Association of 18F- fluorodeoxyglucose uptake with the expression of metabolism-related molecules in papillary thyroid cancer.

OBJECTIVES: 18F-fluorodeoxyglucose positron emission tomography-computed tomography (18F-FDG-PET/CT) is a diagnostic imaging method that is based on the Warburg effect, which is the increased uptake of glucose through aerobic glycolysis in cancer cells. The diagnostic value of 18F-FDG-PET/CT for thyroid cancer is controversial. However, uptake of 18F-FDG and the corresponding maximum standardized uptake value (SUVmax) is expected to reflect the metabolic status of cancer cells. In the present study, we sought to determine the relationship between 18F-FDG uptake and tumor metabolism- associated factors. METHODS: This was a single-center retrospective study. In the present study, SUVmax was compared with the expression of hexokinase 2 (HK2), glucose transporter 1 (GLUT1), vascular endothelial growth factor (VEGF), and glutaminase 1 (GLS1) in 41 patients with thyroid cancer. RESULTS: GLS1 expression was found to be moderately correlated with SUVmax (p < 0.001, r = 0.51), whereas HK2 and VEGF expression were weakly correlated (p = 0.011, r = 0.28, p = 0.008, r = 0.29, respectively) and GLUT1 did not correlate with SUVmax (p = 0.62, r = 0.06). CONCLUSION: Our findings suggest 18F-FDG PET/CT reflects GLS1 expression in thyroid cancer and could be used to select suitable candidates for GLS1 inhibitor treatment.

Enhancer infestation drives tumorigenic activation of inactive B compartment in Epstein-Barr virus-positive nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignant epithelial tumor endemic to Southern China and Southeast Asia. While previous studies have revealed a low frequency of gene mutations in NPC, its epigenomic aberrations are not fully elucidated apart from DNA hypermethylation. Epigenomic rewiring and enhancer dysregulation, such as enhancer hijacking due to genomic structural changes or extrachromosomal DNA, drive cancer progression. METHODS: We conducted Hi-C, 4C-seq, ChIP-seq, and RNA-seq analyses to comprehensively elucidate the epigenome and interactome of NPC using C666-1 EBV(+)-NPC cell lines, NP69T immortalized nasopharyngeal epithelial cells, clinical NPC biopsy samples, and in vitro EBV infection in HK1 and NPC-TW01 EBV(-) cell lines. FINDINGS: In C666-1, the EBV genome significantly interacted with inactive B compartments of host cells; the significant association of EBV-interacting regions (EBVIRs) with B compartment was confirmed using clinical NPC and in vitro EBV infection model. EBVIRs in C666-1 showed significantly higher levels of active histone modifications compared with NP69T. Aberrant activation of EBVIRs after EBV infection was validated using in vitro EBV infection models. Within the EBVIR-overlapping topologically associating domains, 14 H3K4me3(+) genes were significantly upregulated in C666-1. Target genes of EBVIRs including PLA2G4A, PTGS2 and CITED2, interacted with the enhancers activated in EBVIRs and were highly expressed in NPC, and their knockdown significantly reduced cell proliferation. INTERPRETATION: The EBV genome contributes to NPC tumorigenesis through "enhancer infestation" by interacting with the inactive B compartments of the host genome and aberrantly activating enhancers. FUNDING: The funds are listed in the Acknowledgements section.

Peritumoral SPARC expression induced by exosomes from nasopharyngeal carcinoma infected Epstein-Barr virus: A poor prognostic marker.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) cells have high metastatic potential. Recent research has revealed that the interaction of between tumor cells and the surrounding stroma plays an important role in tumor invasion and metastasis. In this study, we showed the prognostic value of expression of SPARC, an extracellular matrix protein with multiple cellular functions, in normal adjacent tissues (NAT) surrounding NPC. In the immunohistochemical analysis of 51 NPC biopsy specimens, SPARC expression levels were significantly elevated in the NAT of EBER (EBV-encoded small RNA)-positive NPC compared to that in the NAT of EBER-negative NPC. Moreover, increased SPARC expression in NAT was associated with a worsening of overall survival. The enrichment analysis of RNA-seq of publicly available NPC and NAT surrounding NPC data showed that high SPARC expression in NPC was associated with epithelial mesenchymal transition promotion, and there was a dynamic change in the gene expression profile associated with interference of cellular proliferation in NAT, including SPARC expression. Furthermore, EBV-positive NPC cells induce SPARC expression in normal nasopharyngeal cells via exosomes. Induction of SPARC in cancer-surrounding NAT cells reduced intercellular adhesion in normal nasopharyngeal structures and promoted cell competition between cancer cells and normal epithelial cells. These results suggest that epithelial cells loosen their own binding with the extracellular matrix as well as stromal cells, facilitating the invasion of tumor cells into the adjacent stroma by activating cell competition. Our findings reveal a new mechanism by which EBV creates a pro-metastatic microenvironment by upregulating SPARC expression in NPC.

Repression of DERL3 via DNA methylation by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV)-associated invasive malignancy. Increasing evidence indicates that epigenetic abnormalities, including DNA methylation, play important roles in the development of NPC. In particular, the EBV principal oncogene, latent membrane protein 1 (LMP1), is considered a key factor in inducing aberrant DNA methylation of several tumour suppressor genes in NPC, although the mechanism remains unclear. Herein, we comprehensively analysed the methylome data of Infinium BeadArray from 51 NPC and 52 normal nasopharyngeal tissues to identify LMP1-inducible methylation genes. Using hierarchical clustering analysis, we classified NPC into the high-methylation, low-methylation, and normal-like subgroups. We defined high-methylation genes as those that were methylated in the high-methylation subgroup only and common methylation genes as those that were methylated in both high- and low-methylation subgroups. Subsequently, we identified 715 LMP1-inducible methylation genes by observing the methylome data of the nasopharyngeal epithelial cell line with or without LMP1 expression. Because high-methylation genes were enriched with LMP1-inducible methylation genes, we extracted 95 high-methylation genes that overlapped with the LMP1-inducible methylation genes. Among them, we identified DERL3 as the most significantly methylated gene affected by LMP1 expression. DERL3 knockdown in cell lines resulted in significantly increased cell proliferation, migration, and invasion. Lower DERL3 expression was more frequently detected in the advanced T-stage NPC than in early T-stage NPC. These results indicate that DERL3 repression by DNA methylation contributes to NPC tumour progression.

Recent Advances in Assessing the Clinical Implications of Epstein-Barr Virus Infection and Their Application to the Diagnosis and Treatment of Nasopharyngeal Carcinoma.

Reports about the oncogenic mechanisms underlying nasopharyngeal carcinoma (NPC) have been accumulating since the discovery of Epstein-Barr virus (EBV) in NPC cells. EBV is the primary causative agent of NPC. EBV-host and tumor-immune system interactions underlie the unique representative pathology of NPC, which is an undifferentiated cancer cell with extensive lymphocyte infiltration. Recent advances in the understanding of immune evasion and checkpoints have changed the treatment of NPC in clinical settings. The main EBV genes involved in NPC are LMP1, which is the primary EBV oncogene, and BZLF1, which induces the lytic phase of EBV. These two multifunctional genes affect host cell behavior, including the tumor-immune microenvironment and EBV behavior. Latent infections, elevated concentrations of the anti-EBV antibody and plasma EBV DNA have been used as biomarkers of EBV-associated NPC. The massive infiltration of lymphocytes in the stroma suggests the immunogenic characteristics of NPC as a virus-infected tumor and, at the same time, also indicates the presence of a sophisticated immunosuppressive system within NPC tumors. In fact, immune checkpoint inhibitors have shown promise in improving the prognosis of NPC patients with recurrent and metastatic disease. However, patients with advanced NPC still require invasive treatments. Therefore, there is a pressing need to develop an effective screening system for early-stage detection of NPC in patients. Various modalities, such as nasopharyngeal cytology, cell-free DNA methylation, and deep learning-assisted nasopharyngeal endoscopy for screening and diagnosis, have been introduced. Each modality has its advantages and disadvantages. A reciprocal combination of these modalities will improve screening and early diagnosis of NPC.

Protein Farnesylation on Nasopharyngeal Carcinoma, Molecular Background and Its Potential as a Therapeutic Target.

Nasopharyngeal carcinoma (NPC) is one of the Epstein-Barr virus (EBV)-associated malignancies. NPC is highly metastatic compared to other head and neck carcinomas, and evidence has shown that the metastatic features of NPC are involved in EBV infection. The prognosis of advanced cases, especially those with distant metastasis, is still poor despite advancements in molecular research and its application to clinical settings. Thus, further advancement in basic and clinical research that may lead to novel therapeutic modalities is needed. Farnesylation is a lipid modification in the C-terminus of proteins. It enables proteins to attach to the lipid bilayer structure of cellular membranes. Farnesylation was initially identified as a key process of membrane association and activation of the RAS oncoprotein. Farnesylation is thus expected to be an ideal therapeutic target in anti-RAS therapy. Additionally, more and more molecular evidence has been reported, showing that proteins other than RAS are also farnesylated and have significant roles in cancer progression. However, although several clinical trials have been conducted in cancers with high rates of ras gene mutation, such as pancreatic carcinomas, the results were less favorable than anticipated. In contrast, favorable outcomes were reported in the results of a phase II trial on head and neck carcinoma. In this review, we provide an overview of the molecular pathogenesis of NPC in terms of the process of farnesylation and discuss the potential of anti-farnesylation therapy in the treatment of NPC.

Estrogen induces the expression of EBV lytic protein ZEBRA, a marker of poor prognosis in nasopharyngeal carcinoma.

Several epidemiological studies have suggested that Epstein-Barr virus (EBV) lytic infection is essential for the development of nasopharyngeal carcinoma (NPC), as the elevation of antibody titers against EBV lytic proteins is a common feature of NPC. Although ZEBRA protein is a key trigger for the initiation of lytic infection, whether its expression affects the prognosis and pathogenesis of NPC remains unclear. In this study, 64 NPC biopsy specimens were analyzed using immunohistochemistry. We found that ZEBRA was significantly associated with a worsening of progression-free survival in NPC (adjusted hazard ratio, 3.58; 95% confidence interval, 1.08-11.87; p = 0.037). Moreover, ZEBRA expression positively correlated with key endocrinological proteins, estrogen receptor α, and aromatase. The transcriptional level of ZEBRA is activated by estrogen in an estrogen receptor α-dependent manner, resulting in an increase in structural gene expression levels and extracellular virus DNA copy number in NPC cell lines, reminiscent of lytic infection. Interestingly, it did not suppress cellular proliferation or increase apoptosis, in contrast with cells treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, indicating that viral production induced by estrogen is not a cell lytic phenomenon. Our results suggest that intratumoral estrogen overproduced by aromatase could induce ZEBRA expression and EBV reactivation, contributing to the progression of NPC.

EBV genome variations enhance clinicopathological features of nasopharyngeal carcinoma in a non-endemic region.

Nasopharyngeal carcinoma (NPC) is caused by infection with Epstein-Barr virus (EBV) and endemic in certain geographic regions. EBV lytic gene, BALF2, closely associates with viral reactivation and BALF2 gene variation, the H-H-H strain, causes NPC in endemic region, southern China. Here, we investigate whether such EBV variations also affect NPC in a non-endemic region, Japan. Viral genome sequencing with 47 EBV isolates of Japanese NPC were performed and compared with those of other EBV-associated diseases from Japan or NPC in Southern China. EBV genomes of Japanese NPC are different from those of other diseases in Japan or endemic NPC; Japanese NPC was not affected by the endemic strain (the BALF2 H-H-H) but frequently carried the type 2 EBV or the strain with intermediate risk of endemic NPC (the BALF2 H-H-L). Seven single nucleotide variations were specifically associated with Japanese NPC, of which six were present in both type 1 and 2 EBV genomes, suggesting the contribution of the type 2 EBV-derived haplotype. This observation was supported by a higher viral titer and stronger viral reactivation in NPC with either type 2 or H-H-L strains. Our results highlight the importance of viral strains and viral reactivation in the pathogenesis of non-endemic NPC.

Epstein-Barr Virus LMP1 Induces Soluble PD-L1 in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy. The principal oncogene of EBV, latent membrane protein 1 (LMP1), induces the expression of programmed death-ligand 1 (PD-L1), which is an immunosuppressive transmembrane protein and a promising therapeutic target for various malignancies. Recent studies have revealed an association between the level of soluble PD-L1 (sPD-L1) and disease progression. However, the role of sPD-L1 in NPC or its relevance to LMP1 has not been elucidated. This study aimed to examine whether LMP1 induces sPD-L1 in vitro and analyze the clinical relevance of LMP1, PD-L1, and sPD-L1 in NPC patients. Analysis of nasopharyngeal cell lines revealed that LMP1 induces both cellular PD-L1 and sPD-L1. Analysis of biopsy specimens from 32 NPC patients revealed that LMP1 expression was significantly correlated with PD-L1 expression. Finally, the serum sPD-L1 level in NPC patients was higher than that in the controls. Moreover, the sPD-L1 level in the advanced stage was higher than that in the early stage. However, LMP1 expression, PD-L1 expression, and sPD-L1 levels were not associated with prognosis. These results suggest that LMP1 induces both sPD-L1 and PD-L1, which are associated with NPC progression.

Effect of early tracheostomy in mechanically ventilated patients.

OBJECTIVE: To investigate the effect of the timing of tracheostomy in patients who required prolonged mechanical ventilation using two methods: analysis of early versus late tracheostomy and landmark analysis. STUDY DESIGN: Retrospective cohort study. METHODS: Patients who were emergently intubated and admitted into the intensive care unit or high dependency unit between January 2011 and August 2016, with or without tracheostomy, were included. In the early and late tracheostomy analysis, all patients were divided into early (≤10 days, n = 88) and late (>10 days, n = 132) groups. In the landmark analysis, 198 patients requiring ventilation for more than 10 days were divided into early tracheostomy (≤10 days, n = 57) and nonearly tracheostomy (>10 days, n = 141) groups. We compared 60-day ventilation withdrawal rate and 60-day mortality. RESULTS: Early tracheostomy was a significant factor for early ventilation withdrawal, as shown by log-rank test results (early and late tracheostomy: P = .001, landmark: P = .021). Multivariable analysis showed that the early group was also associated with a higher chance of ventilation withdrawal in each analysis (early and late tracheostomy: adjusted hazard ratio [aHR] = 1.69, 95% confidence interval [CI] = 1.20-2.39, P = .003; landmark: aHR = 1.61, 95% CI = 1.06-2.38, P = .027). Early tracheostomy, however, was not associated with improved 60-day mortality (early and late tracheostomy: aHR = 0.88, 95% CI = 0.46-1.69, P = .71; landmark: aHR = 1.46; 95% CI = 0.58-3.66; P = .42). CONCLUSION: For patients requiring ventilation, performing tracheostomy within 10 days of admission was independently associated with shortened duration of mechanical ventilation; 60-day mortality was not associated with the timing of tracheostomy. LEVEL OF EVIDENCE: 2b.