中线T细胞淋巴瘤与EB病毒关系的研究

为了解ＥＢ病毒在中线Ｔ细胞淋巴瘤中的感染情况，作者对遵义地区４６例中线Ｔ细胞淋巴瘤进行ＥＢＶ检测，用聚合酶链反应检测ＥＢＶ特征性的ＤＮＡ序列（ＥＢＶＤＮＡ），用ＲＮＡ原位杂交法检测ＥＢＶ编码的ＲＮＡ（ＥＢＥＲ１／２）。结果：ＥＢＶＤＮＡ阳性率为７６．１％（３５／４６）；ＥＢＥＲ１／２阳性率为６７．４％（３１／４６）。鼻腔、鼻咽、口咽Ｔ淋巴瘤ＥＢＥＲ１／２阳性率分别为８８．９％（１６／１８）、７１．４％（５／７）、４７．６％（１０／２１）。在多形细胞性淋巴瘤中，中多形和大多形细胞性ＥＢＥＲ１／２阳性率为８０．０％（１６／２０），小多形细胞性为７３．３％（１１／１５）。结果表明ＥＢＶ感染与中线Ｔ淋巴瘤关系密切，它在该肿瘤的发病中可能起重要作用，中线Ｔ淋巴瘤的ＥＢＶ感染率与解剖学部位有关，多形细胞性淋巴瘤ＥＢＶ感染率较高，特别是中多形和大多形细胞性     

A Strategy for Precision of Genotyping of Epstein-Barr Virus by Polymerase Chain Reaction: Application for Studying Hodgkin's Lymphoma

Previous studies on the genotyping of Epstein-Barr virus (EBV) have been based on the analysis of a single gene locus. The assignment of genotype of an isolate could easily be overlooked with this assay. Our strategy for precision of EBV genotyping has exploited the existence of two families of EBV strains (type A and B) that can be distinguished at three divergent gene loci (EBNA-2, EBNA-3C, and EBER). To precisely determine the genotype of EBV in Hodgkin's disease (HD), we designed primers and simultaneously analysed these three gene loci that distinguish type A and B viruses by the polymerase chain reaction (PCR) technique. The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of PCR-amplified products or the mobility shifts in single-strand conformation polymorphism (SSCP) analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. Fifteen EBV-infected cell lines were analysed and a good correlation between EBNA-2 and EBNA-3C typing results was found. In contrast, approximately 33% of the cell lines analysed maintained type A sequences in EBNA-2 and EBNA-3C genes while carrying type B sequences in the EBER region. Data obtained from analysis of cell lines served as a reference for studying HD samples. EBV DNA was detected in about 70% of HD. Among the EBV-positive samples, 56% were associated with type A virus, 13% with type B. and 31% with dual viral sequences. Thus, type A virus is predominant in HD. Based on the histology, the frequencies of EBV positivity were 83%, 71%, and 33% for mixed cellularity, nodular sclerosis and lymphocyte predominance, respectively. The detection of high frequency of both type A and B sequences in HD may provide a lead in investigating the role of dual viral infection in EBV pathogenesis.     

Detection of Epstein-Barr Virus in Oral Papilloma

Fifty-one cases of malignant and non-malignant oral diseases were investigated for Epstein-Barr virus (EBV). EBV DNA was detected by polymerase chain reaction analysis in 2 of 4 papillomas, but not in other tissues including 36 squamous cell carcinomas and 4 leukoplakias. The copy numbers of EBV DNA in the two positive samples were estimated to be 120 and 36 per cell, respectively. Intense EBV DNA signals were detected on papilloma cells by in situ hybridization. DNAs for the benign and malignant types of human papilloma virus were not detected in papilloma tissues. The present results suggest that EBV is a causative agent of oral papilloma.     

Detection of Epstein-Barr Virus DNA in Reed-Sternberg Cells of Hodgkin's Disease Using the Polymerase Chain Reaction and in situ Hybridization

Thirty-one cases of Hodgkin's disease were examined for the occurrence of Epstein-Barr virus (EBV) genome by using the polymerase chain reaction (PCR) of DNA in formalin-fixed paraffin-embedded tissues and the in situ hybridization technique. The cases were subdivided into 17 cases of nodular sclerosis (NS), nine cases of mixed cellularity (MC), four cases of lymphocyte predominance (LP), and one case of lymphocyte depletion (LD). EBV DNA was detected in eight cases including four cases of NS, three cases of MC and one case of LP. The sensitivity of PCR was higher than that of Southern blot hybridization of DNA from fresh frozen tissue, because Southern blot hybridization using the Bam HI-W fragment of EBV detected virus DNA only in two of three cases which were positive by PCR. The results of in situ hybridization studies confirmed that EBV genome was localized within the nuclei of Reed-Sternberg (RS) cells and their mononuclear variants. Furthermore, double-labeling studies combining in situ hybridization and immunocytochemistry using CD30 (BerH2) and CD15 (LeuM1) as markers of RS cells, as well as pan B-marker (L26) and pan T-marker, CD45RO (UCHL1), were performed to demonstrate the phenotype of EBV DNA-positive cells, confirming that EBV DNA was present in RS cells but not in lymphocytes. The results of this study indicate a significant association between EBV and some cases of Hodgkin's disease.     

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。     

Borna病病毒与淋巴瘤关系的初步研究

[目的]探讨Borna病病毒(BDV)感染与人类淋巴瘤的关系。[方法]采用荧光定量套式逆转录酶聚合酶链式反应(FQ-nRT-PCR),检测了经病理学诊断的非霍奇金淋巴瘤(NHL)14例、霍奇金淋巴瘤(HD)6例及健康人20例的外周血单个核细胞(PBMC)中BDV p24基因片段。[结果]20例淋巴瘤患者与20例健康对照者的BDV p24基因片段检测均为阴性。[结论]淋巴瘤与BDV感染无明显相关性。     

EB 病毒与非霍奇金淋巴瘤——附180 例报道

 目的　探讨 NHL与 EBV感染的相关性。方法　用 ISH方法检测 1 80例 NHL中EBV编码的 RNA( EBER1 /2 )。结果　 ( 1 ) EBER1 /2感染率为 42 .8% ( 77/1 80 ) ,感染率依次为TCL64.8%、NK/T46.2 %、BCL9.5 %。其中 TCL、NK/T与 BCL相比 ,P≤ 0 .0 0 5 ;( 2 )结内、结外EBV感染率依次为 2 2 .2 %、5 3.8% ,有显著差异 ,P<0 .0 0 5 ;( 3)面中线 (鼻腔 /口咽环 ) EBER1 /2感染率 72 .4% ( 5 5 /76) ,多形细胞性 EBER1 /2感染率 70 .1 % ( 5 4 /77)。结论　本地区 NHL与 EBV感染关系密切 ,TCL、NK/T淋巴瘤感染率明显高于 BCL,而且有部位限制性和亚型倾向性.     

定量和定位检测EB病毒在鼻咽癌组织中的感染状态

背景与目的EB病毒(Epstein-Barrvirus,EBV)与鼻咽癌密切相关,但EBV是鼻咽癌的致瘤因素还是仅仅是伴随关系有待进一步研究,而且EBV在鼻咽癌发生过程中的潜伏状态也不明确,本研究通过检测EBV在不同鼻咽组织中的表达变化,为进一步阐明EBV与鼻咽癌发生的关系提供线索。方法应用荧光定量PCR(real-timePCR)技术定量检测47例鼻咽低分化鳞癌患者癌、癌旁及相对正常鼻咽粘膜组织中的平均EBV拷贝数;并采用原位杂交技术(地高辛标记的EBER-1探针)对鼻咽癌、癌旁、对侧正常鼻咽粘膜中的EBV进行定位检测。以10例正常人鼻咽粘膜组织作对照。结果正常人鼻咽粘膜EBV检出率为80%(8/10),平均拷贝数为6.7×102/滋gDNA;100%(47/47)鼻咽癌和癌旁组织以及72.3%(34/47)的对侧正常鼻咽粘膜组织检出EBV,平均拷贝数分别为6.9×105/滋gDNA、1.5×105/滋gDNA、9.8×103/滋gDNA。正常人鼻咽粘膜、鼻咽癌患者对侧正常鼻咽粘膜之间感染EBV的几率、潜伏感染EBV的数量没有显著性差异(P>0.05);鼻咽癌及其癌旁、对侧正常粘膜组织三者之间,鼻咽癌与癌旁组织之间,癌旁和对侧正常粘膜组织之间EBV拷贝数存在显著性差异(P<0.01)。EBER-1原位分子杂交发现,对侧正常粘膜上皮细胞未检测到EBER-1的表达信号,癌旁靠近癌一侧的部分不典型增生细胞检测到EBER-1弱阳性信号,而在癌组织中的所有癌细胞均有EBER-1强阳性信号。结论无论是鼻咽癌患者还是健康人,EBV在鼻咽组织中的潜伏感染是一种普遍现象,但从鼻咽癌患者对侧正常鼻咽粘膜到癌旁再到癌组织EBV感染的量发生了改变,说明鼻咽上皮细胞中EBV感染量和表达信号的改变很可能是EBV致鼻咽癌的重要环节。     

微小病毒B19感染与甲状腺乳头状癌的关系

目的：探讨人类微小病毒B19感染（parvovirus B19，B19）与甲状腺乳头状癌（Papillary thyroid carcinoma,PTC）的关系。方法：对38例手术切除的PTC患者甲状腺组织（其中30例伴有周围正常甲状腺组织）及16例甲状腺腺瘤患者腺瘤旁正常甲状腺组织，分别用巢式聚合酶链反应（nested polymerase chain reaction，nPCR）、原位杂交（in situ hybridization，ISH）和免疫组织化学（immunohistochemistu，IHC）检测B19病毒DNA和病毒蛋白的表达。结果：nPCR扩增出173bp的B19特异性目的条带。ISH和IHC检测出B19病毒DNA和蛋白分别位于肿瘤细胞胞核和胞浆内在PTC中，nPCR、ISH和IHC阳性率分别为97.4％（37／38）、78.9％（30／38）和63.2％（24／38）．而腺瘤旁正常甲状腺组织中，阳性率分别为43．8％（7／16）、12．5％（2／16）和6．25％（1／16）：两者比较差异有统计学意义（3种方法均P〈0．001）：癌旁甲状腺组织ISH和IHC阳性率分别为23.3％（7／30）和10．0％（3／30），与之相对应的30例PTC（ISH：80．0％；IHC：60．0％）相比，差异有统计学意义（两者均P〈0.001）。结论：PTC患者甲状腺肿瘤组织中B19感染率明显高于腺瘤旁和癌旁正常甲状腺组织，提示B19感染在PTC的发生中可能起重要的作用。     

Frequency and genome load of Epstein-Barr virus in 509 breast cancers from different geographical areas

Since the few data exploring a possible association between Epstein-Barr virus (EBV) and breast cancer are conflicting, we investigated this association together with the influences of geographical areas. 509 breast cancers were sampled from areas with varying risks of nasopharynx carcinoma (NPC) such as North Africa (Algeria and Tunisia, high-risk area); southern France (Marseille, intermediate-risk area); and northern Europe (northern France, the Netherlands and Denmark; low-risk areas). Polymerase chain reaction (PCR) of a subregion of EBV BamHIC encoding the EBERs demonstrated that 31.8% of the tumours contained the viral genome. No significant differences were observed among the geographical areas. However, positive samples showed higher loads of the EBV genome in the NPC high- and intermediate-risk areas than in the low-risk areas. EBV type 1 was the dominant strain. In situ hybridization studies using a(35)S-labelled riboprobe for EBER1 and a laser capture microdissection, combined with quantitative PCR, showed that EBV localization was restricted to some tumour epithelial cell clusters. EBV could not be detected in the stroma. Considering the whole population covered, the presence of the EBV genome was not correlated with age, menopausal status, tumour, size, nodal status or histological grade.     

Intratumoral Production of IL-6 in B Cell Chronic Lymphocytic Leukemia and B Lymphomas

Interleukin-6 is a major B lymphocyte growth factor, and may play a role in the proliferation of malignant B lymphocytes. In order to provide arguments supporting such a role, the intratumoral production of IL-6 was studied by in situ hybridization and immunohistochemistry in 53 neoplastic tissues from B cell chronic lymphocytic leukemia or B lymphomas. IL-6-producing cells were detected in all samples but 5. However, the number of IL-6 producing cells was variable amongst the different cases. Increased density of IL-6-producing cells was highly dependent on the presence of malignant immunoblasts within the neoplastic clone. IL-6 was produced in a paracrine way, macrophages and endothelial cells being the main producers of the cytokine while malignant immunoblasts expressed the IL-6 receptor. Taken together, these results suggest that IL-6 may indeed act as a growth factor for malignant cells in some B lymphoproliferations and that this paracrine loop could be the target of new therapeutic approaches.     

Microsatellite Instability and k-ras, p53 Mutations in Thyroid Lymphoma

Patho-epidemiological studies showed that thyroid lymphoma (TL) arises in inflammatory lesions of chronic lymphocytic thyroiditis (CLTH). Replication error (RER) is found in inflammatory lesions and associated cancer, suggesting that chronic inflammation could be a risk factor for neoplastic development through causing RER. To clarify whether RER is involved in the pathogenesis of TL, we examined the microsatellite instability (MSI) in 9 cases with CLTH and 19 with TL, including 10 diffuse large B-cell lymphoma (DLBL), 4 follicle center cell lymphoma, 3 marginal zone B-cell lymphoma of extranodal (MALT) type, and 2 lymphoplasmacytic type. Sixteen distinct microsatellite repeats were analyzed. Mutations of p53 and k- ras genes were also examined. When alterations at 2 or more microsatellite loci were judged as positive, only 5 DLBL cases exhibited MSI. The frequency of MSI in DLBL was significantly higher than that in other types of TL and CLTH ( P < 0.05). Four of 19 cases (21.1%) showed point mutation of the k- ras gene. The k- ras mutations occurred in the cases with DLBL with RER, and four of five cases with RER had a k- ras mutation, indicating a close association between RER and k- ras mutation. p53 mutations were not found in the CLTH. Two of 19 TL cases showed mutations of p53 gene. There was no significant association between RER and p53 mutation. These findings indicate that genomic instability contributes to the progression of TL from low grade to high grade, but not to the development of low grade lymphoma in CLTH lesions.     

EB病毒在儿童白血病患者中的感染状况及其临床意义——附35例报告

背景与目的:Epstein-Barr病毒(EBV)与多种肿瘤的发生密切相关,为此我们探讨在儿童白血病中EBV的感染及其临床意义.方法:采用荧光定量聚合酶链反应(fluorescence quantitative-polymerase chin reaction,FQ-PCR)技术,检测35例儿童白血病[其中急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)26例(初治24例,复治2例);急性非淋巴细胞性白血病(acute nonlymphocytic leukemia,ANLL)8例;慢性淋巴细胞性白血病(chronic lymphocytic leukemia,CLL)1例]及14例正常儿童外周血单核细胞EBV-DNA的含量,并结合患儿的临床表现、泼尼松敏感试验、诱导治疗完全缓解率,分析在儿童白血病患者血中EBV的感染及其临床意义.结果:35例儿童白血病患儿中8例(22.86%)有EBV感染.其中26例ALL中7例(26.92%)EBV感染,EBV-DNA含量为(5.14±6.91)×105 copy/ml;8例ANLL中1例(12.5%)EBV感染,EBV-DNA含量为4.031×103 copy/ml;1例CLL及14例正常对照儿童未检测到EBV-DNA的含量.EBV感染的儿童白血病患儿白细胞数[(144.64±46.41)×109 /L]和肝脾肋下≥5 cm发生率87.5%均高于非EBV感染患儿[(31.04±60.27)×109 /L和7.4%,P＜0.001].感染EBV和非EBV感染的ALL患儿泼尼松疗效者分别为100%、26.32%(P=0.001);诱导治疗完全缓解率分别为28.57%、84.21%(P=0.003).ANLL患儿中,1例感染EBV者的诱导治疗完全缓解率(100%)和早期复发率(100%)较未感染EBV的7例(84.21%,28.57%)高,二者无显著性差异(P＞0.05).结论:EBV感染组儿童白血病患儿肝脾肿大明显,外周血白细胞数明显高于非EBV感染者,EBV感染ALL组对泼尼松敏感试验反应差,诱导治疗获得完全缓解率低.     

Epstein-Barr virus and the origin of Hodgkin lymphoma

Although Epstein-Barr virus (EBV) is present in the malignant Hodgkin/Reed-Sternberg (HRS) cel s of a proportion of cases of classical Hodgkin lymphoma (cHL), how the virus contributes to the pathogene...     

Epstein-Barr Virus-Associated Nodal Malignant Lymphoma in Thailand

Specific subtypes of malignant lymphoma are highly associated with Epstein-Barr virus (EBV) infection. In the ‍present study, the authors evaluated EBV-encoded RNA (EBER) expression by in situ hybridization in 300 cases of ‍malignant lymphomas diagnosed by lymph node biopsy, with 100 cases of reactive lymphoid hyperplasia in lymph ‍nodes as controls, for comparison. There were 100 consecutive cases of classical Hodgkin’s lymphoma (cHL), 100 ‍consecutive cases of non-Hodgkin’s lymphoma, B cell (NHL-B), and 100 consecutive cases of non-Hodgkin’s ‍lymphoma, T cell (NHL-T). EBER expression was detected in 46% of reactive lymphoid hyperplasia cases, but the ‍positively stained cells in those cases constituted less than 5 percent of the total cell populations. When using the ‍presence of EBER in 5 percent or more of the cell population and/or the presence of EBER in the Hodgkin’s Reed- ‍Sternberg’s cells as indicators of positivity, 64% of cHL, 13% of NHL-B, and 51% of NHL-T were found to be ‍positive. The study indicates a strong association of cHL and NHL-T with EBV infection, the link apparently being ‍weaker for NHL-B except for the subtypes of Burkitt’s lymphoma and diffuse large B cell lymphoma. ‍     

Epstein-Barr Virus-associated Post-transplant Non-Hodgkin's Lymphoma: Establishment and Characterization of a New Cell Line

A new human lymphoma cell line derived from pulmonary non-Hodgkin's lymphoma that developed in a renal transplant recipient was established from the patient's pleural effusion and designated PTLC-1. PTLC-1 grew aggressively in suspension, forming very loose clamps with a doubling time of about 18.9 h. The morphological, chromosomal, and immunophenotypic characteristics of the patient's tumor cells and PTLC-1 cells were very similar. PTLC-1 showed a monoclonal rearrangement of IgH gene and was highly tumorigenic in athymic nude mice. In situ hybridization, Southern blot hybridization and polymerase chain reaction demonstrated the presence of Epstein-Barr virus (EBV) genome in the patient's tumor and PTLC-1. PTLC-1 has been maintained in culture for over 60 months. Since EBV has been implicated in the pathogenesis of post-transplant lymphoma, this new cell line should serve as a useful experimental model for studying the etiology and biology of lymphoma developing in organ transplant recipients     

The Characteristics of Epstein-Barr Virus (EBV)-positive Diffuse Large B-Cell Lymphoma: Comparison between EBV+ and EBV- Cases in Japanese Population

We have investigated 114 cases with diffuse large B-cell lymphoma (DLBCL) to clarify the characteristics of DLBCL with Epstein-Barr virus (EBV) infection. Thirteen cases (11.4%) showed EBVencoded RNA 1 (EBER1) signals by RNA in situ hybridization. EBV-encoded latent membrane protein 1 (LMP1) and EBV-encoded nuclear antigen 2 (EBNA2) were expressed in 11 and 4 cases, respectively. Expression of CD30, Bcl-6 and immunoglobulin (Ig) was found in 92%, 31% and 23% with EBV⁺ DLBCL, and in 15%, 79% and 82% with EBV^- DLBCL, respectively. The sequence of rearranged Ig heavy chain (IgH) variable (V) region gene was analyzed in 5 cases with EBV⁺ DLBCL and 61 cases with EBV^- DLBCL. Somatic mutation was found in all cases except one with EBV^- DLBCL. Average mutation frequency was 9.6% in EBV⁺ DLBCL vs. 11.5% in EBV^- DLBCL. The rates of replacement mutation vs. silent mutation (R/S values) in complementarity determining region II and framework region III were 2.7 and 1.5 in EBV⁺ DLBCL, 2.6 and 1.4 in EBV^- DLBCL. Crippling mutation generating a stop codon was found in 2 of 5 cases (40%) with EBV⁺ DLBCL, but none of 61 cases (0%) with EBV^- DLBCL. These findings suggest that EBV⁺ DLBCL and EBV^- DLBCL were both derived from germinal center (GC) or post-GCB cells, and EBV⁺ DLBCL frequently have a non-functional IgH gene owing to crippling mutation.     

Epstein-Barr Virus Gene Expression in Lymphoproliferative Disease

Epstein-Barr virus (EBV) is associated with Burkitt lymphoma, nasopharyngeal carcinoma, and lymphoprolifcrative disease in patients with congenital or acquired immunodeficiencies. B lymphocytes in tissues from these patients often contain EBV genomes. Expression of the EBV genes EBNA-1, EBNA-2, and LMP and the B cell genes CD23, ICAM-1, and LFA-3 has been demonstrated in lymphoblastoid cells in vitro and in B lymphocytes in tissues from patients with lymphoproliferative disease. In contrast, B cells in tissue from patients with Burkitt lymphoma usually show expression of EBNA-1, but not the other viral or B cell genes. Expression of EBNA-1 with variable expression of LMP has been reported in tissue from patients with nasopharyngeal carcinoma. The different patterns of viral and cellular gene expression in these diseases have important implications for their treatment.