单侧唇裂继发鼻畸形修复新法
作者：李文志 凌诒淳 周刚 乔群 杨波
单位：李文志(原中国医学科学院整形外科医院)、杨波 100029 北京安贞医院；凌诒淳、周刚、乔群 中国医学科学院整形外科医院
关键词：单侧唇裂；鼻畸形；修复
癌症990103 【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。
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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
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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).
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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.
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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.
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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.
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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.
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3) Southern blotting: Southern blots were performed according to the method modified from Southern (1975). Gel containing DNA was denatured by soaking in a solution of 1.5 M NaCl and 0.5 M NaOH for 1 hour at room temperature with constant shaking. The gel was then neutralized in a solution containing 1 M Tris HCl (pH 8.0) and 1.5 M NaCl for another hour at room temperature with constant shaking. The gel was inverted so that its original underside was now uppermost and placed on a damp 3 MM paper. A wet Hybond- N- nylon filter (Amersham, United Kingdom) presoaked in 2× SSC buffer for 2～ 3 minutes was placed on top of the gel. Two pieces of whatmann 3-MM paper were wet in 2× SSC and then placed on top of the membrane. A stack of paper towels (5～ 8cm high) was put on the 3-MM paper. A glass plate was placed on top of the stack and was weighed down with a 500 g weight. The capillary transfer of DNA was allowed to proceed for 8～ 24 hours. The towels and the 3 MM filter were then removed above the gel. The position of the gel slots was marked on the filter after the dehydrated gel and filter was turned over. The filter was soaked in 6× SSC at room temperature for 5 min utes, dried at room temperature on a sheet of 3 MM paper and fixed under ultraviolet light transilluminator for 10 minutes. The filter was kept at 4℃ for later use for hybridization with a digoxigenin labelled oligonucleotide probe.
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4) Hybridization
(1) End-labelling of oligonucleotide probe: Oligonucleotide probe (ACAGCTCCTAAGAAGGCACC
3') were labelled with digoxigenin-11-dUTP by Terminal deoxynucleotide Transferase (TDT); the reaction mixture (20 μ l) contained 1 μ g of probe DNA, 5 nmoles digoxigenin-11-dUTP, 5 nmoles dCTP, 20 unites TDT and 10 μ l tailing buffer (5× ). The end-labelling reaction was carried out at 37 ℃ for 3 hours. The labelled probe was put on ice and precipitated by adding 50 μ g tRNA, 1/10 volume 3 M sodium acetate (pH 5.2) and 2 volumes prechilled absolute ethanol. The mixture was incubated at - 70 ℃ for 30 minutes and centrifuged at 13,000 rpm at 4℃ for 15 minutes. The pellet was washed in prechilled 70% ethanol, dried in a Speed Vac concentrator for 15 minutes, resuspended in 1 ml TE buffer (10 mM tris, pH 7.4 and 0.1 mM EDTA and stored at-20 ℃ .
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(2) Hybridization with Dig labelled oligonucleotide probe: Filter was placed in a plastic container, and prehybridized for 1 hour at 45 ℃ in 10 ml hybridization buffer of 5× SSC, 0.1% (W/V) N-lau-roylsarcosine sodium salt (Sigma, St. Louis), 0.02% (W/V) SDS and 0.5% blocking reagent (Boehringer Mannhein).After prehybridization, hybridiztion buffer containing 100 ng of freshly denatured digoxigenin- labelled probe was added and hybridizated overnight in a shaking water bath. After hybridization, the excess probe was removed by 5 stringent washes as follows: twice for 5 minutes at room temperature in 2× SSC and 0.1% (W/V) SDS, twice for 5 minutes at hybridization temperature in 0.1× SSC containing 0.1% (W/V) SDS. Filter can then be used directly for detection of hybridized DNA or stored air- dried for later detection.
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3. In situ hybridization for detecting EBV nuclear small non- polyadenylated RNAs (EBERs) on tissue section slides
1) Preparation of APES (3-aminopropyltriethoxy-silane)-coated slides:
(1) Place slides in 2% APES-acetone for 5～ 10 minutes;
(2) Briefly wash in acetone;
(3) Rinse in distilled water twice;
(4) Dry overnight at 37℃ ;
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(5) Encircle the specimen with the DAKO Pen.
2) Hybridization by use of DAKO Hybridization Detection Kit (K 0046) and FITC-conjugated EBV (E-BER) probe (Y0017). The procedure consists of the following steps:
(1) Deparaffinization and rehydration of paraffin- embedded sections using xylene and a series of graded ethanol.
(2) Pretreatment with 0.1 mg/ml proteinase K.
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(3) Hybridization with FITC-conjugated EBV (EBER) probe at 37℃ for 2 hours.
(4) Detection by use of alkaline phosphatase-con-jugated Rabbit F(ab') Anti-FITC and enzyme substrate, BCIP/NBT. The positive signals are dark blue/black color.
(5) Counterstaining with 2% methyl green solution.
4.Immunohistochemistry
Using DAKO Universal LSAB Kit (K 0681 HRP. Rabbit/Mouse 500 tests) and a variety of primary antibodies for detecting the EBV encoded proteins ( Table 1).
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Table 1 Primary antibodies used Primary antibody
Source
Pretreatment
Working dilution
EBNA-1
OT-1
MW-DW
1/300
EBNA-2
DAKO-M7004
MW-CB
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1/25
LMP-1
DAKO-M0897
MW-DW
1/50
ZEBRA
DAKO-M7005
MW-CB
1/10
EA-D(p138)
OT-13B
MW-DW
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1/400
VCA(p18)
OT-15E
MW-DW
1/150
MA(gp350/220)
OT-6
MW-DW
1/100
MW- CB: Microwave treatment using citrate buffer.
MW- DW: Microwave treatment using distilled water.
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DAKO: DAKO A/S products.
OT: Bioscience Research Unit, Organon Teknika B.V., Boxtel, The Netherlands.
RESULTS
All the patients with KSCC were Cantonese and their sex/age distribution was shown in Table 2. The mean age was 50.82 years old, and the ratio of male to female 1.92:1.
Table 2 Sex/age distribution of 38 KSCC patients
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(Y)
No. of cases
M
F
M+F
31- 35
1
2
3
36- 40
4
2
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41- 45
3
0
3
46- 50
5
2
7
51- 55
3
4
7
56- 60
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2
3
5
61- 65
3
0
3
66- 70
1
0
2
71- 75
1
0
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1
76- 80
1
0
1
Total
25
13
38
The results obtained were summarized in Table 3. Table 3 A summary of results obtained
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Dif: Differentiation of carcinoma cells.
P: Poorly differentated.
M: Moderately differentiated.
W: Well differentiaed.
W-frag: Bam HI W-fragment of EB virus genome.
The epithelial pearls could be found in well differentiated squamous cell carcinoma on H＆ E stained slides.(Fig.1). The poorly developed epithelial pearls and individual keratinizing squamous carcinoma cells could always be found in all of the samples. Both EBNA-1 and EBERs positive carcinoma cells could be found in 37 of 38 specimens by immunohistochemistry (Fig. 2 ) and in situ hybridization technique (Fig. 3), respectively; and the positive rate accounted for 97.37% . The major internal repeat of EBV W fragments could be detected in 32 out of 38 formalin-fixed paraffin-embedded biopsy samples (Fig. 4) by PCR Southern blotting hybridization technique, and the positive rate reached 84.21% . Less than 50% of weakly EBERs-positive carcinoma cells (Fig. 3) could be found in 5 KSCCs which had no positive hybridization blot illustrated by PCR Southern blotting hybridization. One moderately differentiated KSCC (case no. 30) was negative for both PCR Southern blotting hybridization and EBERs in situ hybridization. Not all of the carcinoma cells showed EBERs signals in their nuclei ; 33 out of 37 (89.19% ) EBERs positive specimens had less than 50% of carcinoma cells expressing EBERS.Among these 33 cases, 14 were well and moderately differentiated ( Table 4).
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Fig.1 Well differentiated keratinizing carcinoma of the nasopharynx showing an epithelial pearl and surrounding relatively poorly differentiated carcinoma cells. H＆ E. 5× 20
Fig.2 The poorly differentiated and undifferentiated carcinoma cells located peripherally in an epithelial pearl showing EBNA- 1 positivity. EBNA- 1 immmunostaining. 5× 10.
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Fig.3 The poorly differentiated and undifferentiated carcinoma cells located peripherally in an epithelial pearl showing EBERs positivity. EBERs in situ hybridization; 5× 10.
Fig.4 Most of KSCCs except two (No. 30, D54974 ＆ No. 25, D51849) demonstrated in this figure showing positive blots by PCR Southern Blotting hybridization.
Table 4 Relationship between EBERs+ ve cells and differentiation degree of KSCCs Differentiation
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Of KSCC
Percentage of EBERs positive cells
0%
<25%
26～ 50%
51～ 75%
>76%
Well
0
7
1
0
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0
Moderately
1
4
2
0
0
Poorly
0
14
5
3
1
Total
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1(2.6% )
25(65.8% )
8(21.1% )
3(7.9% )
1(2.6% )
The EBERs positive cells were not always uniform in intensity, being varied from case to case and even cells to cells. Morphologically, the EBERs positive carcinoma cells were undifferentiated or poorly differentiated, and on the other hand the more differentiated or keratinizing carcinoma cells were often EBERs negative. A variable number of EBERs positive cells in 21 out of 37 EBERs positive KSCCs (56.8% ) expressed LMP-1 (Fig. 5). The EBV lytic-cycle gene encoded proteins could also be found in a considerable number of cases ( Table 3), the positive rates were: ZEBRA 29.73% (11/37); EA-D 62.16% (23/37; Fig.6); VCA 48.65% (18/37; Fig.7); and MA 43.24% (16/37; Fig.8). EB viral proteins of three main phases of lytic cycle, namely, immediate early ZEBRA, early EA-D, and late VCA and MA could all be found in 4 cases (case nos. 7, 8, 20, ＆ 24). Besides these 4 cases, there were 5 KSCC specimens (case nos.1, 6, 12, 13, ＆ 26) expressing EA D, VCA and MA. There were 2 KSCCs (case nos.16, and 33) expressing EA-D and VCA. Only 1 KSCC (case no.38) expressed EA-D and MA. There were 3 KSCCs (case nos.22, 25, and 31) expressing VCA and MA. In 7 KSCC specimens ( case nos. 9, 10, 11, 14, 19, 23, ＆ 32), a few of ZEBRA or/and EA-D positive carcinoma cells could be found but no VCA or/and MA positive carcinoma cells detected. It had been confirmed by consecutive sections that ZEBRA or EA-D positive carcinoma cells were often EBERs positive and VCA or MA negative; and VCA and MA were always both positive in the same EBERs negative carcinoma cells.
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Fig.5 A group of poorly differentiated and undifferentiated carcinoma cells in a KSCC specimen showing LMP- 1 positivity.LMP- 1 immunostaining; 5× 10.
Fig.6 Some poorly differentiated and undifferentiated carcinoma cells in a KSCC specimen showing EA- D positivity. EA- D immunostaining; 5× 10.
Fig.7 A few more differentiated carcinoma cells in a KSCC specimen showing VCA positivity. VCA immunostaining; 5× 20.
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Fig.8 Some more differentiated carcinoma cells in a KSCC specimen showing MA positivity. MA immunostaining; 5× 20.
DISCUSSION
An association of EBV with nasopharyngeal non-keratinizing carcinomas (NKCs) was suggested as early as 1979 on grounds of serological studies[8] and subsequently confirmed by demonstration of EBV DNA and EBV-encoded nuclear antigen complex by use of in situ hybridization and anti-complement immunofluorescene, respectively[9 ＆ 10]. Are squamous cell carcinomas or keratinizing squamous cell carcinomas (KSCCs) of the nasopharynx also consistently associated with EB virus? So far as we know there is a discrepancy about this issue down to date. Some authors said “ yes" [4], some reported “ no"[5], and some authors said “ occasionally"[11]. The authors collected 38 KSCCs from the NPC high-incidence area, Guangzhou locale, in the south of China, during the past 10 years and detected EBV DNA and EBERs simultaneously in the formalin-fixed paraffin-embedded biopsy samples by both PCR Southern blotting hybridization and in situ hybridization. The results illustrated that the KSCCs of the nasopharynx were also closely, not occasionally, associated with EB virus as the NKCs did. Among the 38 KSCCs, EB virus could not be demonstrated only in one case (case no.30). It should be emphasized herein that the formalin-fixed paraffin-embedded block of this case had quite a bit of tissue for study, therefore, negative result obtained can not substantially prove that this specimen was actually not infected with EBV. Even if this KSCC specimen really did not contain EB virus, it is also not surprising because of no 100% of NKC samples showing EBV positivity (according to the authors' unpublished data). A positive hybridization blot could not be demonstrated in the 5 KSCCs (case nos. 20, 22, 25, 28, ＆ 32) which contained EBERs positive carcinoma cells. This phenomenon may be interpreted as follows: (1) Though PCR Southern blotting hybridization was a sensitive and specific method for detection of EBV DNA sequences in NPC cells as demonstrated by Chang YS, et al.[12] and Dickens P, et al.[13], the EBV DNA of these 5 KSCC specimens had merely a little quantity (because of a little bit of tissue and only a few EBERs positive carcinoma cells presented) and might be degraded in nature. This may be resulted in a negative reaction. Just as it is, so the results obtained by Hording U, et al.[14] and Akao I, et al.[15] can thus be interpreted. Only 2 out of 14 and 5 out of 7 KSCCs performed by Hording and Akao, respectively, got positive blot. (2) The in situ hybridization technique for detection of EBV encoded small RNAs (EBERs), which are very stable in secondary structure and have relatively long half- life (8- 9 h for EBER- 1 and less than 1 h for EBER- 2 in Daudi cells)[16], was such a kind of more sensitive method[17， 18， 19 ＆ 20] that even a few EBERs positive carcinoma cells could be shown.
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We have observed hundreds of NKC slides stained by EBERs in situ hybridization, and found that the vast majority of carcinoma cells (data not shown) showed positivity. Raab- Traub also said, “ expression of specific viral mRNAs or gene products are consistently detected within all of the tumor cells"[3]. However, 33 out of the 37 EBERs positive KSCCs had less than 50% of carcinoma cells expressing EBERs and all the 14 EBERs positive well- and moderately- differentiated KSCCs were among these 33 cases. It is popularly recognized that regardless of types all the NPCs have more or less squamous differentiation under electron microscopy[1] and the KSCC and NKC, which can be subdivided into differentiated NKC and undifferentiated carcinoma of nasopharyngeal type, may be recognized as a spectrum of squamous differentiation, having a gradually decreased amount of more differentiated or keratinizing carcinoma cells. Basing upon this understanding, we paid our attention to the correlation of EBERs expression and carcinoma cell differentiation. We found that most of the EBERs positive carcinoma cells were undifferentiated or poorly differentiated neoplastic cells, which were the malignant counterparts of basal or suprabasal cells of squamous epithelium under light microscopy; while the more differentiated or keratinizing carcinoma cells, which might be considered as the malignant counterparts of polygonal prickle cells with distinct intercellular bridges or superficially located flattened keratinizing cells of squamous epithelium, were always EBERs negative. Recently, Pathmanathan R, et al.[21] reported the same phenomenon. They said “ In foci of squamous differentiation and keratinization within less differentiated NPC and throughout the expanse of well differentiated squamous cell carcinoma, EBER expression was less abundant". Furthermore, Chen CL, et al.[22] using radioisotope-and biotin-labeled EBV DNA probes for in situ hybridization showed that “ in the keratinizing carcinoma, which was poorly differentiated in this series, the EBV genome was usually detected in anaplastic tumor cells, and not in the keratinizing areas". Zhang J.X. et al.[23] reported the relationship between EBV life cycle and tumour cell differentiation. Basing upon all these findings, we assume that the EB virus was likely to be harbouring in most undifferentiated or poorly differentiated carcinoma cells. Because almost all of the EBERs positive cells were EBNA- 1 positive and a portion of them expressing LMP- 1, this kind of EB virus infection is of latency II in type. It is worth to point out that since the ZEBRA positive carcinoma cells, though only a few in number found on 11 KSCC slides, were undifferentiated in morphology, and the EA-D positive cells found on 23 KSCCs, were either undifferentiated or poorly differentiated too, a few or a small number of the latently EBV-infected undifferentiated/poorly differentiated carcinoma cells might be switched the lytic cycle on and expressing its gene products by some unknown factors. On the other hand, the VCA or MA positive carcinoma cells, found on 22 KSCC specimens were mostly of more differentiated or keratinizing carcinoma cells. Accordingly, we assume that the EB virus harbouring in more differentiated or keratinizing NPC cells is able to express late gene proteins of lytic cycle, such as VCA or MA. In other words, the more differentiated or keratinizing cells are likely to be infected with EB virus in productive cycle.
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Our findings only demonstrate that the lytic cycle products of EB virus could be found in KSCCs of the nasopharynx and can not be interpreted as that a complete lytic cycle of EB virus may occur in every KSCC cells. In fact, most undifferentiated/poorly differentiated carcinoma cells are infected with EB virus in latency; and merely in some given microenvironments, such as in cell differentiation and maturation, the EB virus genome in episomal form might be possible to convert the latent into lytic cycle. We agree the words of Naher H. and Petzoldt D.[24] that “ differentiation and maturation of the epithelial cells is paralleled by active virus production". Even if the EB viruses are undergoing lytic gene expression, most of them possibly occur in abortive form because of the following reasons: (1) The proteins representing three main phases of lytic cycle (immediate early ZEBRA, early EA-D, and late VCA/MA) could only be concurrently found in 4 KSCCs specimens. (2) the ZEBRA+ , EA-D+ , or VCA+ /MA+ cells were not the same ones found on the slides. This findings coincide with what Martel-Renoir D, et al.[25] reported previously. However, a complete lytic cycle of EB virus usually occur in some KSCC specimens because the late gene proteins could be definitely found in some of more differentiated or keratinizing carcinoma cells. Furthermore, as popularly known, the patients with NPC, including NKC ＆ KSCC frequently had an elevated antibodies titer against these late gene proteins. Wolf H et al.[26] had said that “ only terminally differentiated epithelial cells are capable of supporting an efficient lytic cycle of EBV replication". This hypothesis can interpret what we found in the KSCC specimens studied hereof.
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* This work was supported by grants from the National Council of Natural Sciences (NCNS Nos. 39470295 and 39730200-Ⅱ ), The People's Republic of China, and the preliminary results were orally presented, not formally published, at the "Symposium of Epstein- Barr Virus and Nasopharyngeal Cancer" held in Taipei, China, June 9, 1996.
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[11]Della- Torre G, Pilotti S, Donghi R, et al. Epstein- Barr virus genomes in undifferentiated and squamous cell nasopharyngeal carcinomas in Italian patients. Diagn Mol Pathol, 1994, 3:32.
[12]Chang YS, Tyan YS, Liu ST, et al. Detection of Epstein- Barr virus DNA sequences in nasopharyngeal carcinoma cells by enzymatic DNA amplification. J Clin Microbiol, 1990, 28:2398.
[13]Dickens P, Srivastava G, Loke SL, et al. Epstein- Barr virus DNA in nasopharyngeal carcinomas from Chinese patients in Hong Kong. J Clin Pathol, 1992, 45:396.
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[14]Hording U, Nielsen HW, Albeck H, et al. Nasopharyngeal carcinoma: histopathological types and association with Epstein- Barr Virus. Eur J Cancer B Oral Oncol, 1993, 29B:137.
[15]Akao I, Sato Y, Mukai K, et al. Detection of Epstein- Barr virus DNA in formalin- fixed tissue of nasopharyngeal carcinoma using polymerase chain reaction and in situ hybridization. Laryngoscope, 1991, 101:279.
[16]Clemens MJ: Functional significance of the Epstein- Barr virus- encoded small RNAs. Epsrein- Barr Virus Report, 1994, 1;107.
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[17]Hamilton- Dutoit SJ, and Pallesen G. Detection of Epstein- Barr virus small RNAs in routine paraffin sections using non- isotopic RNA/RNA in situ hybridization. Histopathology, 1994, 25(2):101.
[18]Chen CL, Wen WM, Chen JY, et al. Detection of Epstein- Barr virus genome in nasopharyngeal carcinoma by in situ DNA hybridization. Intervirology, 1993, 36(2):91.
[19]Barletta JM, Kingma DW, Ling Y, ChaCharache P, Mann RB, and Ambinder RF. Rapid in situ hybridization for the diagnosis of latent Epstein- Barr virus infection. Mol Cell Probes, 1993, 7(2):105.
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[20]Br Brousset P, Buttet V, Chittal S. Comparison of in situ hybridization using different nonisotopic probes for detection of Epstein- Barr virus in nasopharyngeal carcinoma and immunohistochemical correlation with anti- latent membrane protein antibody. Lab Invest, 1992, 67:457.
[21]Pathmanathan R, Prasad U, Chandrika G, et al. Undifferentiated, nonkeratinizing, and squamous cell carcinoma of the nasopharynx. Variants of Epstein- Barr virus- infected neoplasia. Am J Pathol, 1995, 146; 1355.
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[22]Chen CL, Wen WH, Chen JY, et al. Detection of Epstein- Barr virus genome in nasopharyngeal carcinoma by in situ DNA hybridization. Intervirology, 1993, 36:91.
[23]Zhang, J. X., Chen, H.L., Zong, Y.S., et al. Epstein- Barr virus expression within keratinizing nasopharyngeal carcinoma. J Med Virol, 1998, 55:227.
[24]Naher H, Petzoldt D. Epstein- Barr virus infection- a lympho- and epitheliotropic infection. Hautarzt, 1992, 43:114.
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[25]Martel- Renoir D, Grunewald V, Touitou R, et al. Qualitative analysis of the expression of Epstein- Barr virus lytic genes in nasopharyngeal carcinoma biopsies. J Gen Virol, 1995, 76:1401.
[26]Wolf H, Bogedain C, Schwarzmann F. Epstein- Barr vitus and its interaction with the host. Intervirology, 1993, 35:26.
(收稿：1998-11-10修回：1998-12-08), 百拇医药
单位：李文志(原中国医学科学院整形外科医院)、杨波 100029 北京安贞医院；凌诒淳、周刚、乔群 中国医学科学院整形外科医院
关键词：单侧唇裂；鼻畸形；修复
癌症990103 【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。
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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
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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).
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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.
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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.
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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.
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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.
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3) Southern blotting: Southern blots were performed according to the method modified from Southern (1975). Gel containing DNA was denatured by soaking in a solution of 1.5 M NaCl and 0.5 M NaOH for 1 hour at room temperature with constant shaking. The gel was then neutralized in a solution containing 1 M Tris HCl (pH 8.0) and 1.5 M NaCl for another hour at room temperature with constant shaking. The gel was inverted so that its original underside was now uppermost and placed on a damp 3 MM paper. A wet Hybond- N- nylon filter (Amersham, United Kingdom) presoaked in 2× SSC buffer for 2～ 3 minutes was placed on top of the gel. Two pieces of whatmann 3-MM paper were wet in 2× SSC and then placed on top of the membrane. A stack of paper towels (5～ 8cm high) was put on the 3-MM paper. A glass plate was placed on top of the stack and was weighed down with a 500 g weight. The capillary transfer of DNA was allowed to proceed for 8～ 24 hours. The towels and the 3 MM filter were then removed above the gel. The position of the gel slots was marked on the filter after the dehydrated gel and filter was turned over. The filter was soaked in 6× SSC at room temperature for 5 min utes, dried at room temperature on a sheet of 3 MM paper and fixed under ultraviolet light transilluminator for 10 minutes. The filter was kept at 4℃ for later use for hybridization with a digoxigenin labelled oligonucleotide probe.
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4) Hybridization
(1) End-labelling of oligonucleotide probe: Oligonucleotide probe (ACAGCTCCTAAGAAGGCACC
3') were labelled with digoxigenin-11-dUTP by Terminal deoxynucleotide Transferase (TDT); the reaction mixture (20 μ l) contained 1 μ g of probe DNA, 5 nmoles digoxigenin-11-dUTP, 5 nmoles dCTP, 20 unites TDT and 10 μ l tailing buffer (5× ). The end-labelling reaction was carried out at 37 ℃ for 3 hours. The labelled probe was put on ice and precipitated by adding 50 μ g tRNA, 1/10 volume 3 M sodium acetate (pH 5.2) and 2 volumes prechilled absolute ethanol. The mixture was incubated at - 70 ℃ for 30 minutes and centrifuged at 13,000 rpm at 4℃ for 15 minutes. The pellet was washed in prechilled 70% ethanol, dried in a Speed Vac concentrator for 15 minutes, resuspended in 1 ml TE buffer (10 mM tris, pH 7.4 and 0.1 mM EDTA and stored at-20 ℃ .
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(2) Hybridization with Dig labelled oligonucleotide probe: Filter was placed in a plastic container, and prehybridized for 1 hour at 45 ℃ in 10 ml hybridization buffer of 5× SSC, 0.1% (W/V) N-lau-roylsarcosine sodium salt (Sigma, St. Louis), 0.02% (W/V) SDS and 0.5% blocking reagent (Boehringer Mannhein).After prehybridization, hybridiztion buffer containing 100 ng of freshly denatured digoxigenin- labelled probe was added and hybridizated overnight in a shaking water bath. After hybridization, the excess probe was removed by 5 stringent washes as follows: twice for 5 minutes at room temperature in 2× SSC and 0.1% (W/V) SDS, twice for 5 minutes at hybridization temperature in 0.1× SSC containing 0.1% (W/V) SDS. Filter can then be used directly for detection of hybridized DNA or stored air- dried for later detection.
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3. In situ hybridization for detecting EBV nuclear small non- polyadenylated RNAs (EBERs) on tissue section slides
1) Preparation of APES (3-aminopropyltriethoxy-silane)-coated slides:
(1) Place slides in 2% APES-acetone for 5～ 10 minutes;
(2) Briefly wash in acetone;
(3) Rinse in distilled water twice;
(4) Dry overnight at 37℃ ;
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(5) Encircle the specimen with the DAKO Pen.
2) Hybridization by use of DAKO Hybridization Detection Kit (K 0046) and FITC-conjugated EBV (E-BER) probe (Y0017). The procedure consists of the following steps:
(1) Deparaffinization and rehydration of paraffin- embedded sections using xylene and a series of graded ethanol.
(2) Pretreatment with 0.1 mg/ml proteinase K.
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(3) Hybridization with FITC-conjugated EBV (EBER) probe at 37℃ for 2 hours.
(4) Detection by use of alkaline phosphatase-con-jugated Rabbit F(ab') Anti-FITC and enzyme substrate, BCIP/NBT. The positive signals are dark blue/black color.
(5) Counterstaining with 2% methyl green solution.
4.Immunohistochemistry
Using DAKO Universal LSAB Kit (K 0681 HRP. Rabbit/Mouse 500 tests) and a variety of primary antibodies for detecting the EBV encoded proteins ( Table 1).
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Table 1 Primary antibodies used Primary antibody
Source
Pretreatment
Working dilution
EBNA-1
OT-1
MW-DW
1/300
EBNA-2
DAKO-M7004
MW-CB
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1/25
LMP-1
DAKO-M0897
MW-DW
1/50
ZEBRA
DAKO-M7005
MW-CB
1/10
EA-D(p138)
OT-13B
MW-DW
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1/400
VCA(p18)
OT-15E
MW-DW
1/150
MA(gp350/220)
OT-6
MW-DW
1/100
MW- CB: Microwave treatment using citrate buffer.
MW- DW: Microwave treatment using distilled water.
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DAKO: DAKO A/S products.
OT: Bioscience Research Unit, Organon Teknika B.V., Boxtel, The Netherlands.
RESULTS
All the patients with KSCC were Cantonese and their sex/age distribution was shown in Table 2. The mean age was 50.82 years old, and the ratio of male to female 1.92:1.
Table 2 Sex/age distribution of 38 KSCC patients
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(Y)
No. of cases
M
F
M+F
31- 35
1
2
3
36- 40
4
2
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41- 45
3
0
3
46- 50
5
2
7
51- 55
3
4
7
56- 60
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2
3
5
61- 65
3
0
3
66- 70
1
0
2
71- 75
1
0
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1
76- 80
1
0
1
Total
25
13
38
The results obtained were summarized in Table 3. Table 3 A summary of results obtained
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Dif: Differentiation of carcinoma cells.
P: Poorly differentated.
M: Moderately differentiated.
W: Well differentiaed.
W-frag: Bam HI W-fragment of EB virus genome.
The epithelial pearls could be found in well differentiated squamous cell carcinoma on H＆ E stained slides.(Fig.1). The poorly developed epithelial pearls and individual keratinizing squamous carcinoma cells could always be found in all of the samples. Both EBNA-1 and EBERs positive carcinoma cells could be found in 37 of 38 specimens by immunohistochemistry (Fig. 2 ) and in situ hybridization technique (Fig. 3), respectively; and the positive rate accounted for 97.37% . The major internal repeat of EBV W fragments could be detected in 32 out of 38 formalin-fixed paraffin-embedded biopsy samples (Fig. 4) by PCR Southern blotting hybridization technique, and the positive rate reached 84.21% . Less than 50% of weakly EBERs-positive carcinoma cells (Fig. 3) could be found in 5 KSCCs which had no positive hybridization blot illustrated by PCR Southern blotting hybridization. One moderately differentiated KSCC (case no. 30) was negative for both PCR Southern blotting hybridization and EBERs in situ hybridization. Not all of the carcinoma cells showed EBERs signals in their nuclei ; 33 out of 37 (89.19% ) EBERs positive specimens had less than 50% of carcinoma cells expressing EBERS.Among these 33 cases, 14 were well and moderately differentiated ( Table 4).
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Fig.1 Well differentiated keratinizing carcinoma of the nasopharynx showing an epithelial pearl and surrounding relatively poorly differentiated carcinoma cells. H＆ E. 5× 20
Fig.2 The poorly differentiated and undifferentiated carcinoma cells located peripherally in an epithelial pearl showing EBNA- 1 positivity. EBNA- 1 immmunostaining. 5× 10.
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Fig.3 The poorly differentiated and undifferentiated carcinoma cells located peripherally in an epithelial pearl showing EBERs positivity. EBERs in situ hybridization; 5× 10.
Fig.4 Most of KSCCs except two (No. 30, D54974 ＆ No. 25, D51849) demonstrated in this figure showing positive blots by PCR Southern Blotting hybridization.
Table 4 Relationship between EBERs+ ve cells and differentiation degree of KSCCs Differentiation
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Of KSCC
Percentage of EBERs positive cells
0%
<25%
26～ 50%
51～ 75%
>76%
Well
0
7
1
0
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0
Moderately
1
4
2
0
0
Poorly
0
14
5
3
1
Total
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1(2.6% )
25(65.8% )
8(21.1% )
3(7.9% )
1(2.6% )
The EBERs positive cells were not always uniform in intensity, being varied from case to case and even cells to cells. Morphologically, the EBERs positive carcinoma cells were undifferentiated or poorly differentiated, and on the other hand the more differentiated or keratinizing carcinoma cells were often EBERs negative. A variable number of EBERs positive cells in 21 out of 37 EBERs positive KSCCs (56.8% ) expressed LMP-1 (Fig. 5). The EBV lytic-cycle gene encoded proteins could also be found in a considerable number of cases ( Table 3), the positive rates were: ZEBRA 29.73% (11/37); EA-D 62.16% (23/37; Fig.6); VCA 48.65% (18/37; Fig.7); and MA 43.24% (16/37; Fig.8). EB viral proteins of three main phases of lytic cycle, namely, immediate early ZEBRA, early EA-D, and late VCA and MA could all be found in 4 cases (case nos. 7, 8, 20, ＆ 24). Besides these 4 cases, there were 5 KSCC specimens (case nos.1, 6, 12, 13, ＆ 26) expressing EA D, VCA and MA. There were 2 KSCCs (case nos.16, and 33) expressing EA-D and VCA. Only 1 KSCC (case no.38) expressed EA-D and MA. There were 3 KSCCs (case nos.22, 25, and 31) expressing VCA and MA. In 7 KSCC specimens ( case nos. 9, 10, 11, 14, 19, 23, ＆ 32), a few of ZEBRA or/and EA-D positive carcinoma cells could be found but no VCA or/and MA positive carcinoma cells detected. It had been confirmed by consecutive sections that ZEBRA or EA-D positive carcinoma cells were often EBERs positive and VCA or MA negative; and VCA and MA were always both positive in the same EBERs negative carcinoma cells.
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Fig.5 A group of poorly differentiated and undifferentiated carcinoma cells in a KSCC specimen showing LMP- 1 positivity.LMP- 1 immunostaining; 5× 10.
Fig.6 Some poorly differentiated and undifferentiated carcinoma cells in a KSCC specimen showing EA- D positivity. EA- D immunostaining; 5× 10.
Fig.7 A few more differentiated carcinoma cells in a KSCC specimen showing VCA positivity. VCA immunostaining; 5× 20.
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Fig.8 Some more differentiated carcinoma cells in a KSCC specimen showing MA positivity. MA immunostaining; 5× 20.
DISCUSSION
An association of EBV with nasopharyngeal non-keratinizing carcinomas (NKCs) was suggested as early as 1979 on grounds of serological studies[8] and subsequently confirmed by demonstration of EBV DNA and EBV-encoded nuclear antigen complex by use of in situ hybridization and anti-complement immunofluorescene, respectively[9 ＆ 10]. Are squamous cell carcinomas or keratinizing squamous cell carcinomas (KSCCs) of the nasopharynx also consistently associated with EB virus? So far as we know there is a discrepancy about this issue down to date. Some authors said “ yes" [4], some reported “ no"[5], and some authors said “ occasionally"[11]. The authors collected 38 KSCCs from the NPC high-incidence area, Guangzhou locale, in the south of China, during the past 10 years and detected EBV DNA and EBERs simultaneously in the formalin-fixed paraffin-embedded biopsy samples by both PCR Southern blotting hybridization and in situ hybridization. The results illustrated that the KSCCs of the nasopharynx were also closely, not occasionally, associated with EB virus as the NKCs did. Among the 38 KSCCs, EB virus could not be demonstrated only in one case (case no.30). It should be emphasized herein that the formalin-fixed paraffin-embedded block of this case had quite a bit of tissue for study, therefore, negative result obtained can not substantially prove that this specimen was actually not infected with EBV. Even if this KSCC specimen really did not contain EB virus, it is also not surprising because of no 100% of NKC samples showing EBV positivity (according to the authors' unpublished data). A positive hybridization blot could not be demonstrated in the 5 KSCCs (case nos. 20, 22, 25, 28, ＆ 32) which contained EBERs positive carcinoma cells. This phenomenon may be interpreted as follows: (1) Though PCR Southern blotting hybridization was a sensitive and specific method for detection of EBV DNA sequences in NPC cells as demonstrated by Chang YS, et al.[12] and Dickens P, et al.[13], the EBV DNA of these 5 KSCC specimens had merely a little quantity (because of a little bit of tissue and only a few EBERs positive carcinoma cells presented) and might be degraded in nature. This may be resulted in a negative reaction. Just as it is, so the results obtained by Hording U, et al.[14] and Akao I, et al.[15] can thus be interpreted. Only 2 out of 14 and 5 out of 7 KSCCs performed by Hording and Akao, respectively, got positive blot. (2) The in situ hybridization technique for detection of EBV encoded small RNAs (EBERs), which are very stable in secondary structure and have relatively long half- life (8- 9 h for EBER- 1 and less than 1 h for EBER- 2 in Daudi cells)[16], was such a kind of more sensitive method[17， 18， 19 ＆ 20] that even a few EBERs positive carcinoma cells could be shown.
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We have observed hundreds of NKC slides stained by EBERs in situ hybridization, and found that the vast majority of carcinoma cells (data not shown) showed positivity. Raab- Traub also said, “ expression of specific viral mRNAs or gene products are consistently detected within all of the tumor cells"[3]. However, 33 out of the 37 EBERs positive KSCCs had less than 50% of carcinoma cells expressing EBERs and all the 14 EBERs positive well- and moderately- differentiated KSCCs were among these 33 cases. It is popularly recognized that regardless of types all the NPCs have more or less squamous differentiation under electron microscopy[1] and the KSCC and NKC, which can be subdivided into differentiated NKC and undifferentiated carcinoma of nasopharyngeal type, may be recognized as a spectrum of squamous differentiation, having a gradually decreased amount of more differentiated or keratinizing carcinoma cells. Basing upon this understanding, we paid our attention to the correlation of EBERs expression and carcinoma cell differentiation. We found that most of the EBERs positive carcinoma cells were undifferentiated or poorly differentiated neoplastic cells, which were the malignant counterparts of basal or suprabasal cells of squamous epithelium under light microscopy; while the more differentiated or keratinizing carcinoma cells, which might be considered as the malignant counterparts of polygonal prickle cells with distinct intercellular bridges or superficially located flattened keratinizing cells of squamous epithelium, were always EBERs negative. Recently, Pathmanathan R, et al.[21] reported the same phenomenon. They said “ In foci of squamous differentiation and keratinization within less differentiated NPC and throughout the expanse of well differentiated squamous cell carcinoma, EBER expression was less abundant". Furthermore, Chen CL, et al.[22] using radioisotope-and biotin-labeled EBV DNA probes for in situ hybridization showed that “ in the keratinizing carcinoma, which was poorly differentiated in this series, the EBV genome was usually detected in anaplastic tumor cells, and not in the keratinizing areas". Zhang J.X. et al.[23] reported the relationship between EBV life cycle and tumour cell differentiation. Basing upon all these findings, we assume that the EB virus was likely to be harbouring in most undifferentiated or poorly differentiated carcinoma cells. Because almost all of the EBERs positive cells were EBNA- 1 positive and a portion of them expressing LMP- 1, this kind of EB virus infection is of latency II in type. It is worth to point out that since the ZEBRA positive carcinoma cells, though only a few in number found on 11 KSCC slides, were undifferentiated in morphology, and the EA-D positive cells found on 23 KSCCs, were either undifferentiated or poorly differentiated too, a few or a small number of the latently EBV-infected undifferentiated/poorly differentiated carcinoma cells might be switched the lytic cycle on and expressing its gene products by some unknown factors. On the other hand, the VCA or MA positive carcinoma cells, found on 22 KSCC specimens were mostly of more differentiated or keratinizing carcinoma cells. Accordingly, we assume that the EB virus harbouring in more differentiated or keratinizing NPC cells is able to express late gene proteins of lytic cycle, such as VCA or MA. In other words, the more differentiated or keratinizing cells are likely to be infected with EB virus in productive cycle.
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Our findings only demonstrate that the lytic cycle products of EB virus could be found in KSCCs of the nasopharynx and can not be interpreted as that a complete lytic cycle of EB virus may occur in every KSCC cells. In fact, most undifferentiated/poorly differentiated carcinoma cells are infected with EB virus in latency; and merely in some given microenvironments, such as in cell differentiation and maturation, the EB virus genome in episomal form might be possible to convert the latent into lytic cycle. We agree the words of Naher H. and Petzoldt D.[24] that “ differentiation and maturation of the epithelial cells is paralleled by active virus production". Even if the EB viruses are undergoing lytic gene expression, most of them possibly occur in abortive form because of the following reasons: (1) The proteins representing three main phases of lytic cycle (immediate early ZEBRA, early EA-D, and late VCA/MA) could only be concurrently found in 4 KSCCs specimens. (2) the ZEBRA+ , EA-D+ , or VCA+ /MA+ cells were not the same ones found on the slides. This findings coincide with what Martel-Renoir D, et al.[25] reported previously. However, a complete lytic cycle of EB virus usually occur in some KSCC specimens because the late gene proteins could be definitely found in some of more differentiated or keratinizing carcinoma cells. Furthermore, as popularly known, the patients with NPC, including NKC ＆ KSCC frequently had an elevated antibodies titer against these late gene proteins. Wolf H et al.[26] had said that “ only terminally differentiated epithelial cells are capable of supporting an efficient lytic cycle of EBV replication". This hypothesis can interpret what we found in the KSCC specimens studied hereof.
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* This work was supported by grants from the National Council of Natural Sciences (NCNS Nos. 39470295 and 39730200-Ⅱ ), The People's Republic of China, and the preliminary results were orally presented, not formally published, at the "Symposium of Epstein- Barr Virus and Nasopharyngeal Cancer" held in Taipei, China, June 9, 1996.
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(收稿：1998-11-10修回：1998-12-08), 百拇医药
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