information for OSA patients from the TARGET program. Using a matrix factorization method, we identified two molecular subtypes significantly associated with prognosis, S1 (infiltration type) and S2 (escape type). Both subtypes displayed unique features of functional significance features and cellular infiltration characteristics. We determined that immune and stromal infiltrates were abundant in subtype S1 compare to that in subtype S2. Furthermore, higher expression of immune checkpoint PDCD1LG2 and HAVCR2 was associated with improved prognosis, while a preferable chemotherapeutic response was associated with FAP-positive fibroblasts in subtype S1. Alternatively, subtype S2 is characterized by a lack of effective cytotoxic responses and loss of major histocompatibility complex class I molecule expression. A gene classifier was ultimately generated to enable OSA classification and the results were confirmed using the GSE21257 validation set. Correlations between the percentage of fibroblasts and/or fibrosis and CD8+ cells, and their clinical responses to chemotherapy were assessed and verified based on 47 OSA primary tumors. This study established a new OSA classification system for stratifying OSA patient risk, thereby further defining the genetic diversity of OSA and allowing for improved efficiency of personalized therapy.

VISTA and PD-L1 synergistically predict poor prognosis in patients with extranodal natural killer/T-cell lymphoma.

Although PD-1/PD-L1 blockade therapy confers salutary effects across cancer types, their efficacy in Extranodal Natural killer/T-cell lymphoma (ENKTCL) patients is limited and unpredictable. Here, we comprehensively evaluated the expression profile of a panel of immune-regulatory makers to identify novel prognostic biomarkers and/or therapeutic targets for this malignancy. Using immunohistochemistry and multiplex immunofluorescence, we found that the expression of VISTA (88.1%) was predominantly in CD68+ macrophages and much higher than PD-L1 expression (68.7%) in ENKTCL. B7-H4 and HHLA2 proteins were not detected in ENKTCL. B7-H3 was expressed in minority of ENKTCL patients (13.7%) and mainly colocalized with CD31. A close correlation was detected between VISTA and PD-L1, but they were not co-expressed in the same cells. High expressions of VISTA or PD-L1 were significantly associated with detrimental clinicopathological characteristics, dismal prognosis, and high density of CD8+ TILs, and high VISTA expression was also significantly associated with high density of Foxp3+ TILs. VISTA combined with PD-L1 was an independent prognostic factor for PFS and OS. Moreover, the patients with high VISTA showed a poor response to PD-1 blockades in ENKTCL. In conclusion, these findings provide a rationale for VISTA as an ideal immunotherapeutic target next to PD-L1 for ENKTCL.

Development and Validation of a Model Including Distinct Vascular Patterns to Estimate Survival in Hepatocellular Carcinoma.

Importance: Because of tumor heterogeneity, traditional clinical variables remain insufficient to predict recurrence, which impairs long-term survival among patients undergoing radical hepatectomy for hepatocellular carcinoma (HCC). Vessels encapsulating tumor clusters (VETC) constitute a novel vascular pattern distinct from microvascular invasion (MVI), representing biological aggressiveness of HCC. Objective: To establish a model to estimate individualized recurrence-free survival (RFS) in HCC by integrating VETC and MVI. Design, Setting, and Participants: This prognostic study included 498 patients undergoing radical hepatectomy for HCC from 5 academic centers in China from January 1, 2013, to December 31, 2016, and consisted of 3 cohorts: training (243 [48.8%]), internal validation (122 [24.5%]), and external validation (133 [26.7%]). Follow-up was completed on March 30, 2020, and the data were analyzed from December 1 to 31, 2020. Exposures: VETC, MVI, tumor number, and maximum tumor size. Main Outcomes and Measures: The primary end point was RFS. The risk score for relative recurrence and nomogram for absolute RFS probability were derived from the final model, which contained variables recommended by multivariate least absolute shrinkage and selection operator Cox proportional hazards regression analysis. Their performance was quantified using the Harrell concordance index (C index), the time-dependent area under the receiver operating characteristic curve, and calibration curves and was compared with 6 prognostic systems. Recurrence-free survival was estimated by the Kaplan-Meier method, and RFS curves were compared using a log-rank test. Results: Among the 498 patients, 432 (86.7%) were men; the mean (SD) age at diagnosis was 51.4 (11.3) years. Independent predictors for RFS identified included VETC, MVI, tumor number, and maximum tumor size, which were incorporated into the multivariate model (VMNS model). The C index (0.702; 95% CI, 0.653-0.752) for the VMNS score of the training cohort was significantly higher than those of 6 conventional systems (0.587 [95% CI, 0.535-0.638] to 0.657 [95% CI, 0.606-0.708]). Different recurrence risk groups defined by the VMNS score showed significantly different 2-year RFS (low-risk group, 81.4% [SE, 0.036]; medium-risk group, 62.1% [SE, 0.054]; high-risk group, 30.1% [SE, 0.079]; P < .001). Calibration curves of the VMNS nomogram showed good agreement between the nomogram-predicted RFS probability and actual RFS proportion. The internal and external validation cohorts confirmed the results. Conclusions and Relevance: The VMNS model enabled individualized prognostication of RFS in patients with HCC undergoing curative resection.

ZMYND8 expression combined with pN and pM classification as a novel prognostic prediction model for colorectal cancer: Based on TCGA and GEO database analysis.

BACKGROUND: Zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8 (ZMYND8) is closely correlated with tumor proliferation and invasiveness. However, its prognostic value has not been estimated in colorectal cancer (CRC). OBJECTIVE: We aimed to elucidate the prognostic significance of ZMYND8 expression and the pN and pM classification supplemented by its expression in CRCs. METHODS: The candidate gene ZMYND8 is identified by TCGA database and GEO database, and then we retrospectively evaluated the status and prognostic significance of ZMYND8 expression of 174 patients with CRC. RESULTS: Online data showed high expression of ZMYND8 is closely correlated with worse overall survival. Our study revealed high expression of ZMYND8 in CRC patients was significantly associated with worse overall and disease-free survival (P< 0.05), and was an independently adverse prognostic factor for overall survival (P< 0.001) and disease-free survival (P= 0.001) by univariate and multivariate analysis. C-index to combined prognostic model containing the pN, pM classification supplemented by the status of ZMYND8 expression showed improved predictive ability comparing with the pN and pM classification model (C-index of 0.597 vs. 0.545, respectively). CONCLUSION: The combined prognostic model could improve the ability to determine the clinical outcome of patients with CRC.

Immune Landscape of the Tumor Microenvironment Identifies Prognostic Gene Signature CD4/CD68/CSF1R in Osteosarcoma.

Background: Osteosarcoma (OSA), the most common primary bone malignancy in children and adolescents, is prone to metastases and unfavorable prognosis. Owing to its strong genomic heterogeneity, traditional chemotherapy, or targeted immunotherapy has not effectively improved the related overall survival for decades. Since the landscape of the OSA tumor immune microenvironment is scarcely known, despite it playing a crucial role in predicting clinical outcomes and therapeutic efficacies, we aimed to elucidate its molecular characteristics. Methods: The immune signature of 101 OSA samples was explored using transcriptome profiling and clinical characteristics retrieved from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program. Correlations between the prognostic immune markers and their clinical chemotherapy responses were assessed and verified based on 45 OSA primary tumors. Findings: We identified the heterogeneity underlying tumor immune signature in OSA, and found CD4+ T cells and macrophage markers CD4/IFNGR2/CD68 to be feasible prognostic factors, exerting significantly positive correlation with each other. Specifically, CSF1R, which plays an essential role in the regulation of proliferation and differentiation of macrophages, was found to be a specific signature associated with CD4/CD68, with improved OSA clinical outcomes. Interpretation: The immune landscape based on CD4/CD68/CSF1R gene signatures showed considerable promise for prognostic and therapeutic stratification in OSA patients. A specific immune signature for OSA, abundantly consisting of Th1-polarized CD4+ T cells and CSF1R-related CD68+ macrophages, may improve the predictive efficacy of chemotherapy and improve prognosis in patients with OSA.