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Objective:To investigate the association of plasma EBV-DNA copy number, serum cytokines and B symptoms in patients with extranodal natural killer/T-cell lymphoma, nasal type (ENKTL), unravel the mechanism and assess the prognostic value of clinical indicators.Methods:Clinical data of 173 newly-diagnosed ENKTL patients (116 male, 57 female; median age: 43, 4 to 71 years)were retrospectively analyzed. According to Ann Arbor stage, 126 cases were classified as stage I-II and 47 cases of stage Ⅲ-IV. The primary sites of tumors included nasal cavity (n=100), extranasal upper aerodigestive tract (extranasal UADT, n=34), and extra-upper aerodigestive tract (extra-UADT, n=39). Prior to treatment, 91 patients had B symptoms and 82 cases of without B symptoms. According to plasma EBV-DNA copy levels, all patients were divided into the negative group (n=36), low load group (<10 4 copies/ml, n=73) and high load group (≥10 4 copies/ml, n=64). Serum cytokines including IFN-γ, IL-2, IL-4, IL-6, IL-10 and TNF-α were detected. Correlation analysis was performed by Cochran-Armitage trend test and Spearman correlation analysis. Survival analysis was conducted using univariate and multivariate Cox regression hazard analysis and survival curves were derived from Kaplan-Meier survival analysis. Results:The incidence of B symptoms and fever showed a significant upward trend with the increasing plasma EBV-DNA copy levels. In addition, serum levels of IFN-γ, IL-6 and IL-10 cytokines were higher in patients with B symptoms than those without B symptoms (all P<0.05). Serum IFN-γ, IL-6, and IL-10 levels were also positively correlated with plasma EBV-DNA copy number. The occurrence of B symptoms was associated with high-risk clinical features including advanced stage, primary tumor invasion, regional lymph node involvement, and elevated pre-treatment LDH. Survival analysis showed that stage, B symptoms, plasma EBV-DNA, and the above serum cytokines affected the prognosis of overall survival (OS) and progression-free survival (PFS) (all P<0.05). However, multivariate analysis showed that the occurrence of B symptoms was not an independent prognostic factor of ENKTL patients. Conclusion:This exploratory study suggests that the incidence of B symptoms is associated with increasing levels of EBV-DNA copies and cytokines, and these indicators are also important factors influencing the prognosis of ENKTL patients.
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Nasopharyngeal carcinoma is one of the most common malignant tumors in the southern part of China. The main etiologi-cal factors of nasopharyngeal carcinoma include genetic susceptibility, dietary factors, and Epstein-Barr virus (EBV) infection. EBV de-oxyribonucleic acid (EBV DNA) can be persistently detected in the plasma of patients with nasopharyngeal carcinoma, and its levels correlate with the disease stage. After successful treatment, EBV DNA is rapidly cleared from the plasma of patients;however, signifi-cant increases in plasma EBV DNA levels are observed in patients with recurrent or metastatic diseases after treatment. Accumulating evidence suggests that EBV DNA detection is useful in the early diagnosis and screening, diagnosis of tumor recurrence or distant me-tastasis, prognosis, and tailored treatment of patients with nasopharyngeal carcinoma. This review summarizes the aforementioned progresses to provide a basis for future clinical and research efforts.
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To investigate the presence of integrated Epstein-Barr virus (EBV) DNA in the NK/T cell lymphoma (NKTCL) ge-nome and analyze the integration information in the genome of NKTCL cell lines. Methods: PCR and in situ hybridization were used to detect EBV infection in five EBV (+) NK/T samples and four EBV (-) NK/T samples provided by the biobanks of the First Affiliated Hospi-tal of Zhengzhou University. Whole-genome DNA of the samples was sequenced and subjected to bioinformatics analysis. Whole-ge-nome sequence alignment was used to identify the EBV integration sequence. BLAST analysis was used to compare EBV fasta files of the samples and EBV fasta library. CREST software was used to extract softclip reads, filter all paired reads, and enumerate their distri-bution on chromosomes. The integrated genomics viewer (IGV) was used to compare the distribution of reads in partial regions of chromosome. PCR was used to amplify the high-frequency integration region of the EBV DNA. The amplified fragments were sanger se-quenced. Results: EBV DNA and EBER expression were detected in five EBV (+) NK/T samples but not in the four EBV (-) NK/T samples. Sequencing depth, coverage depth, proportion of coverage, and proportion of alignment all met the requirements for subsequent re-search. Sequence alignment revealed that the captured sequences were viral sequences. Filtered reads were most numerous in EBV (+) NKTCL cell line SNK, YTS, and EBV (+) nasal NKTCL tissue. The reads were non-randomly enriched in chromosome 2. EBV DNA inte-gration in the 400 bp region of chr2:30234084-30234483 caused insertion or deletion in the chr2p23.1 site. Conclusions: EBV DNA is highly integrated in the chr2p23.1 site of EBV (+) NKTCL cells and may affect the expression of related genes.
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Objective It has been confirmed that Epstein-Barr virus ( EBV) is associated with the occurrence and development of the nasopharyngeal carcinoma ( NPC ) . We investigated the clinical significance of plasma concentrations of EBV-DNA in patients with NPC. Methods Since October,2013 to December,2016,471 patients were analyzed. The significantly associated between EBV-DNA before treatment and staging, tumor burden was analyzed. The survival rate of EBV-DNA before and after treatment was calculated. Results The median copies of pretreatment plasma EBV-DNA in patients is 137 copies,( range 0-494000) ,which is correlated with T stage,N stage,M stage,clinical stage and tumor burden load and that is statistically significant. Overall survival ( OS,P=0. 007) ,progression-free survival ( PFS,P=0. 011) and distant metastasis-free survival ( DMFS,P=0. 003) were significantly lower among patients with pretreatment plasma EBV-DNA more than 1300 copies/ml. Patients with detectable plasma EBV-DNA had significantly worse OS (P=0. 016),PFS (P=0. 000) and DMFS (P=0. 000) than patients with undetectable EBV-DNA after treatment. Cox multivariate analyze suggests that T stage and EBV-DNA after treatment were independent prognostic factors for OS,however the plasma EBV-DNA after treatment ( P=0. 006,0. 001) and N stage ( P=0. 037,0. 017) were independent prognostic factors for PFS and DMFS. Conclusions The plasma EBV-DNA level was significantly correlated with staging and tumor load before treatment in patients with NPC,and the prognosis of patients with higher copies before treatment could be worse. The plasma EBV-DNA after treatment is predictive for OS,PFS and DMFS.
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Objective To explore the expression of Epstein-Barr virus DNA(EBV DNA)in the peripheral blood lymphocytes and plasma of patients with EBV-associated diseases.Methods The whole blood samples were collected from 112 patients with suspected EB virus infection diseases,including 14 cases of nasopharyngeal carcinoma(NPC),16 cases of infectious mononucleosis(IM),22 cases of lymphoma,23 cases of autoimmune disease,26 cases of upper respiratory tract infection, and 11 cases of abnormal liver function.The levels of EBV DNA in lymphocytes and plasma of the same sample were detec-ted by fluorescence quantitative PCR(FQ-PCR).Results The EBV DNA positive rates in lymphocytes and plasma of all 112 patients were 83.0%(93/112)and 27.7%(31/112)respectively,with statistically significant difference(χ2=60.02,P<0.01).The positive rate and the load of EB virus DNA in lymphocytes and plasma of 14 patients with nasopharyngeal car-cinoma(NPC)had no statistical difference(χ2=2.25,t=-1.04,all P>0.05).However,patients with lymphoma,infec-tious mononucleosis,upper respiratory tract infection,autoimmune disease or abnormal liver function,the positive rates and the concentration of EBV DNA in the plasma were dramatically lower than those in the peripheral blood lymphocytes,and the difference was statistically significant(χ2=4.17~15.06,all P<0.05;t=3.94~10.45,all P<0.01).Conclusion The detection of EB DNA in peripheral blood lymphocytes of non NPC patients by FQ-PCR might be better than that in plasma. There was no statistical difference between the detection of EBV DNA in lymphocytes and plasma of patients with nasopha-ryngeal carcinoma.Appropriate specimen type could be selected according to clinical consideration.
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BACKGROUND: Epstein-Barr virus (EBV) is known to be the causative agent of infectious mononucleosis and EBV-related malignancies. In this study, we compared the results of three real-time PCR kits for EBV DNA assays. METHODS: A total of 300 whole blood samples submitted for quantitative EBV PCR between January 2013 and September 2014 at Severance Hospital were included. The samples were tested by using the Artus EBV RG PCR Kit (Qiagen, Germany), AccuPower EBV Quantitative PCR Kit (Bioneer, Korea), and Real-Q EBV Kit (BioSewoom, Korea). Samples with discordant results between the three kits were confirmed by direct sequencing. RESULTS: The result concordance rate and kappa coefficient (K) were 86.3% and 0.69 for Artus-AccuPower, 93.3% and 0.85 for Artus-Real-Q, and 92.3% and 0.83 for AccuPower-Real-Q, respectively. The correlations between the three kits were found to be significant, with a correlation coefficient of r=0.854 for Artus-AccuPower, -0.802 for Artus-Real-Q, and -0.977 for AccuPower-Real-Q, respectively (P<0.0001). If the real-time PCR concordant results of 258 samples and the direct sequencing results of 42 real-time PCR discordant samples were assumed to be true, the sensitivity/specificity values were 0.921/0.976 for Artus, 0.902/0.965 for AccuPower, and 0.967/1.000 for Real-Q. CONCLUSIONS: The three real-time PCR kits showed excellent sensitivities and specificities. All these kits would be acceptable for clinical and therapeutic management of EBV. However, some discordant results between the kits indicate the need for caution in clinical diagnosis and staging. Further implementation of standardized methodology would be needed for EBV DNA assays.
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Diagnosis , DNA , Herpesvirus 4, Human , Infectious Mononucleosis , Polymerase Chain Reaction , Real-Time Polymerase Chain ReactionABSTRACT
Objective:To evaluate the efficacy of Epstein-Barr nuclear antigen 1/immunoglobulin A (EBNA1/IgA), BamH1 Z transactivator/IgA (Zta/IgA), capsid antigen/IgA (VCA/IgA), and Epstein-Barr virus deoxyribonucleic acid (EBV-DNA) in detecting different stages of na-sopharyngeal carcinoma (NPC). The relationship between the EBV markers and stages of NPC was also analyzed. Methods:Blood sam-ples of 152 untreated patients with NPC and 675 healthy subjects were collected.ELISA was used to detect the serum levels of EBNA1/IgA, Zta/IgA, and VCA/IgA. Fluorescence quantitative PCR (FQ-PCR) was used to detect the plasma levels of EBV-DNA. ROC and correla-tion analyses were employed to assess the detection assays for NPC diagnosis. The positive rates of EBV markers in NPC patients in dif-ferent stages were analyzed statistically. Results: The positive rates of EBNA1/IgA, Zta/IgA, VCA/IgA, and EBV-DNA in NPC patients were higher than those in the healthy individuals. The expression of EBNA1/IgA was relatively high in early NPC. The sensitivity of EB-NA1/IgA was 77.8%. In advanced NPC, the level of EBV-DNA was high, and the sensitivity of EBV-DNA was 88.8%. The specificity of EBV-DNA and EBNA1/IgA could reach more than 96%. The combination of EBV-DNA and EBNA1/IgA showed the best diagnostic value, with a sensitivity of 92.1%(early stage 82.5%, advanced stage 98.9%) and a specificity of 96.9%. The positive rates of EBV-DNA were positively associated with the NPC clinic stage and N stage. The positives rates of Zta/IgA were positively associated with the NPC N stage. Conclusion:The best single index for NPC screening in an asymptomatic population is EBNA1/IgA. EBV–DNA is an ideal index for auxiliary diagnostics of advanced NPC. The combination of EBV-DNA and EBNA1/IgA shows the best diagnostic value. EBV-DNA is an important index in the stage and illness monitoring of NPC. Zta/IgA can indirectly reflect the character of lymph node metastasis, and it may be useful in assessment of NPC surveillance.
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BACKGROUND AND OBJECTIVES: Extranodal non-Hodgkin's lymphoma (NHL) of the head and neck (H & N) accounts for 10-20% of all cases of NHL. Despite their frequency, the cause of these lymphomas is still poorly understood. Recently, the role of viral origin in NHLs, including Epstein-Barr virus (EBV), as the main cause of sinonasal lymphomas of T/NK cell phenotype and HTLV-1 as a cause of acute T-cell lymphoma/leukemia has been well documented. We investigated the clinicopathologic findings, immunophenotypic profile, and status of EBV and HTLV-1 DNA of patients with H & N lymphoma. MATERIALS AND METHODS: Twenty-seven patients with NHL of H & N region were studied. There were 15 males and 12 females with the median age of 50 years. All patients were reclassified according to the Working formulation (WF) and REAL classificaton. EBV genome DNA and HTLV-1 RNA were surveyed by PCR assay using formalin-fixed and paraffin-embedded tissue blocks. RESULTS: The tonsil was the most commonly involved site (44.4%), followed by nasal cavity (18.5%), nasopharynx (18.5%) and orbit (7.4%). Immunophenotyping revealed 19 cases of B cell lineage, 7 cases of T cell lineage and one case of null cell type. Most of B-cell lymphomas were diffuse large cell lymphomas (58%). Tonsillar lymphomas were all B-cell origin. Four of the five nasal cavity lymphomas and one nasopharyngeal lymphoma showed an angiocentric T/NK cell phenotype with strong association with EBV. EBV genome was detected in 15 of 26 H & N NHLs (57.7%). Seven of 19 B-cell lymphomas (36.8%) and all T/NK or null cell type lymphomas were positive for FBV DNA. However, there was no HTLV-1 positive cases found. CONCLUSIONS: It could be concluded that the high incidence of EBV of angiocentric T/NK-cell lymphomas of the nasal cavity may indicate a probable role of EBV in the development of these lymphomas.