EB病毒的潜伏感染及其编码的癌基因在鼻咽非霍奇金恶性淋巴瘤中的表达及意义

目的：探讨鼻咽非霍奇金恶性淋巴瘤患者中EB病毒的感染情况以及在EB病毒潜伏感染状态下编码的病毒癌基因产物潜伏膜蛋白-1(LMP-1)与鼻咽部非霍奇金恶性淋巴瘤的发生及预后的关系，另外还对该组鼻咽淋巴瘤的组织细胞起源进行了分析。方法：我们选用EBER-1／2 mRNA探针，经原位杂交方法检测了70例鼻咽恶性淋巴瘤和10例鼻咽慢性炎症病例中的EB病毒感染，同时使用免疫组化染色方法，分别标记了LMP-1、CD45R0、CD20及CD56阳性细胞。结果：EB病毒mRNA在鼻咽恶性淋巴瘤患者中的阳性表达率(62、9％)明显高于慢性炎症患者(0％)。LMP-1蛋白在上述两者间也有差异表达，肿瘤组(68．6％)高于炎症组(20．0％)(P=0．003)。本组有12例NK／T细胞淋巴瘤，特征性表达CD56，与LMP-1相关，但不具有特殊的生物学行为．LMP-1阳性高表达率除与鼻咽淋巴瘤患者出现临床B症状相关(P=0．043)外，与临床分期、出现区域淋巴结肿大均不相关，但LMP-1的表达与患者的预后密切相关，在死亡患者组中有LMP-1的高表达。结论：在鼻咽恶性淋巴瘤的发生、发展过程中，EB病毒的感染及其编码的癌蛋白LMP-1的产生起到了致关重要的作用。此外，LMP-1与不良临床特征和预后相关，同时还与CD56( )的NK/T细胞淋巴瘤相关。     

不同类型淋巴瘤438例EB病毒分布特点

目的 探讨不同类型淋巴瘤中EB病毒(EBV)的分布特点.方法 选用免疫组织化学EnVision法标记ALK1、CD3、CD5、CD7、CD10、CD15、CD20、CD23、CD30、CD38、CD43、CD56、CD68、CD79、CD99、CyclinD1、EMA、TdT与EBV潜伏膜蛋白(LMP-1),原位杂交技术标记EBV编码的RNA(EBER).按照2008年WHO淋巴造血系统肿瘤分类标准对存档的438例淋巴瘤组织标本重新分类.结果 在B细胞非霍奇金淋巴瘤(B-NHL)、NK/T细胞淋巴瘤和霍奇金淋巴瘤(HL)中EBER阳性率分别是5.4％(14/261)、16.5％(19/115)和59.7％(37/62),LMP-1阳性率分别为5.4％(14/261)、5.2％(6/115)和59.7％(37/62).弥漫大B细胞淋巴瘤(DLBCL)患者中,EBER在60岁以上组中的阳性率(13.2％,7/53)明显高于60岁及以下组(1.2％,1/81)(P＜0.05);在NK/T细胞淋巴瘤中LMP-1蛋白表达与EBER表达具有不一致性,EBER阳性率明显高于LMP-1(P＜0.05);5例鼻型NK/T细胞淋巴瘤中全部表达EBER;在HL淋巴瘤中LMP-1蛋白表达与EBER表达具有一致性.结论 EBV感染与淋巴瘤类型相关,EBV在鼻型NK/T细胞淋巴瘤中表达率高,HL次之.鉴于EBER与LMP-1在HL中的一致性表达,出于经济学考虑,LMP-1可代替EBER用作EBV的检测.EBV可能在鼻型NK/T细胞淋巴瘤、HL等的发生、发展中有重要作用.     

171例淋巴瘤病变组织中EB病毒表达情况的分析

目的:探讨不同类型淋巴瘤与EB病毒(Epstein-Barr virus,EBV)感染的关系。方法:收集淋巴瘤组织171例,包括弥漫大B细胞淋巴瘤(DLBC)106例;结外NK/T细胞淋巴瘤,鼻型22例;霍奇金淋巴瘤(HL)19例;血管免疫母细胞性T细胞淋巴瘤(AITL)13例;黏膜相关淋巴组织B细胞淋巴瘤(MALT)11例。应用EBV Lmp-1单抗免疫组化(IHC)和生物素标记的EBER1寡核苷酸探针原位杂交(ISH)分析EBV感染与淋巴瘤的关系。结果:淋巴瘤组织中EBV Lmp-1蛋白与EBER1 mRNA总阳性率分别为11.1%(19/171)、25.7%(44/171)。其中AITL为30.8%(4/13)、61.5%(8/13);HL为47.4%(9/19)、57.9%(11/19);结外NK/T细胞淋巴瘤为22.7%(5/22)、81.8%(18/22);DLBC为0.94%(1/106)、5.7%(6/106);MALT为0(0/11)、9.1%(1/11)。结果显示EBV在DLBC及MALT中的表达率低于AITL、HL及结外NK/T细胞淋巴瘤,差异有统计学意义(P0.05);且原位杂交检测EBER1 mRNA比免疫组化检测Lmp-1蛋白更为敏感(P0.01)。结论:EBV感染与淋巴瘤有密切关系,不同类型淋巴瘤与EBV感染的关系有差异。     

High tumor necrosis factor-alpha levels in the patients with Epstein-Barr virus-associated peripheral T-cell proliferative disease/lymphoma

We have attempted to find out any relationships between circulating tumor necrosis factor (TNF)-alpha levels and Epstein-Barr virus (EBV) associated peripheral T-cell and NK-cell proliferative disease/lymphoma (PTPD/L) status. The distribution of TNF-alpha level was significantly higher (P<0.05) in patients than in controls. Patients carrying EBV genome in their peripheral T-cells showed higher TNF-alpha levels than the patients with EBV negative peripheral T-cells (P<0.001). Among patients whose peripheral T-cells were positive for EBV genome, TNF-alpha levels between the wild type LMP-1 gene carriers and the 30-bp deletion type LMP-1 gene carriers were compared and the wild type LMP-1 gene carrier group showed significantly higher TNF-alpha levels (P<0.01). As for the outcome of the patients and TNF-alpha levels, significant differences were observed between dead and alive with disease group (P<0.001), and dead and alive with complete remission group (P<0.01). Since circulating TNF-alpha levels in PTPD/L patients correlate with the disease and EBV infection status, it may be possible that monitoring of the TNF-alpha levels will be a useful prognostic marker.     

Gene mutation analysis of sinonasal lymphomas in Indonesia

Sinonasal lymphomas comprise NK/T-cell (NKTCL) type and B-cell type with unique geographical development. In this study, mutations of p53, K-ras, c-kit, beta-catenin, and bak gene were analyzed using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) followed by direct sequencing in 41 sinonasal lymphomas (27 NKTCL and 14 B-cell type) from Indonesia. In situ hybridization study with EBER-1 probe revealed that 85% of NKTCL cases were EBV positive, but none of B-cell type was EBV positive. Frequency of mutations in p53, K-ras, c-kit, beta-catenin, and bak gene was 62.9%, 0%, 11.1%, 18.5%, and 25.9%, respectively, in NKTCL, and 71.4%, 0%, 23.1%, 21.4%, and 57.1%, respectively, in B-cell cases, showing that mutation frequency in all genes was higher in B-cell than in NKTCL cases. These findings suggest that gene mutations might be the driving-force for B-cell lymphoma, whereas combined EBV infection and gene mutations contribute to NKTCL development in Indonesia.     

Development of EBV-positive T-cell lymphoma following infection of peripheral blood T cells with EBV.

Chronic active Epstein-Barr virus (EBV) infection is manifested clinically by the persistence of infectious mononucleosis-like symptoms or its complications for a prolonged period ranging from one to several years. This syndrome may include severe disease manifestations and can be fatal. The role of EBV in the pathogenesis of chronic active EBV infection has been unclear. We investigated two Japanese patients with severe chronic active EBV infection who subsequently developed EBV-positive T-cell lymphoma. We found that the patients had evidence of EBV infection in the peripheral blood CD4+ T-cells 19 and 3 months, respectively, before the T-cell lymphoma was diagnosed. The lymphomas were infected with monoclonal EBV and expressed the EBV latency genes EBNA-1, LMP-1, and LMP-2A, a virus latency pattern referred to as latency II. Genetic studies showed that the virus detected in the T-cell lymphoma was indistinguishable from the virus in the peripheral blood CD4+ T-cells. These studies support an important pathogenetic role of T-cell infection with EBV in chronic active EBV infection and in the EBV-positive T-cell lymphoma that followed.     

Nasal lymphomas in Japan: a high prevalence of Epstein-Barr virus type A and deletion within the latent membrane protein gene

The majority of nasal lymphomas are of the natural killer (NK)/T cell lineage. We analyzed 33 specimens of nasal lymphoma from Japanese patients for Epstein-Barr virus (EBV). Phenotypic and genetic analyses showed 28 cases with NK/T cell type and 5 cases with B cell type. All NK/T lymphomas were of pleomorphic cell type except 2 large cell (centroblastoid) and one lymphoblastic lymphoma. All cases with nasal B cell lymphoma were of large (centroblastoid) cell type. EBV was detected in all cases of NK/T cell type with the exception of one lymphoblastic case, and was monoclonally integrated in all cases examined (14/14 cases). All but one case had subtype A of EBV infection with 30-base paired deleted LMP-1 gene. One case of B cell lymphoma showed the presence of EBV infection with subtype A and deletion of LMP-1. Our results indicate that the majority of nasal lymphomas in Japanese patients are of the nasal NK/T cell type, have pleomorphic morphology, a high prevalence of EBV with a monoclonal integration, subtype A and deleted LMP-1 gene. In contrast, nasal B cell lymphoma showed monomorphic appearance and rare EBV infection.     

Expression of Etk/Bmx tyrosine kinase in the tumorigenicity of nasopharyngeal epithelium and its relation with EB virus infection and the apoptosis-related protein Bcl-2

We compared Etk/Bmx expression in nasopharyngeal carcinoma (NPC) and non-neoplastic nasopharyngeal lesions in order to learn whether the expression of this non-receptor protein tyrosine kinase is associated with the development of NPC. We also related Etk/Bmx expression to factors resulting from Epstein-Barr virus (EBV) infection. We used immunohistochemistry (IHC) and in situ hybridization to examine 20 non-neoplastic nasopharyngeal lesions and 49 cases of NPC to assess Etk/Bmx, EB virus latent membrane protein-1 (LMP-1), Bcl-2 and EBV-encoded small RNA-1 expression in these samples. Etk/Bmx expression was present in the basal cell nuclei of the nasopharyngeal epithelium in 1/9 (11.1%) cases of chronic nasopharyngitis and 2/11 cases (18.2%) of dysplasia. While 13/49 (26.5%) NPC cases expressed Etk/Bmx, the difference in frequency between the expression of Etk/Bmx in the non-neoplastic and NPC cases was not significant. Etk/Bmx expression was correlated with the presence of EBER-1 immunopositivity in dysplasia and in NPC but not in chronic nasopharyngitis. The presence of Etk/Bmx immunopositivity was independent of the expression of either LMP-1 or Bcl-2 in either the nasopharyngeal carcinoma or the non-neoplastic lesions. This suggests that in some cases of non-neoplastic and neoplastic nasopharyngeal lesions, Etk/Bmx may participate in regulating epithelial differentiation. While EBV-related small RNA-1 may participate in this regulation, neither LMP-1 or Bcl-2 expression appears to be related to Etk/Bmx expression.     

EB病毒LMP-1蛋白与Fas系统在非霍奇金淋巴瘤的表达及意义

目的：研究EB病毒LMP—1、Fas、FasL在NHL的表达及意义。方法：采用免疫组化S-P法对于9例NHL进行LMP-1及Fas系统的检测。结果：LMP—1、Fas、FasL表达在NHL均比对照组明显增高：LMP-1的表达与Fas、FasL呈正相关;FasL低表达,Fas高表达：呈不平衡表达;Fas、FasL的表达与淋巴瘤免疫表型及疾病分期无关。结论：LMP—1可上调Fas系统,Fas系统在感染EBV淋巴细胞的恶性转化可能起重要作用。通过FasL低表达,影响CTL的功能,抵抗肿瘤的免疫监视所致。     

NK/T细胞淋巴瘤基因组中EBV DNA整合检测及分析

  目的  明确NK/T细胞淋巴瘤（NK/T cell lymphoma,NKTCL）基因组中是否存在EB病毒（Epstein-Barr virus,EBV）DNA整合,初步分析NKTCL细胞系基因组中EBV DNA整合信息。  方法  利用PCR法扩增EBV DNA和原位杂交法检测EBER表达,验证由郑州大学第一附属医院生物样本库提供的5例EBV（+）及4例EBV（-）NK/T样本EBV感染情况。测序全基因组DNA样本并进行生物信息学分析。利用全基因组序列比对捕获EBV整合序列;使用Blast比对样本EBV fasta文件与EBV fasta库。采用CREST软件提取softclip reads,过滤paired reads并对过滤后的reads进行染色体分布的统计。使用IGV比对染色体部分区域reads分布情况,采用PCR法扩增EBV DNA高频整合区域并行sanger测序。  结果  5例EBV（+）NK/T样本中均检测出EBV DNA和EBER表达,4例EBV（-）NK/T样本则未能检出。样本的测序深度、覆盖深度、覆盖率和比对率均满足后续研究要求。比对结果显示捕获的序列为病毒序列。EBV（+）NKTCL细胞系SNK、YTS和EBV（+）鼻腔NTKCL组织的reads数目最多,且在2号染色体上呈非随机性富集。chr2:30234084-30234483 400 bp区域存在EBV DNA整合,并导致chr2p23.1位点的插入缺失。  结论  EBV（+）NKTCL细胞在chr2p23.1位点存在EBV DNA的高频整合,提示可能影响相关基因表达。      

Epstein-Barr virus related hemophagocytic syndrome in a T-cell rich B-cell lymphoma.

We report the case of a 30-year-old woman who presented with an EBV related hemophagocytic syndrome. After a few months she developed a T-cell rich B-cell non-Hodgkin's lymphoma with liver involvment. Serological data demonstrated a reactivation of the EBV infection. Tumor progression with liver involvement occured during treatment with conventional chemotherapy. Tumor reduction and disappearence of all masses was seen after starting high-dose sequential chemotherapy, followed by an autologous peripheral blood progenitor transplantation. LMP-1 could be amplified in the tumor material by PCR technology, but no LMP-1 expression could be found in the few malignant B-cells with Reed–Sternberg morphology. Sequence analysis of the carboxy terminal of the LMP-1 region revealed the naturally occuring 30 bp deletion variant of the LMP-1 with multiple point mutations within the NF kb region. Since LMP-1 was not expressed in the malignant tumor cells, no evidence could be found, that EBV participated in the tumorigenesis of this case.     

NK/T细胞淋巴瘤相关基因异常的研究进展

NK/T细胞淋巴瘤(NK/T-cell lymphoma,NKTCL)的发病与EB病毒(epstein-barr virus,EBV)感染、基因异常、染色体异常、信号通路及蛋白表达异常相关.本文就NKTCL相关基因异常的研究进展做一简要综述.NKTCL基因异常包括基因突变和缺失等,可影响细胞增殖、分化、凋亡及信号转导.其中,EBV相关基因LMP1表达产物为LMP1蛋白(latent membrane protein 1),破坏细胞信号传导通路;KIR2DI4、EZH2、DDX3X及c-Myc基因异常通过调节免疫应答、DNA转录等途径影响细胞周期;TP53、JAK3、PTPRK、PRDM1及BCOR基因异常通过编码蛋白、影响细胞内信号转导通路等途径抑制细胞凋亡,进而导致细胞的恶性转化.在基因水平对NKTCL进行深入研究会帮助我们进一步认识NKTCL的发病机制,为疾病的治疗提供新的思路与靶点,完善预后评价体系.     

What we should know about natural killer/T‐cell lymphomas

Natural‐killer/T cell lymphoma (NKTCL) is the most common extranodal lymphoma with highly aggressive clinical outcome. System biology techniques provide novel insights into the pathogenesis, risk stratification, and clinical management in NKTCL. Comparative genomic hybridization analysis reveal most frequent deletion of chromosome 6q21. Whole‐exome sequencing studies identify recurrent somatic gene mutations, involving RNA helicases, tumor suppressors, JAK‐STAT pathway molecules, and epigenetic modifiers. Genome‐wide association study reports strongest association of HLA‐DPB1 rs9277378 with lymphomagenesis. Alterations of oncogenic signaling pathways as well as epigenetic dysregulation of microRNA and long non‐coding RNAs are also observed in NKTCL. Epstein‐Barr virus (EBV) is the major etiology of NKTCL and the pathogenic mechanism remains unclear. Different risk stratification models are proposed based on clinical parameters (IPI, PINK, and PINK‐E, etc.) or biomarkers (Ki67, C‐reactive protein level, and EBV DNA, etc.). Therapeutic strategies vary according to disease stage, including radiotherapy, asparaginase‐based chemotherapy, hematopoietic stem‐cell transplantation, targeted therapy (immune checkpoints inhibitors, and histone deacetylation inhibitors, etc.). Future investigations will be emphasized on EBV‐related pathogenesis of NKTCL, prognostic and therapeutic biomarkers, as well as multi‐center clinical trials, so as to optimize personalized treatment of NKTCL in the era of precision medicine.     

鼻型结外自然杀伤-T细胞淋巴瘤研究进展

鼻型结外自然杀伤(NK)-T细胞淋巴瘤(NKTCL)起源于成熟NK细胞或NK样T细胞,常表达CD3ε和CD56,Epstein-Barr病毒(EBV)常阳性并在发病机制中起重要作用.已经建立了多种NKTCL的细胞株和动物模型并用于实验研究.NKTCL存在多种染色体畸变,最常见的是6q-,但无明显特异性.瘤细胞还存在多种基因表达异常及表观遗传学异常,为将来的靶向治疗提供了潜在的治疗靶点.     

Determination of Frequency of Epstein-Barr Virus in Non-Hodgkin Lymphomas Using EBV Latent Membrane Protein 1 (EBV-LMP1) Immunohistochemical Staining

Background: The presence of Epstein-Barr virus (EBV) in Non-Hodgkin’s lymphoma can be identified byimmunohistochemistry for detection of EBV latent membrane protein (LMP). The role of EBV as an etiologic agentin the development of non-Hodgkin lymphoma has been supported by detection of high levels of latent membraneprotein 1 (LMP-1) expression in tumors. However, no study has been conducted in a Pakistani population up tillnow to determine the frequency of Epstein-Barr virus positivity. The objective of our study was to determine avalue for non-Hodgkin lymphoma patients using EBV LMP-1 immunostaining in our institution. Materials andMethods: This study was carried out at the Department of Histopathology, Armed Forces Institute of Pathology(AFIP), Pakistan from December 2011 to December 2012. It was a cross sectional study. A total of 71 patientswho were diagnosed with various subtypes of NHL after histological and EBV LMP-1 immunohistochemicalevaluation were studied. Sampling technique was non-probability purposive. Statistical analysis was achievedusing SPSS version 17.0. Mean and SD were calculated for quantitative variables like patient age. Frequenciesand percentages were calculated for qualitative variables like subgroup of NHL, results outcome of IHC forEBV and gender distribution. Results: Mean age of the patients was 53.6±16 years (Mean±SD). A total of 50(70.4%) were male and 21 (29.6%) were female. Some 9 (12.7%) out of 71 cases were positive for EBV–LMP-1immunostaining, 2 (22.2%) follicular lymphoma cases, 1 (11.1%) case of T-cell lymphoblastic lymphoma, 4 (44.4%)cases of diffuse large B cell lymphomas, 1 (11.1%) mantle cell lymphoma and 1 (11.1%) angioimmunoblastic Tcell lymphoma case. Conclusion: In our study, frequency of EBV in NHL is 12.7% and is mostly seen in diffuselarge B cell lymphoma. This requires further evaluation to find out whether this positivity is due to co-infectionor has a role in pathogenesis.     

EBV-NK Cells Interactions and Lymphoproliferative Disorders

Epstein-Barr virus (EBV) usually infects epithelial cells in the oropharynx and B lymphocytes asymptomatically. Occasionally, however, EBV infects T-cells and natural killer (NK) cells, and infection of these cells has been associated with the development of leukemias and lymphomas. EBV-positive lymphoproliferative disorders of NK cells have been reported with increasing frequency, but the interactions between EBV and NK cells are not fully understood, in part because NK cells are not usually infected with EBV in vitro. The lymphoma-derived EBV-positive NK cell line, YT, has been useful in the study of EBV infection of NK cells. YT cells express the EBV-associated nuclear antigen (EBNA)-1, the latent membrane protein (LMP)-1, and LMP-2A, but not EBNA-2 and LMP-2B genes. This pattern of latent gene expression is compatible with a type II latency program, normally associated with nasopharyngeal carcinoma, Hodgkin's disease, and T-cell lymphoma. In this report, we summarize recent information on EBV-NK cell interactions and EBV-positive lymphoproliferative disorders of NK cells.     

Interferon regulatory factor 7: a key cellular mediator of LMP-1 in EBV latency and transformation

Interferon regulatory factor 7 (IRF-7) was cloned within the biological context of Epstein-Barr virus (EBV) latency, and has an intimate relation with EBV. EBV latent membrane protein 1 (LMP-1) regulates IRF-7 both by inducing the expression of IRF-7 and by activating IRF-7 protein through phosphorylation and nuclear translocation in a post-translational manner. The activated IRF-7 then functions to regulate both EBV and cellular target genes involved in latency, transformation and immune regulation. IRF-7 appears to be a key cellular latency protein involved in both the pathogenesis and persistence of EBV infection.     

Molecular profile of Epstein-Barr virus infection in HHV-8-positive primary effusion lymphoma.

Primary effusion lymphoma (PEL) selectively involves the serous body cavities, occurs predominantly in immunodeficient patients and is infected consistently by human herpesvirus type-8. PEL is also frequently infected by Epstein-Barr virus (EBV). The precise pathogenetic role of EBV coinfection in PEL is not fully understood. The lymphoma fails to express the EBV transforming proteins EBNA-2 and LMP-1, whereas it expresses EBNA-1 (latency I phenotype). Some studies have hypothesized that other EBV-positive lymphomas expressing the latency I phenotype may associate with specific molecular variants of EBNA-1, although this issue has not been addressed in PEL. On this basis, this study is aimed at a detailed molecular characterization of EBV in PEL. Fifteen EBV positive PEL (12 AIDS-related, one post-transplant, two arising in immunocompetent hosts) were subjected to molecular characterization of the viral genes EBNA-1 and LMP-1, as well as definition of EBV type-1/type-2. The EBNA-1 gene displayed a high degree of heterogeneity in different cases of PEL, with seven distinct recognizable variants and subvariants. A wild-type LMP-1 gene was detected in 10/15 cases, whereas in 5/15 cases the LMP-1 gene harbored a deletion spanning codons 346-355. EBV type-1 occurred in 11/15 PEL whereas EBV type-2 occurred in 4/15 cases. Despite a high degree of genetic variability of the virus in different PEL cases, each single PEL harbored only one EBV variant, consistent with monoclonality of infection and suggesting that infection preceded clonal expansion. Overall, our results indicate that: (1) individual PEL cases consistently harbor a single EBV strain; (2) EBNA-1 displays a high degree of heterogeneity in different PEL cases; (3) no specific EBV genotype preferentially associates with PEL.     

EB病毒感染与淋巴瘤基因组不平衡改变的初步研究

 目的 探讨EB病毒（EBV）感染与淋巴瘤基因组不平衡改变、临床表现、病理类型、疗效和预后的关系。方法 免疫组化检测EBV潜伏膜蛋白（LMP-1）,原位杂交方法检测EBV编码的小RNA（EBERs）表达情况,比较基因组杂交（CGH）进行基因组水平不平衡改变的研究。结果 EBV感染的霍奇金淋巴瘤（HL）和胃黏膜相关淋巴瘤（MALT）型基因组无不平衡改变。外周T淋巴细胞非霍奇金淋巴瘤（NHL）,表现为基因异常的数目多,核型复杂,预后不良。大B细胞NHL、小B细胞淋巴瘤表现为基因组水平缺失与增益的数目少、化疗效果好。结论 EBV感染后的NHL宿主细胞基因组水平改变,扩增与／或缺失数目多,临床表现呈进展性、化疗效果差、预后不良。