腮腺淋巴上皮瘤样癌与EB病毒感染的关系
作者：宗永生 刘克拉 饶慧兰 李智 林汉良 陈国雄 吴文翰
单位：宗永生 刘克拉 饶慧兰 李智 林汉良(中山医科大学病理学教研室，广州 510089)；陈国雄 吴文翰(香港大学微生物学系)
关键词：淋巴上皮瘤样癌；腮腺肿瘤；Epstein-Barr病毒；基因表达
临床与实验病理学杂志990501
摘要 目的：研究Epstein-Barr病毒(EBV)与腮腺淋巴上皮瘤样癌(lymphoepithelioma-like carcinoma, LELC)的关系，并检测瘤细胞内EBV基因编码产物。方法：作者收集了中山医科大学所属病理科1986年1月至1995年12 月间32例腮腺LELCs.。32例LELC石蜡包埋标本再次切片。采用免疫组化和原位核酸杂交法检测瘤细胞内EBV基因表达产物。结果：(1) 在125例腮腺癌中有32例淋巴上皮瘤样癌，占总病例的25.6%(32/125)。(2)所有32例腮腺LELC组织中均有数量不等的EBNA-1和EBERs阳性瘤细胞。(3)27例LELC组织中部分瘤细胞表达LMP-1。(4)所有标本中均未见ZEBRA阳性细胞。(5)32例腮腺LELC组织中EA-D、VCA和MA的阳性表达率分别为71.9%(23/32)、68.8%(22/32)和12.5%(4/32)。结论：(1)在鼻咽癌高发的广州地区，腮腺LELC的发病率也较高。(2)腮腺LELC组织中均有EB病毒感染。(3)EB病毒在腮腺LELC的感染主要为潜伏Ⅱ型，即表达EBNA-1、EBERs和LMP-1。(4)在腮腺LELC瘤细胞内潜伏感染的EB病毒可部分进入溶解周期，从而表达EA-D、VCA和MA。
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分类号 R739.87 文献标识码 A
文章编号 1001-7399(1999)05-0377-05
Association of Epstein-Barr virus with lymphoepithelioma-like carcinoma of the parotid gland
Zong Yongsheng，Liu Kela， Rao Huilan，Lin Hanliang，Li Zhi，(Department of Pathology, Sun Yat-Sen University of Medical Sciences, Guangzhou 510089, China)
Chen Guoxiong， Wu Wenhan
(Department of Microbiology, University of Hong Kong, Hong Kong, China)
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ABSTRACT Purpose To investigate the relationship between Epstein-Barr virus (EBV) and lymphoepithelioma-like carcinoma (LELC) of the parotid gland and detect the gene expression products of EBV harbouring in LELC cells. Methods Thirty-two parotid LELCs were collected from the Departments of Pathology, Sun Yat-sen University of Medical Sciences, Guangzhou, China during the period of January 1986 and December 1995. All the 32 formalin-fixed paraffin-embedded blocks had been consecutively re-sectioned again. Immunohistochemical and in situ nucleic acid hybridization methods for detection of EBV gene encoded products were performed. Results (1) 32 LELCs were found out of 125 parotid gland carcinomas, the frequency was 25.6% (32/125). (2) All of the 32 specimens contained a variable number of EBNA-1 and EBERs positive neoplastic cells. (3) Twenty-seven out of 32 specimens (27/32, 84.4%) had a portion of carcinoma cells expressing LMP-1. (4) No ZEBRA positive cell could be found. (5) EA-D, VCA and MA positivity rates for these 32 parotid LELCs reached to 71.9%(23/32), 68.8%(22/32), and 12.5%(4/32), respectively. Conclusions (1) The parotid gland LELC is frequently to be seen in Guangzhou locale of China, where is a high-incidence area of nasopharyngeal carcinoma (NPC). The parotid gland LELC and NPC are co-prevalent in Guangzhou locale. (2) This disease is also consistently associated with EBV infection. (3) The EBV infection of the parotid gland LELCs is essentially the type of latency Ⅱ, expressing EBNA-1, EBERs and LMP-1. (4) The latent infected EBV harbouring in LELC cells could in part be switched over to lytic cycle, producing EA-D, VCA or/and MA.
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KEY WORDS lymphoepithelioma-like carcinoma; parotid neoplasms; Epstein-Barr virus; gene expression
The lymphoepithelioma-like carcinoma (LELC) or undifferentiated carcinoma with lymphoid stroma of the parotid gland, which is closely associated with Epstein-Barr virus (EBV) infection is frequently developed in Eskimos living in Greenland, where is a well-known high incidence area of nasopharyngeal carcinoma (NPC)〔1～3〕. NPC is also consistently associated with EBV and prevalent in Guangzhou locale of southern China. Its mean standardized incidence or morbidity rate was 18.12/100 000 for both sexes(25.60 in males and 11.05 in females)〔4,5〕. Is the parotid gland LELC also frequently developed in Guangzhou? Does EBV also closely associate with this disease? What is the EBV encoded gene expression pattern in the neoplastic cells of this tumour? The ob-jective of this study was focussing on these issues.
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1 MATERIALS AND METHODS
Thirty-two parotid gland LELCs from the archives of the Departments of Pathology, Sun Yat-sen University of Medical Sciences during the period of January 1986 and December 1995 were collected for this study. The patient had unilateral enlargement of the parotid gland and no any symptoms or signs suspicious for NPC. The tumours studied were solitary and solid and not a bilateral, multiple and cystic disease. Multiple sections of the surgical specimens had been taken and the residual glandular tissue within the tumour mass could be found microscopically, indicating that the carcinoma of the patients was arising from the parotid gland itself and not a metastatic one.
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DAKO Hybridization Detection Kit (Code No K0046) and Dako EBV (EBER) probe (Code No Y0017) were used for in situ hybridization. Methyl green was used as counterstaining. DAKO LSAB Kit/HRP, Rabbit/Mouse, Universal (Code No K0681) was used for immunohistochemical detection, and the primary antibodies used were listed in table 1.
Table 1 Primary antibodies used Primary antibody
Antigen
recognized
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Working
dilution
EBV OT-1x
EBNA-1
1∶300
EBV OT17-2
LMP-1
1∶100
DAKO EBV BZLF1 (Clone BZ.1)
ZEBRA
1∶40
EBV OT13-B(p138)
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EA-D
1∶400
EBV OT15E (p18)
VCA
1∶200
EBV OT-6 (gp350/220Kd)
MA
1∶100
DAKO: DAKO A/S products
OT: Organon-Teknika products, kindly provided by Dr Jaap M Middeldorp, Netherlands
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In addition, routine immunohistochemical method was also used for detection of cytokeratin, T cell and B cell using DAKO primary antibodies M3515, M0742, and M0774, respectively.
Criteria for evaluating positive grade were as follows: Negative (-), no positive cell found; Grade 1(1+), 1%～25% positive cells found; Grade 2(2+), 26%～50% positive cells found; Grade 3(3+), 51%～75% positive cells found; Grade 4 (4+), 76%～100% positive cells found.
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2 RESULTS
We found 32 LELCs in 125 parotid gland carcinomas; the frequency accounted for 25.6%(32/125). Eighteen cases were male and 14 female; the ratio of male to female was 1.29:1. The patients age was ranged from 16 to 68 years old. The mean age was 45.75 years old and the median age 47.0 years old. The peak of age group was 40～49 years old. Eighteen LELCs were developed in right parotid gland, 14 in left. The duration of having an enlarged cervical mass before visiting doctor ranged from 2 months to 7 years, averagely 18 months.
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The histological features of this tumour were identical with those of nasopharyngeal non-keratinizing carcinoma, especially of undifferentiated carcinoma of nasopharyngeal type, which is characterized by nests of too poorly differentiated or undifferentiated neoplastic cells with indistinct cell borders or syncytial appearance (Fig 1). Sometimes, the LELC cells might have a few scanty intercellular bridges or eosinophilic cytoplasm, being similar to poorly differentiated squamous carcinoma cells in many ways. The neoplastic cells were always admixed with abundant infiltrating lymphocytes, of which T cells were the predominant ones (Fig 2). The results of EBV encoded gene expression in these 32 LELCs were summarized in table 2.
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Fig 1 Lymphoepithelioma-like carcinoma of the parotid gland showing the histological features identical with those of undifferentiated carcinoma of nasopharyngeal type. HE×50
Fig 2 Abudant T lymphocytes infiltrating within the LELCnests of the parotid gland LELC. UCHL-1 IHC×50
Table 2 EBV encoded gene expression in 32 parotid gland LELCs Case No
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Site
EBERs
EBNA-1
LMP-1
ZEBRA
EA-D
VCA
MA
1
M/53
LP
4+
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4+
3+
-
1+
1+
-
2
M/45
RP
3+
3+
1+
-
1+
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1+
-
3
F/43
LP
3+
3+
2+
-
2+
1+
-
4
M/63
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RP
3+
3+
2+
-
1+
1+
1+
5
M/47
LP
3+
3+
1+
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-
1+
-
-
6
M/65
LP
3+
3+
2+
-
2+
1+
-
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7
M/49
LP
1+
1+
1+
-
1+
1+
-
8
M/67
LP
4+
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4+
2+
-
1+
-
-
9
M/42
LP
2+
2+
1+
-
-
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-
-
10
F/58
RP
3+
3+
1+
-
2+
-
-
11
F/40
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RP
4+
4+
2+
-
1+
1+
1+
12
F/39
RP
3+
3+
-
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-
1+
1+
-
13
M/65
LP
4+
4+
2+
-
1+
-
-
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14
M/37
RP
4+
4+
2+
-
2+
1+
-
15
M/57
LP
3+
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3+
1+
-
-
-
-
16
F/68
RP
3+
3+
-
-
1+
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1+
-
17
F/41
LP
2+
2+
1+
-
1+
1+
-
18
F/48
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RP
2+
2+
-
-
-
1+
1+
19
M/42
RP
1+
1+
1+
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-
1+
-
-
20
F/36
RP
2+
2+
1+
-
1+
1+
-
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21
M/18
RP
2+
2+
2+
-
2+
1+
-
22
F/49
RP
1+
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1+
-
-
-
1+
-
23
F/52
LP
1+
1+
1+
-
2+
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1+
-
24
F/21
RP
3+
3+
2+
-
2+
1+
-
25
F/68
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RP
2+
2+
1+
-
-
-
-
26
M/31
LP
3+
3+
1+
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-
-
1+
-
27
M/38
RP
3+
3+
1+
-
-
1+
1+
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28
F/45
LP
1+
1+
1+
-
1+
1+
-
29
M/16
RP
2+
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2+
1+
-
2+
1+
-
30
M/47
RP
2+
2+
1+
-
2+
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1+
-
31
F/35
LP
2+
2+
1+
-
-
-
-
32
M/39
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RP
1+
1+
-
-
-
-
-
RP: Right parotid gland
LP: Left parotid gland
EB virus encoded small non-polyadenylated RNAs (EBERs) positive carcinoma cells could be found in all of the 32 specimens. Most of the neoplastic cells showed strong nuclear signal (Fig 3). Sometimes, the intensity and distribution of EBERs signal might be varied from cell to cell. In such situation the distribution of EBERs signal seemed identical with that of heterochromatin seen on H&E slides (Fig 4). The tumour infiltrating lymphocytes and residual benign glandular epithelial cells were negative for EBERs. The EBERs positive neoplastic cells were certainly EBNA-1 positive.
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Fig 3 Almost all of the carcinoma cells showing EBERs signals(3+～4+),while the surrounding lymphocytes being negative. EBERs-ISH×50
Fig 4 The intensity and distribution of EBERs signals were similar to those of nuclear heterochromatins. EBERs-ISH×100
The LMP-1 positive carcinoma cells could be found in 27 out of 32 LELC specimens (27/32, 84.4%). Positive brown granules were detected cyto-membranously in the neoplastic cells (Fig 5). It should be emphasized that only a small portion of the carcinoma cells (less than 50%) showed LMP-1 positivity. Among these 27 LMP-1 positive cases, 26 were of grade 1～2 (26/27, 96.3%), and only 1 specimen had more than 51% LMP-1 positive cells.
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FIg 5 The parotid gland LELC cells showing cytoplasmic LMP-1 positivity. LMP-1 IHC×100
No ZEBRA positive cell could be found in these 32 LELC specimens observed. A scattering of (1%～25%, 14 cases) or a small number of (26%～50%, 9 cases) EA-D positive cells (Fig 6) were found within the carcinoma nests in 23 out of 32 LELCs (23/32, 71.9%). These EA-D positive cells were often relatively small in size. A few VCA positive neoplastic cells (Fig 7) could be found in 22 specimens (22/32, 68.8%) and several MA (gp350/220) positive carcinoma cells (Fig 8) could merely be found in 4 specimens (4/32, 12.5%).
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FIg 6 The EA-D positive cells were scattered within the LELC nest. EA-D IHC×50
FIg 7 Some of the parotid gland LELC cells showing cytoplasmic VCA positivity. VCA IHC×50
FIg 8 Afew LELC cells of the parotid gland showing MA positivity. MA IHC×100
3 DISCUSSION
As mentioned above, the parotid gland LELC and NPC were both frequently seen in Greenland natives〔1～3〕. Abdulla and Mian reviewed English and Arabic literatures up to 1996 and found the total number of reported cases of salivary gland LELC to be 103 with predominant involvement of the parotid gland〔6〕. Guangzhou locale is a high-incidence area of NPC〔4,5〕. Does parotid gland LELC be frequently seen in Guangzhou locale too? Our results showed that there were 32 LELCs among 125 parotid gland carcinomas previously diagnosed as poorly differentiated or undifferentiated carcinomas during the past ten years. So, the parotid gland LELC and NPC are co-prevalent in Guangzhou locale.
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The median age (47.0 years old), mean age (45.75 years old) and age group peak (30～49 years old)) of the patients with parotid gland LELC were identical to those of NPC patients〔4〕. However, the ratio of male to female in the parotid gland LELCs was different to that of NPCs arising in Guangzhou locale (1.29:1 versus 2.21:1)〔4〕. It seems that the LELC of the parotid gland was not predominantly prevalent in males as the NPC did. The ratio of male to female in 14 salivary gland LELCs reported by Takashi was 1:7〔7〕, further implicating that the female was likely to be suffered from this disease. The average duration of unilateral parotid gland enlargement in these 32 LELCs was 18 months, which was also similar to 16 months reported by Takashi〔7〕.
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The salivary gland LELCs in Greenland were closely associated with EBV infection〔1～3〕. Iezzoni et al. said that the association of EBV with the salivary patients gland LELC is restricted to Asian patients〔8〕. However, the white people could suffer this disease with EBV infection also〔9〕. Does the parotid gland LELCs in Guangzhou locale also closely related with EBV? Our results demonstrated that the majority of carcinoma cells in all of the 32 LELCs showed nuclear EBERs and EBNA-1 signal, which could not be found within the stromal lymphocytes and adjacent non-tumourous glandular cells. It should be emphasized that all of the 32 parotid gland LELCs were solitary and solid in nature, having no symptoms and signs of NPC and therefore not metastatic NPCs. Furthermore, they were not bilateral, multiple and cystic; and therefore might not be induced by HIV as reported by Terry et al〔10〕. The above findings reasonably confirm a fact that there is an existence of a consistent and specific association between EBV and neoplastic cells of the parotid gland LELCs arising in residents of Guangzhou locale. Chan et al. reported 5 Chinese salivary gland LELCs in Hong Kong〔11〕. Their findings coincided with what we found. We assume that the EB virus probably plays a very important role in the pathogenesis of this disease. This conclusion coincided with what Lanier et al. had reported in Greenland〔12〕.In addition, We found that neither EBERs nor EBNA-1 signal could be seen in 8 so-called benign lymphoepithelial lesions (data not shown). After making a survey of surgical salivary gland tumours, Hsu〔13〕 and Tsa〔14〕 suggested that this virus may not play a role in the pathogenesis of most tumors of the major salivary gland except LELCs. So, We also confirm whether EBV exists in so-called lymphoepithelial islands of the parotid glands is a critical marker for distinguishing benignancy and malignancy as reported by Takashi〔7〕.
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In regard to EBV infection and its gene expression, what are the similarities and differences between LELC of the parotid gland and NPC? The EBV harbouring in either the neoplastic cells of parotid gland LELC or of NPC essentially express EBNA-1, EBERs and LMP-1, illustrating that the EBV infection of these two tumours is in the same latency Ⅱ type. However, the LMP-1 expression rate of these 32 parotid gland LELCs reached 84.4%(27/32) which is obviously higher than 30%～65% reported in the literature〔15,16〕. Leung et al. reported 10 cases of the salivary gland LELCs (eight parotid and two submandibular) in Hong Kong Chinese, all of which showed EBERs and LMP-1 positivity〔17〕 .Is there really existence of different LMP-1 expression rates between parotid gland LELC and NPC? How to interpret this difference? Is this discrepancy due to different detection methods used? This issue remains to be further studied.
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The EA-D expression rate was 71.9%(23/32) which is also higher than our unpublished datum of NPC (48.8%, 60/123). Furthermore, according to our comparative observation, the EA-D positive carcinoma cells in the parotid gland LELCs were always more numerous than in NPCs. The VCA and MA expression rates were 68.8%(22/32) and 12.5% (4/32), respectively. Worthy to note is that only a small number or even a few neoplastic cells showed VCA or MA positivity. These findings indicate that the protein products encoded by EBV lytic genes could be expressed in a part of the parotid gland LELC cells, and the EBV harbouring in LELC cells were able to switch over to lytic cycle from latency status. Why the immediate early gene BZLF1 product, ZEBRA was not to be detected? We think that this might be resulting from the short half-life of ZEBRA protein itself. Considering the gradual decreasing of the expression rates from EA-D(71.9%) to VCA (68.8%) and then to MA(12.5%), the authors assume that the lytic cycle of EBV harbouring in neoplastic cells was of abortive form, being not able to complete its production cycle. Martel-Renoir et al.〔18〕 has also confirmed this abortive lytic cycle happened in nasopharyngeal carcinoma. Recently, Zhang et al. found expression of lytic cycle products encoded by EBV within keratinizing nasopharyngeal carcinoma cells〔19〕. This report further supports the viewpoint of Zhangs paper.
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基金项目：国家自然科学基金重点项目(No 39730200-Ⅱ)
作者简介：宗永生，男，71岁，教授，博士生导师
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16，Young LS, Dawson CW, Clark D et al. Epstein-Barr virus gene expression in nasopharyngeal carcinoma. J Gen Virol, 1988;69:1051～1065
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(Figure seen illustration page 55)
收稿日期：1999-05-07, http://www.100md.com
单位：宗永生 刘克拉 饶慧兰 李智 林汉良(中山医科大学病理学教研室，广州 510089)；陈国雄 吴文翰(香港大学微生物学系)
关键词：淋巴上皮瘤样癌；腮腺肿瘤；Epstein-Barr病毒；基因表达
临床与实验病理学杂志990501
摘要 目的：研究Epstein-Barr病毒(EBV)与腮腺淋巴上皮瘤样癌(lymphoepithelioma-like carcinoma, LELC)的关系，并检测瘤细胞内EBV基因编码产物。方法：作者收集了中山医科大学所属病理科1986年1月至1995年12 月间32例腮腺LELCs.。32例LELC石蜡包埋标本再次切片。采用免疫组化和原位核酸杂交法检测瘤细胞内EBV基因表达产物。结果：(1) 在125例腮腺癌中有32例淋巴上皮瘤样癌，占总病例的25.6%(32/125)。(2)所有32例腮腺LELC组织中均有数量不等的EBNA-1和EBERs阳性瘤细胞。(3)27例LELC组织中部分瘤细胞表达LMP-1。(4)所有标本中均未见ZEBRA阳性细胞。(5)32例腮腺LELC组织中EA-D、VCA和MA的阳性表达率分别为71.9%(23/32)、68.8%(22/32)和12.5%(4/32)。结论：(1)在鼻咽癌高发的广州地区，腮腺LELC的发病率也较高。(2)腮腺LELC组织中均有EB病毒感染。(3)EB病毒在腮腺LELC的感染主要为潜伏Ⅱ型，即表达EBNA-1、EBERs和LMP-1。(4)在腮腺LELC瘤细胞内潜伏感染的EB病毒可部分进入溶解周期，从而表达EA-D、VCA和MA。
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分类号 R739.87 文献标识码 A
文章编号 1001-7399(1999)05-0377-05
Association of Epstein-Barr virus with lymphoepithelioma-like carcinoma of the parotid gland
Zong Yongsheng，Liu Kela， Rao Huilan，Lin Hanliang，Li Zhi，(Department of Pathology, Sun Yat-Sen University of Medical Sciences, Guangzhou 510089, China)
Chen Guoxiong， Wu Wenhan
(Department of Microbiology, University of Hong Kong, Hong Kong, China)
, 百拇医药
ABSTRACT Purpose To investigate the relationship between Epstein-Barr virus (EBV) and lymphoepithelioma-like carcinoma (LELC) of the parotid gland and detect the gene expression products of EBV harbouring in LELC cells. Methods Thirty-two parotid LELCs were collected from the Departments of Pathology, Sun Yat-sen University of Medical Sciences, Guangzhou, China during the period of January 1986 and December 1995. All the 32 formalin-fixed paraffin-embedded blocks had been consecutively re-sectioned again. Immunohistochemical and in situ nucleic acid hybridization methods for detection of EBV gene encoded products were performed. Results (1) 32 LELCs were found out of 125 parotid gland carcinomas, the frequency was 25.6% (32/125). (2) All of the 32 specimens contained a variable number of EBNA-1 and EBERs positive neoplastic cells. (3) Twenty-seven out of 32 specimens (27/32, 84.4%) had a portion of carcinoma cells expressing LMP-1. (4) No ZEBRA positive cell could be found. (5) EA-D, VCA and MA positivity rates for these 32 parotid LELCs reached to 71.9%(23/32), 68.8%(22/32), and 12.5%(4/32), respectively. Conclusions (1) The parotid gland LELC is frequently to be seen in Guangzhou locale of China, where is a high-incidence area of nasopharyngeal carcinoma (NPC). The parotid gland LELC and NPC are co-prevalent in Guangzhou locale. (2) This disease is also consistently associated with EBV infection. (3) The EBV infection of the parotid gland LELCs is essentially the type of latency Ⅱ, expressing EBNA-1, EBERs and LMP-1. (4) The latent infected EBV harbouring in LELC cells could in part be switched over to lytic cycle, producing EA-D, VCA or/and MA.
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KEY WORDS lymphoepithelioma-like carcinoma; parotid neoplasms; Epstein-Barr virus; gene expression
The lymphoepithelioma-like carcinoma (LELC) or undifferentiated carcinoma with lymphoid stroma of the parotid gland, which is closely associated with Epstein-Barr virus (EBV) infection is frequently developed in Eskimos living in Greenland, where is a well-known high incidence area of nasopharyngeal carcinoma (NPC)〔1～3〕. NPC is also consistently associated with EBV and prevalent in Guangzhou locale of southern China. Its mean standardized incidence or morbidity rate was 18.12/100 000 for both sexes(25.60 in males and 11.05 in females)〔4,5〕. Is the parotid gland LELC also frequently developed in Guangzhou? Does EBV also closely associate with this disease? What is the EBV encoded gene expression pattern in the neoplastic cells of this tumour? The ob-jective of this study was focussing on these issues.
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1 MATERIALS AND METHODS
Thirty-two parotid gland LELCs from the archives of the Departments of Pathology, Sun Yat-sen University of Medical Sciences during the period of January 1986 and December 1995 were collected for this study. The patient had unilateral enlargement of the parotid gland and no any symptoms or signs suspicious for NPC. The tumours studied were solitary and solid and not a bilateral, multiple and cystic disease. Multiple sections of the surgical specimens had been taken and the residual glandular tissue within the tumour mass could be found microscopically, indicating that the carcinoma of the patients was arising from the parotid gland itself and not a metastatic one.
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DAKO Hybridization Detection Kit (Code No K0046) and Dako EBV (EBER) probe (Code No Y0017) were used for in situ hybridization. Methyl green was used as counterstaining. DAKO LSAB Kit/HRP, Rabbit/Mouse, Universal (Code No K0681) was used for immunohistochemical detection, and the primary antibodies used were listed in table 1.
Table 1 Primary antibodies used Primary antibody
Antigen
recognized
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Working
dilution
EBV OT-1x
EBNA-1
1∶300
EBV OT17-2
LMP-1
1∶100
DAKO EBV BZLF1 (Clone BZ.1)
ZEBRA
1∶40
EBV OT13-B(p138)
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EA-D
1∶400
EBV OT15E (p18)
VCA
1∶200
EBV OT-6 (gp350/220Kd)
MA
1∶100
DAKO: DAKO A/S products
OT: Organon-Teknika products, kindly provided by Dr Jaap M Middeldorp, Netherlands
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In addition, routine immunohistochemical method was also used for detection of cytokeratin, T cell and B cell using DAKO primary antibodies M3515, M0742, and M0774, respectively.
Criteria for evaluating positive grade were as follows: Negative (-), no positive cell found; Grade 1(1+), 1%～25% positive cells found; Grade 2(2+), 26%～50% positive cells found; Grade 3(3+), 51%～75% positive cells found; Grade 4 (4+), 76%～100% positive cells found.
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2 RESULTS
We found 32 LELCs in 125 parotid gland carcinomas; the frequency accounted for 25.6%(32/125). Eighteen cases were male and 14 female; the ratio of male to female was 1.29:1. The patients age was ranged from 16 to 68 years old. The mean age was 45.75 years old and the median age 47.0 years old. The peak of age group was 40～49 years old. Eighteen LELCs were developed in right parotid gland, 14 in left. The duration of having an enlarged cervical mass before visiting doctor ranged from 2 months to 7 years, averagely 18 months.
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The histological features of this tumour were identical with those of nasopharyngeal non-keratinizing carcinoma, especially of undifferentiated carcinoma of nasopharyngeal type, which is characterized by nests of too poorly differentiated or undifferentiated neoplastic cells with indistinct cell borders or syncytial appearance (Fig 1). Sometimes, the LELC cells might have a few scanty intercellular bridges or eosinophilic cytoplasm, being similar to poorly differentiated squamous carcinoma cells in many ways. The neoplastic cells were always admixed with abundant infiltrating lymphocytes, of which T cells were the predominant ones (Fig 2). The results of EBV encoded gene expression in these 32 LELCs were summarized in table 2.
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Fig 1 Lymphoepithelioma-like carcinoma of the parotid gland showing the histological features identical with those of undifferentiated carcinoma of nasopharyngeal type. HE×50
Fig 2 Abudant T lymphocytes infiltrating within the LELCnests of the parotid gland LELC. UCHL-1 IHC×50
Table 2 EBV encoded gene expression in 32 parotid gland LELCs Case No
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Site
EBERs
EBNA-1
LMP-1
ZEBRA
EA-D
VCA
MA
1
M/53
LP
4+
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4+
3+
-
1+
1+
-
2
M/45
RP
3+
3+
1+
-
1+
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1+
-
3
F/43
LP
3+
3+
2+
-
2+
1+
-
4
M/63
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RP
3+
3+
2+
-
1+
1+
1+
5
M/47
LP
3+
3+
1+
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-
1+
-
-
6
M/65
LP
3+
3+
2+
-
2+
1+
-
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7
M/49
LP
1+
1+
1+
-
1+
1+
-
8
M/67
LP
4+
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4+
2+
-
1+
-
-
9
M/42
LP
2+
2+
1+
-
-
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-
-
10
F/58
RP
3+
3+
1+
-
2+
-
-
11
F/40
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RP
4+
4+
2+
-
1+
1+
1+
12
F/39
RP
3+
3+
-
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-
1+
1+
-
13
M/65
LP
4+
4+
2+
-
1+
-
-
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14
M/37
RP
4+
4+
2+
-
2+
1+
-
15
M/57
LP
3+
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3+
1+
-
-
-
-
16
F/68
RP
3+
3+
-
-
1+
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1+
-
17
F/41
LP
2+
2+
1+
-
1+
1+
-
18
F/48
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RP
2+
2+
-
-
-
1+
1+
19
M/42
RP
1+
1+
1+
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-
1+
-
-
20
F/36
RP
2+
2+
1+
-
1+
1+
-
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21
M/18
RP
2+
2+
2+
-
2+
1+
-
22
F/49
RP
1+
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1+
-
-
-
1+
-
23
F/52
LP
1+
1+
1+
-
2+
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1+
-
24
F/21
RP
3+
3+
2+
-
2+
1+
-
25
F/68
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RP
2+
2+
1+
-
-
-
-
26
M/31
LP
3+
3+
1+
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-
-
1+
-
27
M/38
RP
3+
3+
1+
-
-
1+
1+
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28
F/45
LP
1+
1+
1+
-
1+
1+
-
29
M/16
RP
2+
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2+
1+
-
2+
1+
-
30
M/47
RP
2+
2+
1+
-
2+
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1+
-
31
F/35
LP
2+
2+
1+
-
-
-
-
32
M/39
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RP
1+
1+
-
-
-
-
-
RP: Right parotid gland
LP: Left parotid gland
EB virus encoded small non-polyadenylated RNAs (EBERs) positive carcinoma cells could be found in all of the 32 specimens. Most of the neoplastic cells showed strong nuclear signal (Fig 3). Sometimes, the intensity and distribution of EBERs signal might be varied from cell to cell. In such situation the distribution of EBERs signal seemed identical with that of heterochromatin seen on H&E slides (Fig 4). The tumour infiltrating lymphocytes and residual benign glandular epithelial cells were negative for EBERs. The EBERs positive neoplastic cells were certainly EBNA-1 positive.
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Fig 3 Almost all of the carcinoma cells showing EBERs signals(3+～4+),while the surrounding lymphocytes being negative. EBERs-ISH×50
Fig 4 The intensity and distribution of EBERs signals were similar to those of nuclear heterochromatins. EBERs-ISH×100
The LMP-1 positive carcinoma cells could be found in 27 out of 32 LELC specimens (27/32, 84.4%). Positive brown granules were detected cyto-membranously in the neoplastic cells (Fig 5). It should be emphasized that only a small portion of the carcinoma cells (less than 50%) showed LMP-1 positivity. Among these 27 LMP-1 positive cases, 26 were of grade 1～2 (26/27, 96.3%), and only 1 specimen had more than 51% LMP-1 positive cells.
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FIg 5 The parotid gland LELC cells showing cytoplasmic LMP-1 positivity. LMP-1 IHC×100
No ZEBRA positive cell could be found in these 32 LELC specimens observed. A scattering of (1%～25%, 14 cases) or a small number of (26%～50%, 9 cases) EA-D positive cells (Fig 6) were found within the carcinoma nests in 23 out of 32 LELCs (23/32, 71.9%). These EA-D positive cells were often relatively small in size. A few VCA positive neoplastic cells (Fig 7) could be found in 22 specimens (22/32, 68.8%) and several MA (gp350/220) positive carcinoma cells (Fig 8) could merely be found in 4 specimens (4/32, 12.5%).
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FIg 6 The EA-D positive cells were scattered within the LELC nest. EA-D IHC×50
FIg 7 Some of the parotid gland LELC cells showing cytoplasmic VCA positivity. VCA IHC×50
FIg 8 Afew LELC cells of the parotid gland showing MA positivity. MA IHC×100
3 DISCUSSION
As mentioned above, the parotid gland LELC and NPC were both frequently seen in Greenland natives〔1～3〕. Abdulla and Mian reviewed English and Arabic literatures up to 1996 and found the total number of reported cases of salivary gland LELC to be 103 with predominant involvement of the parotid gland〔6〕. Guangzhou locale is a high-incidence area of NPC〔4,5〕. Does parotid gland LELC be frequently seen in Guangzhou locale too? Our results showed that there were 32 LELCs among 125 parotid gland carcinomas previously diagnosed as poorly differentiated or undifferentiated carcinomas during the past ten years. So, the parotid gland LELC and NPC are co-prevalent in Guangzhou locale.
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The median age (47.0 years old), mean age (45.75 years old) and age group peak (30～49 years old)) of the patients with parotid gland LELC were identical to those of NPC patients〔4〕. However, the ratio of male to female in the parotid gland LELCs was different to that of NPCs arising in Guangzhou locale (1.29:1 versus 2.21:1)〔4〕. It seems that the LELC of the parotid gland was not predominantly prevalent in males as the NPC did. The ratio of male to female in 14 salivary gland LELCs reported by Takashi was 1:7〔7〕, further implicating that the female was likely to be suffered from this disease. The average duration of unilateral parotid gland enlargement in these 32 LELCs was 18 months, which was also similar to 16 months reported by Takashi〔7〕.
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The salivary gland LELCs in Greenland were closely associated with EBV infection〔1～3〕. Iezzoni et al. said that the association of EBV with the salivary patients gland LELC is restricted to Asian patients〔8〕. However, the white people could suffer this disease with EBV infection also〔9〕. Does the parotid gland LELCs in Guangzhou locale also closely related with EBV? Our results demonstrated that the majority of carcinoma cells in all of the 32 LELCs showed nuclear EBERs and EBNA-1 signal, which could not be found within the stromal lymphocytes and adjacent non-tumourous glandular cells. It should be emphasized that all of the 32 parotid gland LELCs were solitary and solid in nature, having no symptoms and signs of NPC and therefore not metastatic NPCs. Furthermore, they were not bilateral, multiple and cystic; and therefore might not be induced by HIV as reported by Terry et al〔10〕. The above findings reasonably confirm a fact that there is an existence of a consistent and specific association between EBV and neoplastic cells of the parotid gland LELCs arising in residents of Guangzhou locale. Chan et al. reported 5 Chinese salivary gland LELCs in Hong Kong〔11〕. Their findings coincided with what we found. We assume that the EB virus probably plays a very important role in the pathogenesis of this disease. This conclusion coincided with what Lanier et al. had reported in Greenland〔12〕.In addition, We found that neither EBERs nor EBNA-1 signal could be seen in 8 so-called benign lymphoepithelial lesions (data not shown). After making a survey of surgical salivary gland tumours, Hsu〔13〕 and Tsa〔14〕 suggested that this virus may not play a role in the pathogenesis of most tumors of the major salivary gland except LELCs. So, We also confirm whether EBV exists in so-called lymphoepithelial islands of the parotid glands is a critical marker for distinguishing benignancy and malignancy as reported by Takashi〔7〕.
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In regard to EBV infection and its gene expression, what are the similarities and differences between LELC of the parotid gland and NPC? The EBV harbouring in either the neoplastic cells of parotid gland LELC or of NPC essentially express EBNA-1, EBERs and LMP-1, illustrating that the EBV infection of these two tumours is in the same latency Ⅱ type. However, the LMP-1 expression rate of these 32 parotid gland LELCs reached 84.4%(27/32) which is obviously higher than 30%～65% reported in the literature〔15,16〕. Leung et al. reported 10 cases of the salivary gland LELCs (eight parotid and two submandibular) in Hong Kong Chinese, all of which showed EBERs and LMP-1 positivity〔17〕 .Is there really existence of different LMP-1 expression rates between parotid gland LELC and NPC? How to interpret this difference? Is this discrepancy due to different detection methods used? This issue remains to be further studied.
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The EA-D expression rate was 71.9%(23/32) which is also higher than our unpublished datum of NPC (48.8%, 60/123). Furthermore, according to our comparative observation, the EA-D positive carcinoma cells in the parotid gland LELCs were always more numerous than in NPCs. The VCA and MA expression rates were 68.8%(22/32) and 12.5% (4/32), respectively. Worthy to note is that only a small number or even a few neoplastic cells showed VCA or MA positivity. These findings indicate that the protein products encoded by EBV lytic genes could be expressed in a part of the parotid gland LELC cells, and the EBV harbouring in LELC cells were able to switch over to lytic cycle from latency status. Why the immediate early gene BZLF1 product, ZEBRA was not to be detected? We think that this might be resulting from the short half-life of ZEBRA protein itself. Considering the gradual decreasing of the expression rates from EA-D(71.9%) to VCA (68.8%) and then to MA(12.5%), the authors assume that the lytic cycle of EBV harbouring in neoplastic cells was of abortive form, being not able to complete its production cycle. Martel-Renoir et al.〔18〕 has also confirmed this abortive lytic cycle happened in nasopharyngeal carcinoma. Recently, Zhang et al. found expression of lytic cycle products encoded by EBV within keratinizing nasopharyngeal carcinoma cells〔19〕. This report further supports the viewpoint of Zhangs paper.
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基金项目：国家自然科学基金重点项目(No 39730200-Ⅱ)
作者简介：宗永生，男，71岁，教授，博士生导师
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(Figure seen illustration page 55)
收稿日期：1999-05-07, http://www.100md.com