肺癌中EB病毒的原位杂交检测

为观察EB病毒在肺癌组织中的分布并探讨EB病毒感染与肺癌的关系，对87例肺癌组织中经多聚酶链反应（PCR）检测52例EB病毒阳性的石蜡包埋组织，用原位杂交技术进行定位检测。癌组织及癌旁肺组织原位杂交EB病毒阳性率分别为37．9％（33／87）和11．5％（IO／87）；中、低分化癌与高分化癌EB病毒阳性率有显著差异，强阳性病例均为分化较差的肿瘤；累及胸膜、肋骨、隔肌的肺癌，其EB病毒阳性率明显高于未侵及者；随着EB病毒感染的加重，淋巴细胞浸润增多。结果表明：EB病毒感染与肺癌细胞的生物学特性及肺癌的发展有关。     

原发性肝细胞癌中肝干细胞标记物的检测

目的研究肝干细胞标记物在原发性肝细胞癌中的表达特征。方法45例原发性肝细胞癌手术切除标本经10％中性福尔马林固定、常规石蜡包埋,用免疫组化EliVision两步法检测AFP、GST-1T、CK19、c-kit单克隆抗体的表达。结果 c-kit、AFP、GST-π、CK19阳性率分别为13．3％（6／45）、53．3（24／45）、60．0％（27／35）、37．8％（17／45）;癌旁组织也不同程度表达肝干细胞标记;血anti-HBc阳性、癌组织AFP阳性患者癌组织CK19阳性率分别高于血anti-HBc阴性、癌组织AFP阴性患者。结论原发性肝细胞癌和癌旁组织中均有部分细胞表达肝干细胞标志,可能与肝细胞癌的发展有关。     

胃癌组织HLA－I类和DR分子免疫组化研究

本文应用免疫组化法对６４例胃癌、癌旁组织和６例胃溃疡大致正常胃粘膜冰冻和石蜡切片进行了染色．结果表明，正常胃粘膜和癌旁胃粘膜上皮细胞ＨＬＡ－Ｉ类分子表达阳性，其着色较均一，ＨＬＡ－ＤＲ染色均阴性．胃癌细胞Ｉ类分子表达缺失（２７／６４例），与癌旁上皮比较差异显著（Ｐ＜０．０１）。粘液细胞癌和低分化癌Ｉ类分子缺失率显著高于高分化癌（Ｐ＜０．０２５）．此外，发生肿瘤转移的病例Ｉ类分子缺失率（１２／１５例）显著高于无转移组（１／５例，Ｐ＜０．０２５）．ＤＲ分子在癌组织表达阳性，其阳性率高达５３．１％（３４／６４例）．低分化癌ＤＲ分子阳性率亦显著高于高分化癌和中分化癌，未分化癌ＤＲ分子阳性率亦显著高于高分化癌（Ｐ＜０．０１～０．０５）．提示（１）ＨＬＡ－Ｉ类分子表达缺失可能与癌细胞逃避宿主免疫监视发生润浸生长和转移有关；（２）分化程度不同的癌组织ＨＬＡ－Ｉ类分子表达差异显著，提示癌细胞分化可能影响Ｉ、Ⅱ类分子表达和肿癌抗原呈递；（３）ＨＬＡ－Ｉ类和ＤＲ分子表达异常可能是上皮恶性转变的标志之一．     

结肠粘膜,腺瘤和腺癌组织中Ki—ras癌基因突变的检测

对４１例石蜡包埋，同时含在可分割的正常粘膜，癌旁腺瘤和腺的手术切除结肠标本；１０例石蜡包埋，内窥镜摘除的腺瘤标本，分别用限制性片段长度多态性，单链构象多态性和Ｓ１核酸酶保护技术对Ｋｉ－ｒａｓ癌基因第１２和第６１密码子突变进行了检测，结果发现：正常粘膜中无Ｋｉ－ｒａｓ癌基因突变“腺癌组织中有５１％（２１／４１例），癌旁腺瘤中有２２％（９／４１例）非癌旁腺癌瘤中有１０％（１／１０例）有Ｋｉ－ｒａｓ癌基     

PTEN/MMAC1蛋白在42例原发性肺癌中的表达研究

目的 研究抑癌基因PTEN／MMAC1在原发性肺癌中的表达状况及其临床意义。方法 应用SP免疫组织化学方法检测42例手术切除肺癌标本PTEN的表达。结果 （1）肺癌组PTEN蛋白表达总缺失率为40．48％，小细胞癌表达缺失率75．00％，鳞癌31．58％，腺癌33．33％。肺癌细胞的分化程度不同，其PTEN表达缺失存在明显差别，高分化和低分化肺癌PTEN表达缺失率分别为13．33％，72．73％（P＜0．01）；鳞癌高分化组缺失率为12．50％，低分化组缺失率为66．67％（P＜0．05）；腺癌高分化组缺失率为14．29％，低分化组缺失率为80．00％（P＜0．05）；本组有淋巴结转移组与无淋巴结转移组缺失率分别为66．67％，25．93％，两者之间有显著差异（P＜0．01），不同的临床分期间PTEN缺失率亦有显著的差别，Ⅲa期缺失率较高。生存时间≤12月，＜60月，＞12月，≥60月，PTEN蛋白缺失率分别为71．43％，25．00％，14．29％，三者之间差异显著（P＜0．05）。结论 肺癌PTEN／MMAC1在蛋白质水平的表达缺失可能有助于其预后的判断。     

食管癌与单纯性疱疹病毒的关系

目的：观察单纯性疱疹病毒（ＨＳＶ）在食管癌组织中的分布并探讨其与食管癌的关系。方法：对５２ 例食管癌石蜡包埋组织用免疫组化和原位杂交技术进行定位观察。结果：癌组织ＨＳＶ 免疫组化阳性率ＨＳＶ１ 为５３－８ ％ ,ＨＳＶ２ 为５７－７％ ;癌旁食管粘膜ＨＳＶ 为６５－５ ％ ,其中ＨＳＶ１（１２／２９）４１％ ,ＨＳＶ２（１６／２９）５５－２％ 。原位杂交癌阳性率为５３－８ ％ ,癌旁粘膜为５８－６％ 。ＨＳＶ 阳性率与癌组织的分化程度和淋巴结转移相关（ Ｐ＜ ０－０５） ,但与癌组织间质淋巴细胞浸润的强度无关（ Ｐ＞０－０５）。结论：ＨＳＶ感染可能影响食管癌的分化和癌细胞的生物学特性。     

肺癌患者EBV感染、LMP1和Bcl-2表达状况的研究

目的 研究肺癌患者癌组织中EB病毒（Epstein-Barr virus,EBV）感染、EBV潜伏膜蛋白1 （latent membrane protein 1,LMP1）和B淋巴细胞瘤/白血病-2基因（B cell lymphoma/Leukemia-2,Bcl-2）的表达及意义。方法 采用原位杂交法（in situ hybridization,ISH）检测肺癌组织标本中EBV编码的小RNA（ EBER1）,免疫组化法（immunohistochemistry,IHC）检测Bcl-2和LMP1的表达,以GD-6多媒体彩色病理图像分析系统进行形态学定量,以图像的平均面积（average area,AA）和积分吸光度（Integral optical density,IA）表示表达量的多少。结果 在108例肺癌癌组织中36例EBV阳性,阳性率为33.3％;LMP1阳性表达7例,阳性率6.5％。EBV阳性肺癌组中Bcl-2表达显著高于EBV阴性组,其AA分别为58014.23±6918.45和38156.22±4096.79,其IA分别为11.00±1.48和8.03±0.78,差异有统计学意义。进一步分析LMPI和Bcl-2表达的关系,可见LMP1可使Bcl-2表达率增加,但定性分析和定量分析差异无统计学意义。结论 EBV感染使Bcl-2表达增加,可能在肺癌的发生发展中有一定作用。在肺癌组织中EBV可能不是通过LMP1来影响Bcl-2的表达,具体机制有待进一步研究。     

凝血酶受体-1在肺癌组织中的表达及其与转移的关系

目的研究凝血酶受体（PAR）-1在肺癌组织中的表达及其与肺癌侵袭、转移的关系。方法免疫组织化学sP法、形态学计量及逆转录-聚合酶链反应（RT-PCR）检测肺癌原发灶和转移灶组织（36例液氮冻存肺癌组织、80例石蜡包埋肺癌组织）中PAR-1的表达。结果具有侵袭和转移部位的癌巢、脉管内癌栓、癌周围的肺泡上皮不典型增生灶及支气管腺导管上皮不典型增生灶均呈现较强的阳性反应。肺癌组织PAR-1蛋白表达阳性率为73．8％（59／80例）;转移组85．7％（48／56）与非转移组45,8％（11／24）之间差异有统计学意义（P〈0．05）。转移和非转移（P〈0．05）、原发灶和转移灶（P〈0．05）、肿瘤组织和肺组织（P〈0．01）各组之间PAR．1蛋白含量差异有统计学意义;而肿瘤大小、组织学类型和组织学分化各组间差异无统计学意义（P〉0．05）。肺癌组织PAR．1mRNA表达阳性率63．9％（23／36例）;转移组78．3％（18／23例）与非转移组38．5％（5／13）之间差异有统计学意义（P〈0．05）。结论PAR-1过度表达与肺癌的转移表型、组织发生及恶性表型有关;PAR-1可能是肺癌转移过程中发挥重要作用的因素之一。     

人肝癌及肝硬变中IGF—2及HBxAg表达的对比研究

对６０例肝细胞癌、癌旁肝组织和４７例肝硬变石蜡切片进行ＩＧＦ－２和ＨＢｘＡｇ免疫组化染色。结果表明，在ＨＢｘＡｇ阳性的肝癌和肝硬变中ＩＧＦ－２阳性率分别为１００％（３２／３２例）和９４．６％（３５／３７例），而ＨＢｘＡｇ阴性组分别为８２．１％（２３／２８例）和６０％（６／１０例）。ＩＧＦ－２在肝癌中的阳性强度明显高于癌旁肝和肝硬变。ＩＧＦ－２的分布形态有：（１）胞浆包涵体样、（２）全胞浆、（３）核     

唾腺淋巴上皮样癌与EBV的相关性

目的 探讨唾腺淋巴上皮样癌与EB病毒的关系及病理诊断价值。方法 采用PCR法检测唾腺淋巴上皮样癌组织中EB病毒，免疫组织化学检测EB病毒潜伏膜蛋白1(latent membrane protein-1)的蛋白表达。结果 PCR检测EBV／IR3区域DNA阳性率为81．8％(9／11)，免疫组化检测EB病毒LMP-1阳性表达率54．5％(6／11)；EB病毒和LMP-1蛋白表达同时检出率为45％(5／11)。结论 唾腺淋巴上皮样癌的发生、发展与EB病毒感染密切相关，PCR检测石蜡包埋组织中的EB病毒具有操作简单、灵敏度高等优点。     

Incidence of latent infection of Epstein–Barr virus in lung cancers—an analysis of EBER1 expression in lung cancers by in situ hybridization

To evaluate the presence of Epstein–Barr virus (EBV) in lung cancers of Japanese patients, 81 lung cancers were examined using a highly sensitive in situ hybridization (ISH) method, employing an antisense oligonucleotide probe for EBV-encoded small nuclear RNA-1 (EBER). EBER1 expression was demonstrated in one poorly differentiated squamous cell carcinoma associated with marked lymphoid stroma (PDSCC-LS), two well differentiated adenocarcinomas, and two moderately differentiated squamous cell carcinomas, but was not detectable in other lung cancers, including small cell carcinomas. Unlike lymphoepithelioma-like undifferentiated carcinoma (LELC) of the lung, the PDSCC-LS consisted of poorly differentiated cells with distinct cell borders and nuclei with a coarse chromatin pattern and some prominent nucleoli. Most of the cancer cells expressed intense EBER1 signals. Although small to moderate numbers of cells positive for EBER1 were present in two adenocarcinomas and two squamous cell carcinomas, EBER1 signals varied in intensity and number in these four cases. Although polymerase chain reaction (PCR) and Southern blot hybridization with a ³²P-labelled probe internal to the primers were conducted to detect the EBV genome in 24 lung cancers, including five EBER1-positive cases, the genome was found to be positive in the five cases with EBER1-positive staining, including the PDSCC-LS, two adenocarcinomas and two squamous cell carcinomas, but not in the other cases. This study indicates that the morphological features of EBV-associated lung cancers are not restricted to the typical LELC type.     

Association of Epstein-Barr virus with lymphoepithelioma-like carcinoma of the lung in southern China

Having reviewed the data on 3,663 consecutive cases of primary lung carcinoma in southern China, we found that 32 cases could meet the criteria for lymphoepithelioma-like carcinoma (LELC) of the lung. To study the relationship between pulmonary LELC and Epstein-Barr virus (EBV) infection, we used in situ hybridization and immunohistochemistry techniques to detect the EBV-encoded small nonpolyadenylated RNA (EBER), latent membrane protein 1 (LMP1), and viral capsid antigen (VCA) in 32 cases of LELC and 19 cases of non-LELC lung carcinoma. Of the 32 cases, 30 (94%) showed EBER positivity. Of the 30 EBER-positive pulmonary LELC cases, 16 and 7 expressed LMP1 and VCA, respectively. In contrast with LELC, none of the 19 cases of non-LELC lung carcinoma showed EBER-, LMP1-, or VCA-positive signals in carcinoma cells. The results demonstrate that there is a close relationship between EBV infection and pulmonary LELC. EBV infection may have an essential role in the tumorigenesis of pulmonary LELC. EBV latent infection is the main status in pulmonary LELC except for individual EBV entering into a lytic cycle.     

Detection of latent Epstein-Barr virus (EBV) DNA in paraffin sections of nasopharyngeal carcinomas expressing no EBV-encoded small RNAs using in situ PCR

Summary.  In situ hybridization (ISH) with EBER 1 (Epstein-Barrvirus (EBV)-encoded small RNA1) probes is widely used for in situ detection of EBV-infected cells. ISH with an EBER1 probe showed that 10 of 40 NPC cases were negative for EBER1 expression. For in situ detection of EBV DNA, we used in situ PCR method which can detect one copy of EBV DNA per cell. Of the 10 EBER1-negative cases, three cases including one each of well- and poorly differentiated carcinomas and undifferentiated carcinoma were EBV DNA-positive by in situ PCR. The remaining seven were truly negative for the presence of EBV DNA. All the EBV genome-negative NPC cases examined here were histologically classified as poorly differentiated or undifferentiated carcinomas which are known to be closely associated with EBV, indicating the existence of EBV DNA-negative NPC cases, regardless of histological type or differentiation. These results indicate that there are EBV genome-positive NPC cases expressing no EBER1 and that in situ PCR can be suitable for in situ detection of EBV-infected cells, especially those expressing no EBER1 in paraffin sections. Received April 14, 1997 Accepted June 5, 1997     

EB病毒潜伏感染在不同部位T细胞淋巴瘤中的表达

目的：探讨不同部位Ｔ细胞淋巴瘤（ＴＭＬ）与ＥＢ病毒（ＥＢＶ）感染的关系。方法：对１００例不同部位的ＴＭＬ（结内４０例、结外６０例），采用原位杂交法检测肿瘤细胞ＥＢＶ编码的ＲＮＡ（ＥＢＥＲ１／２）。结果：（１）１００例中ＥＢＥＲ１／２检出率４８％（４８／１００），结内ＴＭＬ检出率３０％（１２／４０），结外ＴＭＬ检出率６０％（３６／６０），结内、结外ＥＢＶ检出率差异有非常显著意义（Ｐ＜０．０１）。（２）结外呼吸道ＴＭＬＥＢＥＲ１／２检出率鼻腔、鼻咽、口咽和肺分别是８８．９％（１６／１８）、７１．４％（５／７）、４７．６％（１０／２１）、３３．３％（１／３）。（３）胃肠道、软组织ＴＭＬＥＢＥＲ１／２检出率分别是１６．７％（１／６）、３３．３％（１／３）。结论：ＥＢＶ感染在不同部位ＴＭＬ中的表达具有明显部位限制性，特别是鼻腔、鼻咽的ＴＭＬ与ＥＢＶ关系最密切，其ＥＢＥＲ１／２检出率明显高于身体其它部位Ｔ细胞淋巴瘤     

Detection of EBV in nasopharyngeal carcinoma by quantum dot fluorescent in situ hybridization

Aims

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia and is frequently associated with Epstein–Barr virus (EBV) infection. The primary aim of this study was to improve the method of EBV detection by exploring quantum dots in FISH detection, and compare QD-based FISH with conventional ISH.

Materials and methods

Biopsy specimens were retrospectively retrieved from 35 NPC patients as paraffin-embedded tissue blocks. QD-FISH was developed to detect the presence of EBV encoded small RNA (EBER) using biotin-labeled EBER oligonucleotide probe indirectly labeled with streptavidin-conjugated quantum dots. Conventional ISH was also performed using a commercial kit to assess concordance between the two methods.

Results

All the 35 NPC cases were nonkeratinizing carcinoma (7 differentiated and 28 undifferentiated subtypes). EBER-positive signals were detected in 91.43% (32/35) and 80% (28/35) cases by QD-FISH and ISH, respectively. There was no significant difference in the number of EBER-positive cases by the two methods. A moderate concordance was found between QD-FISH and ISH for EBER status (κ = 0.55). Four EBER-negative cases by ISH showed EBER-positive signals when detected by QD-FISH.

Conclusions

EBV is closely associated with NPC in Chinese patients. QD-FISH is a novel effective method for EBER detection, and has a moderate concordance with conventional ISH.     

Epstein-Barr virus (EBV) infection and p53 protein expression in gastric carcinoma

In the presented studies p53 protein expression was evaluated in samples of gastric carcinoma originating from 32 selected adult patients (with documented diagnosis of adenocarcinoma of the stomach and without the presence of Helicobacter pylori infection). Among the patients 14 individuals carried EBV-positive gastric carcinoma (group 1) while the 18 remaining patients carried EBV-negative gastric carcinoma (group 2). EBV infection was detected testing the tissue material for the presence of EBER by RNA in situ hybridization (ISH) and testing sera of the patients for EBV-specific antibodies. Expression of p53 protein was analysed using immunohistochemistry. Presence of p53 protein was noted in 9 (64.3%) cases of EBV-positive gastric cancer (group 1) and in 10 (55.5%) cases of EBV-negative gastric cancer (group 2). No significant differences were detected in the frequencies of p53 protein expression in the two studied groups. The results permit to conclude that abnormalities in p53 in gastric cancer are independent of EBV infection, even if EBV may participate in development of the tumour.