RESUMEN
BACKGROUND: Population screening of asymptomatic persons with Epstein-Barr virus (EBV) DNA or antibodies has improved the diagnosis of nasopharyngeal carcinoma and survival among affected persons. However, the positive predictive value of current screening strategies is unsatisfactory even in areas where nasopharyngeal carcinoma is endemic. METHODS: We designed a peptide library representing highly ranked B-cell epitopes of EBV coding sequences to identify novel serologic biomarkers for nasopharyngeal carcinoma. After a retrospective case-control study, the performance of the novel biomarker anti-BNLF2b total antibody (P85-Ab) was validated through a large-scale prospective screening program and compared with that of the standard two-antibody-based screening method (EBV nuclear antigen 1 [EBNA1]-IgA and EBV-specific viral capsid antigen [VCA]-IgA). RESULTS: P85-Ab was the most promising biomarker for nasopharyngeal carcinoma screening, with high sensitivity (94.4%; 95% confidence interval [CI], 86.4 to 97.8) and specificity (99.6%; 95% CI, 97.8 to 99.9) in the retrospective case-control study. Among the 24,852 eligible participants in the prospective cohort, 47 cases of nasopharyngeal carcinoma (38 at an early stage) were identified. P85-Ab showed higher sensitivity than the two-antibody method (97.9% vs. 72.3%; ratio, 1.4 [95% CI, 1.1 to 1.6]), higher specificity (98.3% vs. 97.0%; ratio, 1.01 [95% CI, 1.01 to 1.02]), and a higher positive predictive value (10.0% vs. 4.3%; ratio, 2.3 [95% CI, 1.8 to 2.8]). The combination of P85-Ab and the two-antibody method markedly increased the positive predictive value to 44.6% (95% CI, 33.8 to 55.9), with sensitivity of 70.2% (95% CI, 56.0 to 81.4). CONCLUSIONS: Our results suggest that P85-Ab is a promising novel biomarker for nasopharyngeal carcinoma screening, with higher sensitivity, specificity, and positive predictive value than the standard two-antibody method. (Funded by the National Key Research and Development Program of China and others; ClinicalTrials.gov number, NCT04085900.).
Asunto(s)
Anticuerpos Antivirales , Detección Precoz del Cáncer , Herpesvirus Humano 4 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Proteínas Virales , Humanos , Anticuerpos Antivirales/inmunología , Estudios de Casos y Controles , Herpesvirus Humano 4/inmunología , Inmunoglobulina A , Tamizaje Masivo , Carcinoma Nasofaríngeo/diagnóstico , Carcinoma Nasofaríngeo/inmunología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/virología , Estudios Prospectivos , Estudios Retrospectivos , Biomarcadores/análisis , Proteínas Virales/inmunología , Epítopos/inmunologíaRESUMEN
Genetic variants in Epstein-Barr virus (EBV) have been strongly associated with nasopharyngeal carcinoma (NPC) in South China. However, different results regarding the most significant viral variants, with polymorphisms in EBER2 and BALF2 loci, have been reported in separate studies. In this study, we newly sequenced 100 EBV genomes derived from 61 NPC cases and 39 population controls. Comprehensive genomic analyses of EBV sequences from both NPC patients and healthy carriers in South China were conducted, totaling 279 cases and 227 controls. Meta-analysis of genome-wide association study revealed a 4-bp deletion downstream of EBER2 (coordinates, 7188-7191; EBER-del) as the most significant variant associated with NPC. Furthermore, multiple viral variants were found to be genetically linked to EBER-del forming a risk haplotype, suggesting that multiple viral variants might be associated with NPC pathogenesis. Population structure and phylogenetic analyses further characterized a high risk EBV lineage for NPC revealing a panel of 38 single nucleotide polymorphisms (SNPs), including those in the EBER2 and BALF2 loci. With linkage disequilibrium clumping and feature selection algorithm, the 38 SNPs could be narrowed down to 9 SNPs which can be used to accurately detect the high risk EBV lineage. In summary, our study provides novel insight into the role of EBV genetic variation in NPC pathogenesis by defining a risk haplotype of EBV for downstream functional studies and identifying a single high risk EBV lineage characterized by 9 SNPs for potential application in population screening of NPC.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Genoma Viral , Herpesvirus Humano 4 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Femenino , Humanos , Masculino , China/epidemiología , Pueblos del Este de Asia , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/genética , Variación Genética , Estudio de Asociación del Genoma Completo , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/genética , Filogenia , Polimorfismo de Nucleótido SimpleRESUMEN
Nasopharyngeal carcinoma (NPC) risk prediction models based on Epstein-Barr virus (EBV)-antibody testing have shown potential for screening of NPC; however, the long-term stability is unclear. Here, we investigated the kinetics of two EBV-antibody NPC risk scores within the Taiwan NPC Multiplex Family Study. Among 545 participants with multiple blood samples, we evaluated the stability of a 2-marker enzyme-linked immunosorbent assay score and 13-marker multiplex serology score using the intra-class correlation coefficient (ICC) by fitting a linear mixed model that accounted for the clustering effect of multiple measurements per subject and age. We also estimated the clustering of positive tests using Fleiss's kappa statistic. Over an average 20-year follow-up, the 2-marker score showed high stability over time, whereas the 13-marker score was more variable (p < .05). Case-control status is associated with the kinetics of the antibody response, with higher ICCs among cases. Positive tests were more likely to cluster within the same individual for the 2-marker score than the 13-marker score (p < .05). The 2-marker score had an increase in specificity from ~90% for single measurement to ~96% with repeat testing. The 13-marker score had a specificity of ~73% for a single measurement that increased to ~92% with repeat testing. Among individuals who developed NPC, none experienced score reversion. Our findings suggest that repeated testing could improve the specificity of NPC screening in high-risk NPC multiplex families. Further studies are required to determine the impact on sensitivity, establish optimal screening intervals, and generalize these findings to general population settings in high-risk regions.
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Anticuerpos Antivirales , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Humanos , Taiwán/epidemiología , Herpesvirus Humano 4/inmunología , Anticuerpos Antivirales/sangre , Masculino , Femenino , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/epidemiología , Infecciones por Virus de Epstein-Barr/virología , Adulto , Persona de Mediana Edad , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/sangre , Neoplasias Nasofaríngeas/epidemiología , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/inmunología , Carcinoma Nasofaríngeo/diagnóstico , Carcinoma Nasofaríngeo/sangre , Carcinoma Nasofaríngeo/epidemiología , Cinética , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Adulto Joven , Factores de Riesgo , AncianoRESUMEN
Nasopharyngeal carcinoma (NPC) carcinogenesis and malignant transformation are intimately associated with Epstein-Barr virus (EBV) infection. A zinc-fingered transcription factor known as Krüppel-like factor 5 (KLF5) has been shown to be aberrantly expressed in a number of cancer types. However, little is known about the regulatory pathways and roles of KLF5 in EBV-positive NPC. Our study found that KLF5 expression was significantly lower in EBV-positive NPC than in EBV-negative NPC. Further investigation revealed that EBER1, which is encoded by EBV, down-regulates KLF5 via the extracellular signal-regulated kinase (ERK) signalling pathway. This down-regulation of KLF5 by EBER1 contributes to maintaining latent EBV infection in NPC. Furthermore, we uncovered the biological roles of KLF5 in NPC cells. Specifically, KLF5 may influence the cell cycle, prevent apoptosis, and encourage cell migration and proliferation - all of which have a generally pro-cancer impact. In conclusion, these findings offer novel strategies for EBV-positive NPC patients' antitumour treatment.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Factores de Transcripción de Tipo Kruppel , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Humanos , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Sistema de Señalización de MAP Quinasas , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/metabolismo , Latencia del Virus , ARN ViralRESUMEN
The Epstein-Barr Virus (EBV) is involved in the etiology of multiple hematologic and epithelial human cancers. EBV+ tumors employ multiple immune escape mechanisms, including the recruitment of immunosuppressive regulatory T cells (Treg). Here, we show some EBV+ tumor cells express high levels of the chemokines CCL17 and CCL22 both in vitro and in vivo and that this expression mirrors the expression levels of expression of the EBV LMP1 gene in vitro. Patient samples from lymphoblastic (Hodgkin lymphoma) and epithelial (nasopharyngeal carcinoma; NPC) EBV+ tumors revealed CCL17 and CCL22 expression of both tumor cell-intrinsic and -extrinsic origin, depending on tumor type. NPCs grown as mouse xenografts likewise showed both mechanisms of chemokine production. Single cell RNA-sequencing revealed in vivo tumor cell-intrinsic CCL17 and CCL22 expression combined with expression from infiltrating classical resident and migratory dendritic cells in a CT26 colon cancer mouse tumor engineered to express LMP1. These data suggest that EBV-driven tumors employ dual mechanisms for CCL17 and CCL22 production. Importantly, both in vitro and in vivo Treg migration was effectively blocked by a novel, small molecule antagonist of CCR4, CCR4-351. Antagonism of the CCR4 receptor may thus be an effective means of activating the immune response against a wide spectrum of EBV+ tumors.
Asunto(s)
Quimiocina CCL17/inmunología , Quimiocina CCL22/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Neoplasias/inmunología , Neoplasias/virología , Linfocitos T Reguladores/inmunología , Animales , Infecciones por Virus de Epstein-Barr/complicaciones , Herpesvirus Humano 4 , Xenoinjertos , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/virología , Humanos , Ratones , Carcinoma Nasofaríngeo/inmunología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/virologíaRESUMEN
Metabolic reprogramming induced by Epstein-Barr virus (EBV) often mirrors metabolic changes observed in cancer cells. Accumulating evidence suggests that lytic reactivation is crucial in EBV-associated oncogenesis. The aim of this study was to explore the role of metabolite changes in EBV-associated malignancies and viral life cycle control. We first revealed that EBV (LMP1) accelerates the secretion of the oncometabolite D-2HG, and serum D-2HG level is a potential diagnostic biomarker for NPC. EBV (LMP1)-driven metabolite changes disrupts the homeostasis of global DNA methylation and demethylation, which have a significantly inhibitory effect on active DNA demethylation and 5hmC content. We found that loss of 5hmC indicates a poor prognosis for NPC patients, and that 5hmC modification is a restriction factor of EBV reactivation. We confirmed a novel EBV reactivation inhibitor, α-KG, which inhibits the expression of EBV lytic genes with CpG-containing ZREs and the latent-lytic switch by enhancing 5hmC modification. Our results demonstrate a novel mechanism of which metabolite abnormality driven by EBV controls the viral lytic reactivation through epigenetic modification. This study presents a potential strategy for blocking EBV reactivation, and provides potential targets for the diagnosis and therapy of NPC.
Asunto(s)
Metilación de ADN , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Activación Viral , Humanos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/complicaciones , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Matriz Viral/genética , Epigénesis Genética , Progresión de la EnfermedadRESUMEN
BACKGROUND: Lytic Epstein-Barr virus (EBV) infection plays a major role in the pathogenesis of nasopharyngeal carcinoma (NPC). For patients with recurrent or metastatic NPC and resistant to conventional therapies, adoptive cell therapy using EBV-specific cytotoxic T cells (EBV-CTLs) is a promising option. However, the long production period (around 3 to 4 weeks) and low EBV-CTL purity (approximately 40% of total CD8 T cells) in the cell product limits the application of EBV-CTLs in clinics. Thus, this study aimed to establish a protocol for the rapid production of EBV-CTLs. METHODS: By culturing peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors with EBV-specific peptides and interleukin (IL)-2, IL-15, and interferon α (IFN-α) for 9 days, we identified that IL-15 can enhance IL-2-mediated CTL activation and significantly increase the yield of CTLs. RESULTS: When IFN-α was used in IL-2/IL-15-mediated CTL production from days 0 to 6, the productivity of EBV-CTLs and EBV-specific cytotoxicity significantly were reinforced relative to EBV-CTLs from IL-2/IL-15 treatment. Additionally, IFN-α-induced production improvement of virus-specific CTLs was not only the case for EBV-CTLs but also for cytomegalovirus-specific CTLs. CONCLUSION: We established a novel protocol to rapidly expand highly pure EBV-CTLs from PBMCs, which can produce EBV-CTLs in 9 days and does not require feeder cells during cultivation.
Asunto(s)
Herpesvirus Humano 4 , Linfocitos T Citotóxicos , Humanos , Linfocitos T Citotóxicos/inmunología , Herpesvirus Humano 4/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/virología , Interleucina-2/metabolismo , Interleucina-2/farmacología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Interleucina-15/metabolismo , Interferón-alfa/metabolismo , Citotoxicidad Inmunológica , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/inmunología , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/patología , Activación de Linfocitos/inmunología , Inmunoterapia Adoptiva/métodosRESUMEN
PURPOSE: Patients with recurrent or metastatic nasopharyngeal carcinoma (RM-NPC) have proven benefit from anti-programmed cell death 1 (anti-PD-1) monotherapy. Here, we retrospectively analyze the association of plasma Epstein-Barr virus (EBV) DNA load and tumor viral lytic genome with clinical outcome from 2 registered phase I trials. METHODS: Patients with RM-NPC from Checkmate 077 (nivolumab phase I trial in China) and Camrelizumab phase I trial between March 2016 and January 2018 were enrolled. Baseline EBV DNA titers were tested in 68 patients and EBV assessment was performed in 60 patients who had at least 3 post-baseline timepoints of EBV data and at least 1 post-baseline timepoint of radiographic assessment. We defined "EBV response" as 3 consecutive timepoints of load below 50% of baseline, and "EBV progression" as 3 consecutive timepoints of load above 150% of baseline. Whole-exome sequencing was performed in 60 patients with available tumor samples. RESULTS: We found that the baseline EBV DNA load was positively correlated with tumor size (spearman p < 0.001). Both partial response (PR) and stable disease (SD) patients had significantly lower EBV load than progression disease (PD) patients. EBV assessment was highly consistent with radiographic evaluation. Patients with EBV response had significantly improved overall survival (OS) than patients with EBV progression (log-rank p = 0.004, HR = 0.351 [95% CI: 0.171-0.720], median 22.5 vs. 11.9 months). The median time to initial EBV response and progression were 25 and 36 days prior to initial radiographic response and progression, respectively. Patients with high levels of EBV lytic genomes at baseline, including BKRF2, BKRF3 and BKRF4, had better progression-free survival (PFS) and OS. CONCLUSION: In summary, early clearance of plasma EBV DNA load and high levels of lytic EBV genes were associated with better clinical outcome in patients with RM-NPC receiving anti-PD-1 monotherapy.
Asunto(s)
ADN Viral , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Recurrencia Local de Neoplasia , Nivolumab , Carga Viral , Humanos , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/tratamiento farmacológico , Carcinoma Nasofaríngeo/sangre , Carcinoma Nasofaríngeo/patología , Masculino , Femenino , Persona de Mediana Edad , ADN Viral/sangre , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/sangre , Neoplasias Nasofaríngeas/patología , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/sangre , Estudios Retrospectivos , Adulto , Recurrencia Local de Neoplasia/virología , Nivolumab/uso terapéutico , Genoma Viral , Anciano , Anticuerpos Monoclonales Humanizados/uso terapéutico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Pronóstico , Resultado del TratamientoRESUMEN
O-Glycan synthesis enzyme glucosaminyl (N-acetyl) transferase 3 (GCNT3) is closely related to the occurrence and development of various cancers. However, the regulatory mechanism and function of GCNT3 in nasopharyngeal carcinoma (NPC) are still poorly understood. This study aims to explore the regulatory mechanism of EBV-encoded latent membrane protein 2A (LMP2A) on GCNT3 and the biological role of GCNT3 in NPC. The results show that LMP2A can activate GCNT3 through the mTORC1 pathway, and there is a positive feedback between the mTORC1 and GCNT3. GCNT3 regulates EMT progression by forming a complex with ZEB1 to promote cell migration. GCNT3 can also promote cell proliferation. These findings indicate that targeting the LMP2A-mTORC1-GCNT3 axis may represent a novel therapeutic target in NPC.
Asunto(s)
Movimiento Celular , Proliferación Celular , N-Acetilglucosaminiltransferasas , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Proteínas de la Matriz Viral , Humanos , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Transición Epitelial-Mesenquimal/genéticaRESUMEN
BACKGROUND: Treatment of Epstein-Barr virus(EBV)-positive nasopharyngeal carcinoma (NPC) with cisplatin/5-fluorouracil (5-FU) induction chemotherapy, followed by radiochemotherapy and subsequent interferonß, has yielded high survival rates in children, adolescents, and young adults. A previous study has shown that reduction of radiation dose from 59.4 to 54.0â¯Gy appears to be safe in patients with complete response (CR) to induction chemotherapy. As immune checkpoint-inhibitors have shown activity in NPC, we hypothesize that the addition of nivolumab to standard induction chemotherapy would increase the rate of complete tumor responses, thus allowing for a reduced radiation dose in a greater proportion of patients. METHODS: This is a prospective multicenter phase 2 clinical trial including pediatric and adult patients with their first diagnosis of EBV-positive NPC, scheduled to receive nivolumab in addition to standard induction chemotherapy. In cases of non-response to induction therapy (stable or progressive disease), and in patients with initial distant metastasis, treatment with nivolumab will be continued during radiochemotherapy. Primary endpoint is tumor response on magnetic resonance imaging (MRI) and positron emission tomography (PET) after three cycles of induction chemotherapy. Secondary endpoints are event-free (EFS) and overall survival (OS), safety, and correlation of tumor response with programmed cell death ligand 1 (PD-L1) expression. DISCUSSION: As cure rates in localized EBV-positive NPC today are high with standard multimodal treatment, the focus increasingly shifts toward prevention of late effects, the burden of which is exceptionally high, mainly due to intense radiotherapy. Furthermore, survival in patients with metastatic disease and resistant to conventional chemotherapy remains poor. Primary objective of this study is to investigate whether the addition of nivolumab to standard induction chemotherapy in children and adults with EBV-positive NPC is able to increase the rate of complete responses, thus enabling a reduction in radiation dose in more patients, but also offer patients with high risk of treatment failure the chance to benefit from the addition of nivolumab. TRIAL REGISTRATION: EudraCT (European Union Drug Regulating Authorities Clinical Trials Database) No. 2021-006477-32.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Quimioterapia de Inducción , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Nivolumab , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quimioradioterapia/métodos , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/diagnóstico , Carcinoma Nasofaríngeo/tratamiento farmacológico , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/terapia , Nivolumab/uso terapéutico , Nivolumab/efectos adversos , Estudios Prospectivos , Resultado del Tratamiento , Estudios Multicéntricos como Asunto , Ensayos Clínicos Fase II como AsuntoRESUMEN
The study presents an analysis of the diagnostic and treatment protocol for a patient with a first episode of nasopharyngeal carcinoma who also has Sjogren's syndrome and Epstein-Barr Virus (EBV) positive cerebrospinal fluid, as detected through metagenomic next-generation sequencing (mNGS). It reviews existing literature to examine the connections between EBV and various conditions including Sjogren's syndrome, encephalitis or meningitis, and nasopharyngeal carcinoma, emphasizing the importance of EBV positive cerebrospinal fluid. The study focuses on a case from the Eighth Medical Center of the General Hospital of the People's Liberation Army, where a patient was admitted with headaches as the primary symptom on March 3, 2021. This patient had a history of Sjogren's syndrome and was later diagnosed with nasopharyngeal carcinoma. The research involved reviewing both domestic and international databases for cases related to cerebrospinal fluid EBV positive encephalitis or meningitis, and nasopharyngeal carcinoma. It aimed to aggregate data on demographics, initial symptoms, treatment methods, and patient outcomes. Findings suggest that positive cerebrospinal fluid EBV is linked to autoimmune diseases, viral encephalitis or meningitis, and nasopharyngeal carcinoma, albeit infrequently in the context of Sjogren's syndrome. Notably, EBV positive cerebrospinal fluid is commonly associated with recurrent nasopharyngeal carcinoma rather than initial episodes. The study concludes that for patients with an immune condition, exhibiting symptoms like headaches or cranial nerve issues, or in cases where nasopharyngeal carcinoma is suspected, early testing through cerebrospinal fluid mNGS or EBV DNA is recommended. This approach facilitates risk assessment, prognosis determination, and the creation of individualized treatment plans.
Asunto(s)
Herpesvirus Humano 4 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Síndrome de Sjögren , Humanos , Síndrome de Sjögren/líquido cefalorraquídeo , Síndrome de Sjögren/diagnóstico , Síndrome de Sjögren/complicaciones , Síndrome de Sjögren/virología , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/diagnóstico , Carcinoma Nasofaríngeo/líquido cefalorraquídeo , Herpesvirus Humano 4/aislamiento & purificación , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/líquido cefalorraquídeo , Infecciones por Virus de Epstein-Barr/líquido cefalorraquídeo , Infecciones por Virus de Epstein-Barr/diagnóstico , Infecciones por Virus de Epstein-Barr/complicaciones , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Saliva sampling is a non-invasive method, and could be performed by donors themselves. However, there are few studies reporting biomarkers in saliva in the diagnosis of NPC. A total of 987 salivary samples were used in this study. First, EBV DNA methylation was profiled by capture sequencing in the discovery cohort (n = 36). Second, a q-PCR based method was developed and five representative EBV DNA CpG sites (11 029 bp, 45 849 bp, 57 945 bp, 66 226 bp and 128 102 bp) were selected and quantified to obtain the methylated density in the validation cohort1 (n = 801). Third, a validation cohort2 (n = 108) was used to further verify the differences of EBV methylation in saliva. A significant increase of EBV methylation was found in NPC patients compared with controls. The methylated score of EBV genome obtained by capture sequencing could distinguish NPC from controls (sensitivity 90%, specificity 100%). Further, the methylated density of EBV DNA CpG sites revealed by q-PCR showed a good diagnostic performance. The sensitivity and specificity of detecting a single CpG site (11 029 bp) could reach 75.4% and 99.7% in the validation cohort1, and 78.2% and 100% in the validation cohort2. Besides, the methylated density of the CpG site was found to decrease below the COV in NPC patients after therapy, and increase above the COV after recurrence. Our study provides an appealing alternative for the non-invasive detection of NPC without clinical setting. It paves the way for conducting a home-based large-scale screening in the future.
Asunto(s)
Metilación de ADN , Infecciones por Virus de Epstein-Barr , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Saliva/química , Biopsia , Humanos , Carcinoma Nasofaríngeo/diagnóstico , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/virología , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/diagnóstico , Herpesvirus Humano 4 , Estudios de Casos y Controles , ADN Viral/genética , Islas de CpGRESUMEN
Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this workflow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.
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Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Empalme del ARN , ARN Largo no Codificante/genética , ARN Mensajero/genética , Programas Informáticos , Benchmarking , Línea Celular Tumoral , Biología Computacional/métodos , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Exones , Herpesvirus Humano 4/patogenicidad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Intrones , Secuenciación de Nanoporos , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/virología , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Análisis de Secuencia de ARNRESUMEN
Epstein-Barr virus (EBV) is associated with several malignant diseases, including Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), certain types of lymphomas, and a portion of gastric cancers. The virus-encoded oncoprotein, LMP1, induces the epithelial-to-mesenchymal transition (EMT), leading to cancer stem cell formation. In the current study, we investigated how LMP1 contributes to cancer stem cell development in NPC. We found that LMP1 plays an essential role in acquiring cancer stem cell (CSC) characteristics, including tumor initiation, metastasis, and therapeutic resistance by activating the PI3K/mTOR/Akt signaling pathway. We dissected the functions of distinct signaling (mTORC1 and mTORC2) in the acquisition of different CSC characteristics. Side population (SP) formation, which represents the chemotherapy resistance feature of CSC, requires mTORC1 signaling. Tumor initiation capability is mainly attributed to mTORC2, which confers on NPC the capabilities of proliferation and survival by activating mTORC2 downstream genes c-Myc. Both mTORC1 and mTORC2 enhance cell migration and invasion of NPC cells, suggesting that mTORC1/2 coregulate metastasis of NPC. The revelation of the roles of the mTOR signaling pathways in distinct tumorigenic features provides a guideline for designing efficient therapies by choosing specific mTOR inhibitors targeting mTORC1, mTORC2, or both to achieve durable remission of NPC in patients. IMPORTANCE LMP1 endows NPC to gain cancer stem cell characteristics through activating mTORC1 and mTORC2 pathways. The different mTOR pathways are responsible for distinct tumorigenic features. Rapamycin-insensitive mTORC1 is essential for CSC drug resistance. NPC tumor initiation capacity is mainly attributed to mTORC2 signaling. mTORC1 and mTORC2 coregulate NPC cell migration and invasion. The revelation of the roles of mTOR signaling in NPC CSC establishment has implications for novel therapeutic strategies to treat relapsed and metastatic NPC and achieve durable remission.
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Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Proliferación Celular/genética , Supervivencia Celular/genética , Infecciones por Virus de Epstein-Barr/fisiopatología , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Carcinoma Nasofaríngeo/fisiopatología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/fisiopatología , Neoplasias Nasofaríngeas/virología , Células Madre Neoplásicas/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, α-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (~0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC.
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Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/virología , Interacciones Huésped-Patógeno/inmunología , Neoplasias Nasofaríngeas/virología , Carcinoma/metabolismo , Carcinoma/virología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Epitelio/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Humanos , Carcinoma Nasofaríngeo/virología , ARN Viral/genéticaRESUMEN
Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and contributes to both B-cell and epithelial-cell malignancies. EBV-infected epithelial cell tumors, including nasopharyngeal carcinoma (NPC), are largely composed of latently infected cells, but the mechanism(s) maintaining viral latency are poorly understood. Expression of the EBV BZLF1 (Z) and BRLF1 (R) encoded immediate-early (IE) proteins induces lytic infection, and these IE proteins activate each other's promoters. ΔNp63α (a p53 family member) is required for proliferation and survival of basal epithelial cells and is over-expressed in NPC tumors. Here we show that ΔNp63α promotes EBV latency by inhibiting activation of the BZLF1 IE promoter (Zp). Furthermore, we find that another p63 gene splice variant, TAp63α, which is expressed in some Burkitt and diffuse large B cell lymphomas, also represses EBV lytic reactivation. We demonstrate that ΔNp63α inhibits the Z promoter indirectly by preventing the ability of other transcription factors, including the viral IE R protein and the cellular KLF4 protein, to activate Zp. Mechanistically, we show that ΔNp63α promotes viral latency in undifferentiated epithelial cells both by enhancing expression of a known Zp repressor protein, c-myc, and by decreasing cellular p38 kinase activity. Furthermore, we find that the ability of cis-platinum chemotherapy to degrade ΔNp63α contributes to the lytic-inducing effect of this agent in EBV-infected epithelial cells. Together these findings demonstrate that the loss of ΔNp63α expression, in conjunction with enhanced expression of differentiation-dependent transcription factors such as BLIMP1 and KLF4, induces lytic EBV reactivation during normal epithelial cell differentiation. Conversely, expression of ΔNp63α in undifferentiated nasopharyngeal carcinoma cells and TAp63α in Burkitt lymphoma promotes EBV latency in these malignancies.
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Infecciones por Virus de Epstein-Barr/complicaciones , Herpesvirus Humano 4/patogenicidad , Queratinocitos/virología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/virología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Latencia del Virus , Diferenciación Celular , Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/virología , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Activación ViralRESUMEN
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.
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Infecciones por Virus de Epstein-Barr , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Transactivadores , Aromatasa , Receptor alfa de Estrógeno , Estrógenos , Herpesvirus Humano 4/patogenicidad , Humanos , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/virología , Transactivadores/genéticaRESUMEN
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.
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Infecciones por Virus de Epstein-Barr , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , China/epidemiología , Infecciones por Virus de Epstein-Barr/complicaciones , Genoma Viral , Herpesvirus Humano 4/genética , Humanos , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/virologíaRESUMEN
Epstein-Barr virus (EBV) infection is closely linked to several human malignancies including endemic Burkitt's lymphoma, Hodgkin's lymphoma and nasopharyngeal carcinomas (NPC). Latent membrane protein 2 (LMP-2) of EBV plays a pivotal role in pathogenesis of EBV-related tumors and thus, is a potential target for diagnosis and targeted therapy of EBV LMP-2+ malignant cancers. Affibody molecules are developing as imaging probes and tumor-targeted delivery of small molecules. In this study, four EBV LMP-2-binding affibodies (ZEBV LMP-212, ZEBV LMP-2132, ZEBV LMP-2137, and ZEBV LMP-2142) were identified by screening a phage-displayed LMP-2 peptide library for molecular imaging and targeted therapy in EBV xenograft mice model. ZEBV LMP-2 affibody has high binding affinity for EBV LMP-2 and accumulates in mouse tumor derived from EBV LMP-2+ xenografts for 24 h after intravenous (IV) injection. Subsequent fusion of Pseudomonas exotoxin PE38KDEL to the ZEBV LMP-2 142 affibody led to production of Z142X affitoxin. This fused Z142X affitoxin exhibits high cytotoxicity specific for EBV+ cells in vitro and significant antitumor effect in mice bearing EBV+ tumor xenografts by IV injection. The data provide the proof of principle that EBV LMP-2-speicifc affibody molecules are useful for molecular imaging diagnosis and have potentials for targeted therapy of LMP-2-expressing EBV malignancies.
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Herpesvirus Humano 4 , Inmunotoxinas/uso terapéutico , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/terapia , Proteínas de la Matriz Viral/metabolismo , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inmunotoxinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Imagen Molecular , Carcinoma Nasofaríngeo/diagnóstico por imagen , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/diagnóstico por imagen , Neoplasias Nasofaríngeas/virología , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/terapia , Biblioteca de Péptidos , Unión Proteica , Proteínas de la Matriz Viral/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nasopharyngeal carcinoma (NPC) is the most common malignant tumor of the nasopharynx. Although NPC is not endemic in India, higher incidences were observed in its North-Eastern regions particularly Sikkim, Nagaland, Manipur, and Mizoram. Early detection of NPC is difficult because the nasopharynx is not readily amenable to clinical examination and symptoms of NPC are nonspecific. The development of suitable biomarkers for early diagnosis of NPC as well as accurate monitoring of treatment response is needed urgently. In this exploratory pilot study, we have investigated the clinical significance of assessing plasma Epstein-Barr virus (EBV) DNA load at diagnosis and during treatment. We found that EBV DNA is detectable at diagnosis in the majority of patients with nonendemic NPC and the absolute copy number of circulating EBV DNA per milliliter increases progressively with the stage of the disease. The viral load declined significantly with induction chemotherapy and definitive chemoradiation but showed a sharp rise at relapse. Patients with EBV DNA levels ≥1500 copies/ml had a higher risk of disease progression or relapse when compared with patients who had EBV DNA <1500 copies/ml at baseline. Estimation of plasma EBV DNA may serve as an excellent noninvasive tool to monitor disease extent, response to therapy, and for better prediction of future relapse or progression-free survival in a nonendemic NPC patient population.