Lymphoepithelial carcinoma ofthe Epstein-Barrvirus · 1Clin Pathol 1995;48:1022-1027 Lymphoepithelial carcinoma ofthe salivary gland: in situ detection ofEpstein-Barrvirus S YLeung,

1 Clin Pathol 1995;48: 1022-1027

Lymphoepithelial carcinoma of the salivarygland: in situ detection of Epstein-Barr virus

S Y Leung, L P Chung, S T Yuen, C M Ho, M P Wong, S Y Chan

AbstractAim-To examine the role ofEpstein-Barrvirus (EBV) in lymphoepithelial car-cinoma ofthe salivary gland in Hong KongChinese.Methods-Ten cases of lymphoepithelialcarcinoma of the salivary gland (eightparotid and two submandibular) were

examined. In situ hybridisation was usedto localise EBER RNA, immunohisto-chemical methods to detect expression oflatent membrane protein 1 (LMP-1) inEBV positive tumours, and Southern blotanalysis to examine the clonality of EBVin the two cases where frozen tissue wasavailable.Results-None of the cases had a historyof Sjogren's syndrome or histological evi-dence of a benign lymphoepithelial lesion.The IgA antibody titre against EBV viralcapsid antigen was elevated in four cases.

All cases were EBV positive by in situhybridisation, with a strong uniform pos-

itive signal in the epithelial cells, andall cases expressed LMP-1. Southern blotanalysis revealed that the clonal episomalform of the virus was present. Two of thethree female patients in this series alsodeveloped carcinoma of cervix. One ofthese carcinomas had histological featuresof a lymphoepithelioma-like carcinomabut was EBV negative.Conclusions-A consistent association be-tween EBV and lymphoepithelial car-cinoma of the salivary gland was found.The presence of the virus in a clonal epi-somal form, and the expression of LMP-1viral oncoprotein is further evidence ofthe role of EBV in the oncogenesis of thistumour.(JT Clin Pathol 1995;48:1022-1027)

Department ofPathology,The University ofHong Kong,Queen Mary HospitalCompound, HongKongS Y LeungL P ChungS T YuenM P WongS Y Chan

Department ofSurgeryCM Ho

Correspondence to:Dr S Y Leung.Accepted for publication24 April 1995

Keywords: Epstein-Barr virus, lymphoepithelial car-cinoma, salivary gland.

Epstein-Barr virus (EBV) has been dem-onstrated in carcinomas of various organs, in-cluding the nasopharynx,14 salivary gland,5lung,89 thymus,'0 and stomach." 12 Most ofthese EBV associated tumours have char-acteristic histological features, such as syncytialsheets of undifferentiated malignant cells in a

dense lymphoid infiltrate. Tumours with thisbasic histological pattern were commonly re-

ferred to as lymphoepitheliomas. In the salivarygland the terms lymphoepithelial carcinoma,malignant lymphoepithelial lesion or un-

differentiated carcinoma with lymphoid stromaare used preferentially. Tumours with similar

morphological appearances arising from organsother than the nasopharynx and salivary glandare commonly termed lymphoepithelioma-like carcinomas. Recently, EBV has also beendemonstrated in a minority of conventionalgastric adenocarcinomas.""'3 There seems tobe a strong ethnic variation in the extent ofEBV involvement in lymphoepitheliomas ofdifferent organs. For instance, EBV is found inmost lymphoepitheliomas of the nasopharynxirrespective of the ethnic group,"4 but forlymphoepithelial carcinoma of the salivarygland, the association is less consistent. Eski-mos in Greenland have a high incidence oflymphoepitheliomas in both the salivary glandand nasopharynx'5-18 and familial clustering ofthese two tumours is commonly observed.'517An association between this group of tumoursand EBV was suggested by serological andnucleic acid hybridisation studies.5`719 A recentin situ hybridisation study demonstrated thepresence of the virus in all lymphoepithelialcarcinomas of the salivary gland in Eskimos6but the lymphoepithelial carcinomas of the twowhite patients in that series were EBV negative.In another series lymphoepithelial carcinomasof the salivary gland from four white patientswere also EBV negative."The Chinese have a high incidence oflympho-

epithelioma of the nasopharynx, but in con-trast to Eskimos, lymphoepithelial carcinomaof the salivary gland is uncommon. A previousstudy on Hong Kong Chinese with lympho-epithelial carcinoma of the salivary gland sug-gested that EBV was present given the highprevalence of elevated serum IgA titres againstthe EBV capsid antigen.'9 A single case reporton a Chinese patient also reported the presenceof EBV on Southern blotting and immuno-histochemistry.7 Here, we report an associationbetween EBV and lymphoepithelial carcinomaof the salivary gland in Hong Kong Chinese.

MethodsTen Hong Kong Chinese patients (seven menand three women aged from 31 to 72 years(median 41 5 years)) with lymphoepithelial car-cinoma of the salivary gland were included inthe study. Clinical data were retrieved from thehospital records. The cases were reviewed andrepresentative paraffin wax blocks from thetumour and surrounding non-neoplastic glandswere retrieved. A lymphoepithelioma-like car-cinoma arising from the uterine cervix in oneof the patients (case 4) was also included. ADNA fragment containing EBER-1 and EBER-2 (EBV nucleotides 6661-7119) was preparedusing the polymerase chain reaction and cloned

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EBV and lymphoepithelial carcinoma of the salivary gland

into the Bluescript vector, from which anti-sense RNA probes were generated by in vitrotranscription. In situ nucleic acid hybridisationwas performed as described previously.9"4 Di-goxigenin and/or 35S labelled probes were used.Two cases ofnasopharyngeal carcinoma knownto be positive for EBV were used as positivecontrols. All EBV negative cases were probedfor K and k light chains to confirm the integrityof the RNA."4

Fresh tumour tissue from case 8 and thecervical lymph node metastases from case 7were snap frozen in liquid nitrogen and storedat -70°C. High molecular weight DNA was

extracted from the frozen blocks, digested withBamHl, separated on a 0-8% agarose gel, andtransferred onto a Hybond-N membrane. Thecell line B95-8, harbouring the lytic form ofEBV, was used as a positive control. Hy-bridisation studies, using a XhoI fragment ofthe terminal repeat region of EBV genome as

a probe for clonal analysis, were performed.2'The hybridisation conditions were as previouslydescribed.9 For immunohistochemical staining,a cocktail of monoclonal antibodies against theEBV latent membrane protein (LMP-1) (CS1-4, diluted 1 in 50; Dako, Glostrup, Den-mark) was used on paraffin wax sections pre-pared from all of the EBV positive cases.

Standard immunohistochemical methods in-corporating Streptavidin-biotin peroxidasewere applied. The slides were pretreated in a

microwave oven (95°C for nine minutes incitrate buffer (pH 6-0)) to facilitate antigenretrieval. Cell blocks prepared from Raji andB95-8 cell lines, harbouring 50 copies and thelytic form of EBV, respectively, were used as

positive controls. A case of middle ear squam-ous cell carcinoma, known to be EBV negative,was used as negative control. For all the cases,negative controls were also obtained by re-

placing the primary antibody by Tris bufferedsaline.

ResultsCLINICAL FINDINGS

The clinical details of the 10 patients are pre-sented in the table. Eight of the tumours were

located in the parotid gland and two in thesubmandibular gland. There was no history of

Sjogren's syndrome and the possibility of a

metastasis from a primary nasopharyngeal car-

cinoma was excluded by direct examinationand biopsy of the nasopharynx. Four patientshad elevated serum IgA levels against EBVcapsid antigen. The follow up period rangedfrom two to 163 months and most patientswere alive and well at the end of the followup period, except for one who died of livermetastasis nine months after surgery. Case 3developed a solitary lung metastasis 31 monthsafter surgery. A wedge resection of the lunglesion was performed, and he is currently alivewith no residual disease 31 months after thesecond operation. Case 4 developed a localrecurrence one, five and nine years after theinitial surgery; the first two recurrences were

successfully treated by radiotherapy alone; a

surgical resection was performed for the thirdrecurrence. The patient is alive with no residualdisease more than four years after the lastsurgery. This patient also developed a lympho-epithelioma-like carcinoma ofthe uterine cervixone year before her third recurrence; thetumour was successfully treated by radio-therapy and Wertheim hysterectomy. Case 9also had a history of carcinoma of the uterinecervix treated by radiotherapy before de-velopment of the salivary gland lympho-epithelial carcinoma. Unfortunately, thepathological material was not available for re-

view in that case.

PATHOLOGYThe 10 lymphoepithelial carcinomas had sim-ilar morphology with a recurrent pattern ofislands of primitive malignant epithelial cellssurrounded by dense lymphoid infiltrate, givinga low power appearance of a jigsaw puzzlepattern. In some areas the tumour cells werespindle shaped, giving rise to a fasciculargrowth pattern in a desmoplastic stroma. Thecytoplasmic borders were usually indistinct,resulting in a syncytial appearance. The nucleiwere oval and vesicular with one or severaldistinct nucleoli (fig 1A). Permeation oflymph-oid cells into the epithelial islands was prom-inent and follicles with germinal centres were

frequently seen in the surrounding stroma.Definite squamous differentiation was not seen.

Clinical features of the 10 patients with lymphoepithelial carcinoma of the salivary glandCase Sex/age TNM Treatment ofno. (years) Site Staging4 Treatment Recurrence recurrence Outcome

1 F/57 Parotid gland T4N2bMO Radical parotidectomy+ RND +RT Liver None DOD, 9m2 M/41* Submandibular T2NOMO Submandibulectomy+RT None None AW, 63 m

gland3 M/42 Parotid gland T2NOMO Total conservative parotidectomy + RT Lung (31 m postop) Wedge resection AW, 62 m4 F/36t Parotid gland TINOMO Superficial parotidectomy+RT Local (12, 60, 108m RT for 1st and 2nd AW, 163m

postop) recurrence; resection for3rd recurrence

5 M/31* Parotid gland TlNOMO Superficial parotidectomy +RT None None AW, 38m6 M/36 Parotid gland T2NOMO Total conservative parotidectomy+RT None None AW, 10m7 M/32* Submandibular T2NlMO Submandibulectomy + excision of LN+RT None None AW, 3 m

gland8 M/45* Parotid gland T3NlMO Radical parotidectomy + excision of LN + RT None None AW, 2 m9 F/72t Parotid gland NA NA NA NA NA10 M/66 Parotid gland NA NA NA NA NA

RND = radical neck dissection; RT = radiotherapy; LN= lymph node; AW = alive and well; DOD = died of disease, Postop = postoperative; m = months; NA= notavailable.* These patients had elevated IgA antibody titres to EBV capsid antigen.t1These patients also had cervical carcinoma.t AJCC 1988."

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Figure 1 (A) Lymphoepithelial carcinoma of the parotidgland (case 2). Syncytial clusters of malignant cellssurrounded by dense lymphoid infiltrate are evident. Thenuclei are vesicular with central prominent nucleoli.(Haematoxylin and eosin, original magnification x 300.)(B) In situ hybridisation using a digoxigenin labelledEBER probe. The nuclei of the malignant epithelial cellsare strongly positive (original magnification x 120).

The inflammatory infiltrate was confined to a

narrow zone around the tumour, outside ofwhich the salivary gland tissue was normal.There were no benign lymphoepithelial lesions.The carcinoma of the uterine cervix in case

4 was composed of syncytial sheets of poorlydifferentiated malignant cells associated withan intense lymphocytic and plasma cell in-filtrate in the stroma. In some areas permeationof the epithelial island by lymphocytes addedfurther to the typical picture of a lympho-epithelioma (fig 2A). The malignant cells hadscanty pale cytoplasm and elongated dark nuc-

lei with small nucleoli. Glandular formationswere detected in some areas, with cytoplasmicmucin production in the glands. The mor-

phology of this cervical carcinoma was similarto the EBV associated poorly differentiatedadenocarcinoma with an intense lymphoid in-filtrate in the stomach-that is, lympho-epithelioma-like carcinoma of the stomach.

IN SITU HYBRIDISATIONEBV EBER RNA was strongly expressed inthe malignant epithelial cells in all the 10lymphoepithelial carcinomas of the parotid andsubmandibular glands (fig 1B) while scatteredEBV positive reactive lymphoid cells were

noted in three cases (fig 3). All normal salivaryglandular epithelium was negative. Thelymphoepithelioma-like carcinoma of the uter-

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Figure 2 (A) Lymphoepithelioma-like carcinoma of thecervix (case 4). Syncytial sheets ofpoorly differentiatedmalignant cells associated with dense lymphoid infiltratewhich percolate the epithelial islands are evident.(Haematoxylin and eosin, original magnification x 198.)(B) In situ hybridisation using a 35S labelled EBERprobe. The malignant cells were EBV RNA negative(original magnification x 198).

ine cervix was also EBV negative (fig 2B).There were scattered K or X light chain positiveplasma cells, confirming the integrity of theRNA in this case.

EBV VIRAL ONCOPROTEIN EXPRESSIONAll cases of salivary gland lymphoepithelial car-cinoma expressed LMP-1 in the malignant epi-thelial cells with both membranous andgranular cytoplasmic staining patterns. Therewas variable staining intensity in differentmalignant cells within a given tumour and thenumber of tumour cells which were positivefor LMP-1 ranged from 50% to nearly 100%in different tumours (figs 4 and 5).

SOUTHERN BLOT HYBRIDISATIONA single band of, respectively, 12-5 and 9-4kilobases was detected in cases 7 and 8, in-dicating that the virus was present in clonalepisomal form in the malignant epithelial cells(fig 6). Smaller fragments, indicative of multi-clonality or the linear form of the virus (as seenin the B95-8 cell line), were not detected.

DiscussionIn common with Greenland Eskimos all lympho-epithelial carcinomas of the salivary gland fromHong Kong Chinese examined so far were EBV



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Figure 3 In situ hyboidisation using a 'S 1eprobe (case 1). Scattered lymphocytes showin,signals are present. The non-neoplastic saliva~acini are negative (original magnification x,

positive. Eskimos have high incidernasopharyngeal carcinoma (12.7 fc9(2 for women) and anaplasticcaOthe salivary gland (3i4 for men awomen),6 both of which are linked iwith EBV. In Hong Kong Ghinesehigh incidence of nasopharyngeal(28 -5I100 000 men and 11-2/1100 0(and yet the incidence of malignanof the salivary gland is low (0t7 fo0-5 for women).2 Moreover, lymplcarcinoma constitutes only a mincsalivary gland malignant neoplasipopulation. In contrast, whites haycidence of both tumours and the fgland lymphoepithelial carcinomaspatients studied so far were all EBVThese findings suggest that there isinteraction between genetic faivironmental factors and the susceepithelial cells of individual organs iin the oncogenic process.

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Figure 5 All tumour cells are homogeneously positive forLMP-1 (case 10) (x 240).

)r men and After primary infection, EBV can remainhoepithelial latent in B lymphocytes24 (scattered reactive)rity of the lymphocytes expressing EBV EBER RNA in ams in our few of the cases presented here). Latent EBVre a low in- is polyclonal and is present in the B lym-.ew salivary phocytes in episomal form. As there are farfrom white more EBV positive malignant cells comparednegative.,O with lymphocytes, the virus in the lymphocytesa complex may not have been picked up on clonal analysis.

ctors, en- EBV is present in the clonal episomal formptibility of in most EBV associated malignant neoplasms,to the virus examples ofwhich include Burkitt's lymphoma,

Hodgkin's lymphoma and lymphoepitheliomasarising in various organs."' 252 Our results areconsistent with those of previous studies.52'This suggests that infection with EBV precedesthe oncogenic process, and that the virus mayplay a role in malignant progression. EBV canimmortalise B lymphocytes through a complexprocess requiring several viral proteins in-

- cluding EBV nuclear antigens (EBNAs) andLMPs. EBNA-2 is important in the trans-formation of B lymphocytes24 and LMP-1 hasbeen reported to have transforming propertiesin rodent fibroblastoid cell lines.27 Expressionof LMP-1 in B lymphocytes up-regulates bcl-2expression, thus preventing apoptosis.28 Inhuman keratinocytes LMP-1 deregulates epi-

:>*t thelial growth and inhibits differentiation,2930with the epithelial cells showing the char-acteristics of transformation including loss ofcontact inhibition, spindling, a tendency to

;,>S proliferate into multilayer clusters, and de-creased cytokeratin expression. Moreover,

e"¢4 there is an impaired cellular response to differ-- '.wfi entiation signals. Thus, a role for LMP-1 in a

; multistep pathogenetic process of lympho-epithelioma formation may be inferred fromthe undifferentiated morphology ofthe tumour.

g for LMP-I Previous immunohistochemical studies inr

both f nasopharyngeal carcinomas found that LMP-6) (x 240). 1 was expressed in 22 to 78% of cases.3132

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Leunig, Chung, Ylue7o, Ho, Wonig, Chani

Figure 6 Southern blot analysis. All DNAsamples were digested with BamHlrestriction endonuclease, separated on a

0-8% agarose gel, and hybridised withXhoI EBVfragment. C = control (B95-8cell line); 8 = case 8 yielding a single 9 4kilobase (kb) band; 7 = case 7yieldintg a

single 12 5 kb band. These single bandsindicate that the clonal episomal form of thevirus is present.

LMP-1 was also expressed in more than 90%of cases of EBV associated Hodgkin's disease33and in a small percentage of EBV associatedsinonasal carcinomas,34 but not in Burkitt'slymphoma.3' Few data are available concerningthe expression of LMP-1 in other EBV as-

sociated carcinomas. In the present seriesLMP- 1 was expressed in all 10 lympho-epithelial carcinomas of the salivary gland,with a heterogeneous level of expression insome cases. Heterogeneous LMP-1 expressionwas also observed in a lymphoblastoid cellline,36 37 expression of which could be induced byTPA (1 2-0-tetradecanoyl-phorbol- 1 3-acetate)and butyrate or by the addition of fetal calfserum. However, why the expression is more

constant in lymphoepithelial carcinomas of thesalivary gland but not in nasopharyngeal car-

cinoma is not known. It is possible that thereis enhanced expression of the protein in thelocal environment of the salivary gland, whichmay be important for maintaining the growthand phenotype of the tumour.

It is interesting that two of our three femalepatients also developed carcinoma of uterinecervix. The cervical carcinoma of one of thepatients (case 4) showed lymphoepithelioma-like carcinoma features, including a poorlydifferentiated morphology with only rare gland-ular formation and a dense lymphoid infiltratein the stroma. The simultaneous presence oftwo tumours of lymphoepithelioma type in thesame patient, one EBV positive and the otherEBV negative, is intriguing. Although cervicalepithelium has been demonstrated as one ofthe potential sites of EBV infection,38 andlymphoepithelioma-like carcinoma has been re-

ported in the cervix, the latter has not beenshown to harbour EBV.39 It is unfortunate that

the cervical carcinoma in case 9 was not avail-able for study, and we do not know whetherthe morphology was similar to that of case 4.Taking into account the rarity of lympho-epithelial carcinoma of the salivary gland inour population, the association between it andcarcinoma of the uterine cervix in two of thecases in our series is unusual and may suggestthe existence of additional unknown factor(s)in the aetiology of lymphoepithelioma-like car-cinoma.

In conclusion, we have shown that lympho-epithelial carcinomas of the salivary gland inHong Kong Chinese uniformly harbour latentEBV. The presence of the virus in clonal epi-somal form and the expression of LMP-1 inthe infected malignant epithelial cells furtherunderlines the importance of EBV in the onco-genetic process.

We thank Dr Elaine Gwi, Pathology Institute, Kwong WahHospital, Hong Kong, for providing the pathological specimenfrom case 5. We also thank Dr R J Collins, Department ofPathology, Queen Mary Hospital, Hong Kong, for reading themanuscript.

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Lymphoepithelial carcinoma ofthe Epstein-Barrvirus · 1Clin Pathol 1995;48:1022-1027 Lymphoepithelial carcinoma ofthe salivary gland: in situ detection ofEpstein-Barrvirus S YLeung,