【摘要】目的：探讨中国南方鼻咽癌高发区鼻咽鳞状细胞癌或称角化性鳞状细胞癌(Keratinizing squamous cell carcinoma,KSCC)是否也如非角化性癌那样与EB病毒感染有密切的关系。方法：应用PCRSouthernblotting、原位分子杂交和免疫组化法检测38例鼻咽鳞状细胞癌组织中EB病毒DNA及其产物EBNA－1,EBNA－2,EBERs,LMP－1,ZEBRA,EA－D,VCA和MA的表达。结果：（1）37例鳞状细胞癌组织中有EBERs阳性癌细胞出现，阳性率达97.4％(37/38)；（2）在37例鳞状细胞癌中的大部分未分化和低分化癌细胞表达EBNA－1和EBERs，21例（56.8％,21/37）组织内有LMP－1表达；（3）其中23例（62.2％,23/37）组织中只有少数癌细胞表达ZEBRA或/和EA－D；（4）22例(59.5％,22/37)组织中分化较好的或角化性癌细胞表达VCA或/和MA。结论：（1）中国南方地区鼻咽鳞状细胞癌与非角化性癌一样与EB病毒的感染有着较恒定而非偶然的关系；（2）鳞状细胞癌组织中大多数未分化和低分化癌细胞均以II型潜伏状态感染EB病毒，主要表达EBNA－1、EBERs和LMP－1,其中一小部分癌细胞也表达溶解期流产型蛋白ZEBRA和EA－D；（3）EB病毒在大部分分化好的和角化性癌细胞中则能够完成其复制周期并表达溶解晚期蛋白VCA或/和MA。

中图号：R739.63；R730.2；R730.231.3

Association of Epstein-Barr Virus With Nasopharyngeal

Squamous Cell Carcinoma in the South of China^＊

Zong Yongsheng ¹ Zhang Jinxia ¹ Chan KwokHung ²

Yung Chunwai ²Middeldorp, J.M³ and Ng Munhon ²

1. Department of Pathology, Sun Yat sen University of Medical Sciences, Guangzhou, China

2. Department of Microbiology, University of Hong Kong, Hong Kong, China

3. Department of Pathology, Free University Hospital, The Netherlands.

【Abstract】 Objective: To investigate whether the nasopharyngeal squamous cell carcinoma or keratinizing squamous cell carcinoma (KSCC) is also closely associated with Epstein－Barr virus infection as nasopharyngeal non－keratinizing carcinoma does.Methods: The Epstein－Barr virus (EBV) DNA and its gene expression products, including EBNA－1, EBNA－2, EBERs, LMP－1, ZEBRA, EA－D, VCA and MA were detected in 38 nasopharyngeal KSCCs using PCR Southern blotting hybridization, in situ hybridization and immunohistochemistry.Results:(1) The EBERs positive cancer cells could be found in 37 KSCCs (97.4％ , 37/38). (2) Most undifferentiated and poorly differentiated cancer cells of these 37 KSCCs expressed EBNA－1 and EBERs; and LMP－1 positive cells could be found in 21 specimens (56.8％ , 21/37). (3) A few of ZEBRA positive or/and EA－ D positive cancer cells were found in 23 out of 37 KSCCs (62.2％ , 23/37). (4) Several of more differentiated or keratinizing cancer cells expressed VCA or/and MA in 22 of 37 KSCCs (59.5％ , 22/37).Conclusions:(1) KSCCs of the nasopharynx in the south of China are also consistently, not occasionally, associated with EBV infection as non－keratinizing carcinomas (NKCs) do; (2) The majority of undifferentiated and poorly differentiated cancer cells in KSCCs are always infected with EBV in type II latency, mainly expressing EBNA－1, EBERs, and LMP－1, and a small number of them may express the abortive immediate early and early viral proteins (ZEBRA ＆ EA－D)； (3) The more differentiated or keratinizing cancer cells infected with EBV in KSCCs might complete its production cycle and express late viral proteins (VCA or/and MA).

Key words: nasopharyngeal neoplasm；Epstein－Barr virus；Squamous cell carcinoma；Lytic infection.

According to the WHO's classification^[1] the nasopharyngeal carcinoma (NPC) has been divided into two major types, namely squamous cell carcinoma or keratinizing squamous cell carcinoma (KSCC) and non－keratinizing carcinoma (NKC). It is well known that the NKC, including the differentiated non－ keratinizing carcinoma and undifferentiated carcinoma of nasopharyngeal type, was closely related to EB virus infection^{[2, 3]}, while the association of EBV with KSCC remains inconclusive. Raab－Traub N, et al. said that KSCC contained EBV DNA^[4], and on the other hand Niedobitek G, et al. got a negative result^[5，6]. Zong et al^.[7] had demonstrated EBV DNA in 3 KSCCs using in situ hybridization technique on cryostat sections. This paper reports the results of detecting EBV DNA, EBV encoded small RNAs (EBERs), EBNA－1, EBNA2, LMP－1, ZEBRA, EA－D, VCA and MA in 38 formalin－fixed paraffin－embedded KSCC specimens by use of PCR Southern blotting hybridization, in situ hybridization and immunohistochemistry and is intended to clarify whether the KSCCs also harbour EB virus as NKCs do and illustrate the possible relationship between tumour cell differentiation and EB virus expression within nasopharyngeal squamous cell carcinoma.

MATERIAL AND METHODS

1. Specimens and records

Thirty eight formalin－fixed paraffin－embedded nasopharyngeal biopsy samples and clinical records of patients with nasopharyngeal squamous cell carcinoma were collected from the Departments of Pathology, Sun Yat－ sen University of Medical Sciences, Guangzhou, during the period of January, 1986 and December, 1995. All the biopsies were taken from the untreated patients. The microscopic features on H＆ E slides coincided with the WHO's standards for diagnosing them as KSCC. Among these 38 cases, 8 could be recognized as well-differentiated, 7 moderately differentiated, and 23 poorly differentiated. The patients' clinical records were reviewed.

2. PCR Southern blotting hybridization

1) DNA preparation: 1 ml of xylene was added to each tube containing about 60 μ m of paraffin sections and mixed at room temperature for about 30 minutes. The tissue and any residual paraffin was pelleted by centrifugation in a microfuge at 13,000 rpm for 5 minu－tes. The xylene from each sample was removed with clean and plugged Pasteur pipette. This process was repeated once. 1 ml of 100％ ethanol was added to each tube and mixed with the tissue for 30 minutes. The mixture was centrifuged in microfuge at 13,000 rpm for 5 minutes and the ethanol was removed by means of clean Pasteur pipette. The ethanol wash was repeated once. After the last wash, the samples were dried under vacuum with the tubes covered by perforated parafilm across the top of the tube until the ethanol had been evaporated completely. 100 μ l of digestion buffer (50 mM Tris pH 8.0, 1 mM EDTA, 0.5％ Tween 20) containing 200 μ g/ml of proteinase K to the extracted, dried samples. The mixture was incubated for 3 hours at 55 ℃ and then boiling 7 minutes for inactivating the protease.

2) PCR: PCR was carried out in 17 mM (NH4)2 SO4, 67 mM Tris (pH 8.8), 1 mM β－ mercaptoethanol, 6.7 μ M EDTA, 17 μ g BSA/ml, 6 mM MgCl2 and 1.25 unit of Amplitag (Perkin Elmer), 50 pmol of primers and 1 μ g of DNA in a reaction volume of 100 μ l. The primers used were derived from the major internal repeat of B95－8, BamHI W positions 1398－1418, 5' GCCAGAGGTAAGTGGACTTT－3' and 1639－1619, 5'－TGGAGAGGTCAGGTTACTTA－3'. The cycles used were as follows: 94 ℃ 30 seconds, 55 ℃ 1 minute, 72 ℃ 1 minute (30 cycles). This was followed by an extension at 72 ℃ for 10－minute using 7C－1 tempcycler (Perkin Elmer). B95－8 DNA was used as positive control and DNA from the EBV－negative cell lines BJAB, Ramos, and Molt 4 was used as negative control. The size of positive product was 240bp.

3) Southern blotting: Southern blots were performed according to the method modified from Southern (1975). Gel containing DN