Epstein-Barr Virus and Jaw Tumors in North Nigeria

JONATHAN SHEPHERD, BDS, MSC, FDSRCS,’ COLIN G. WOODWARD, BSC, PHD,t AND LYNN TURNBULL, BSCS

Nigerian patients with tumors of the jaw were compared with controls in respect of antibodies to the viral capsid antigens of Epstein-Barr Virus (EBV) and of total immunoglobulin levels. Immunoglobulin levels did not differ between patients and controls but increased two weeks postsurgery. Immunoglobulin A (IgA) antibodies to EBV were detected in a small number of patients, and mean titers of IgG antibodies to EBV were lower in the patient group, indicating a lack of association between EBV and tumor formation. An association was noted between the presence of Hepatitis B surface antigen, and depressed antibody titers to EBV in patients with tumors. Of 78 patients studied, 12% were Hepatitis B surface antigen positive.

Cancer 59:1150-1153, 1987.

PSTEIN-BARR VIRUS (EBV) HAS BEEN SHOWN to be as- E sociated with both Burkitt’s lymphoma’ and naso- pharyngeal carcinoma (NPC),2-4 two distinct tumor types affecting the upper end of the alimentary tract. Burkitt’s lymphoma, although essentially a systemic disease, usually begins as a rapidly growing tumor of the mandible or maxilla, destroying bone, and causing loosening of teeth. Systemic involvement is common, but radiographic stud- ies have demonstrated jaw involvement in 95% of pa- t i e n t ~ . ~ EBV specific immunoglobin A (IgA) serum anti- bodies have proved to be an outstanding feature of NPC,’ and EBV nuclear antigen (EBNA), and EBV DNA have been identified in tumor tissue from NPC and Burkitt’s lymphoma patients; but not consistently in tumor tissue from patients with other types of cancer.6 Jaw tumors, similar to ameloblastomas, have been experimentally produced in mice following exposure to polyoma virus’.’; and particles sharing similarities with the paramyrovirus have been reported in a human ameloblastoma? In view of the established association of EBV with two perioral neoplasms, we have examined the association of this virus with a series of other perioral tumors, principally amelo- blastoma. This article describes the results of a serologic investigation of the relationship of antibodies to EBV

From the *Department of Oral Medicine and Oral Surgery, University of Bristol Dental Hospital, Bristol, UK, t h b l i c Health Laboratory, Leeds, UK, and $University Department of Immunology, Leeds General Infir- mary, Leeds, UK.

Requests for reprints: Mr. J. Shepherd, Department of Oral Medicine and Oral Surgery, University of Bristol Dental Hospital, Lower Maudlin Street, Bristol, Avon, UK.

The authors thank Professor E. 0. Adekeye and Dr. V. Karunagaran for allowing investigation of their patients, and Dr. M. H. Hambling and Professor G. Gowland for assistance and advice. They also thank Professor A. B. Rickinson for advice.

Accepted for publication October 24, 1986.

capsid antigen, to various tumors affecting the jaw in a group of North Nigerian patients.

Methods Patients and Specimens

During the first 6 months of 198 1 serum samples were collected from patients with various tumors of the mouth and jaws, who were attending the Maxillo Facial Unit, Ahmadu Bello University Hospital, Kaduna, Nigeria. Serum was collected before the commencement of treat- ment, from 48 patients, and also from a group of 30 con- trol subjects, matched for age, sex, and federal state of abode. Control patients were hospital visitors accompa- nying tumor patients, and patients with fractured man- dibles or maxillae. Serum was also collected from 15 pa- tients two weeks after jaw resection. Serum samples were stored at 4°C until June 198 I , when they were transported to, either the Public Health Laboratory or the University Department of Immunology, Leeds, UK, for examination.

Viral Serology Upon receipt in the laboratory, samples were stored at

-20°C until tested. Hepatitis B surface antigen (HBsAg) was determined by enzymelinked immunoassay (EIA; Auszyme, Abbott Laboratories, Wokingham, UK), and reverse passive hemagglutination (Hepatest, Wellcome Diagnostics, Beckenham, UK), and antibodies to hepatitis B core antigen by EIA (Corzyme, Abbott Laboratories, Wokingham, UK). Hepatitis B ‘e’ antigen (HBeAg), or antibody to HBeAg, was determined by EIA using reagents supplied by the Central Public Health Laboratory, Col- indale, London.

Antibodies to EBV capsid antigens (VCA) were deter- mined by immunofluorescence. Persistently infected cell lines (EB3 or P3HR) were grown in RPMI 1640 medium,

1150

No. 6 EPSTEIN-BARR VIRUS AND JAW TUMORS - Shepherd et al. 1151

supplemented with 10% fetal calf serum (both Flow Lab- oratories, Rockville, MD). Seven days before antigen preparation the cells were transferred to arginine free basal medium Eagle (BME; Wellcome Diagnostics), containing 25% fetal calf serum. To prepare slides for the fluorescence tests, cells were centrifuged and resuspended at 5 X lo6 cells/ml in phosphate buffered saline (PBS). Aliquots (25 pl) were spotted on to polytetra fluoroethylene coated slides (Hendley-Essex, Loughton, UK), allowed to dry at 37°C; fixed for 10 minutes in acetone at room tempera- ture, and stored at -20°C before use. Dilutions of test sera starting at 1:8 were made in PBS, and 25 pl was spotted on to EB3 cells (for IgG and IgA determination) or P3HRl cells (for IgM determination). Slides were in- cubated at 37°C for 2 hours, washed twice with PBS, air- dried, and 25 p1 of fluorescein labeled antihuman IgG, IgA, or IgM added (Wellcome). After a further hour at 37”C, and two washes in PBS, the slides were examined with a Reichart-Jung Microstar (American Optical Co., Slough, UK) fluorescent microscope at a magnification of 200X. The fluorescence of unknown sera was compared with that of a known control, and was graded 0, 1 or 2, according to increasing intensity; titers are expressed as the reciprocal of the highest dilution giving a score of 2. Sera showing EBV IgM positivity, were tested for rheu- matoid factor by a latex agglutination kit (Wellcome Di- agnostics), and only those sera unreactive for rheumatoid factor were classified as positive for subsequent evaluation. A control serum prepared from a pool of sera from pa- tients suffering from nasopharyngeal carcinoma gave the following titers: IgG antibodies to VCA, 1: 1024; IgA an- tibodies to VCA, 1:32; IgM antibodies to VCA, not de- tected. A similar pool from patients suffering from Burk- itt’s lymphoma gave values of: IgG antibodies to VGA, 1512; IgA antibodies to VCA, not detected; IgM anti- bodies to VCA, not detected.

Total levels of IgG, IgM, IgA, and IgE were estimated by the single radial immunodiffusion technique (Mancini

TABLE 1. Histologic Diagnosis and Nos. of Patients

No. of Histoloclic diagnosis uatients

Ameloblastoma Squamous cell carcinoma Pleomorphic adenoma Ossifying fibroma Adenocarcinorna Lymphoma Pindborg tumor Antral carcinoma Myeloma Chondrosarcoma Myxoma

Total

19 9 5 4 2 2 1 2 1 2 1

48

test) at the University Department of Immunology, Leeds General Infirmary.

Histology

Tissue for histologic examination was obtained by bi- opsy preoperatively, or at the time of surgery, placed in 10% formol saline, and despatched to the Department of Dental Science, Royal College of Surgeons of England. Sections were stained with hematoxylin and eosin, and examined by light microscopy.

Analysis of Results

Statistical analysis of results was camed out by means of the Mann-Witney, nonparametric test, except for com- parison of pre and postoperative immunoglobulin levels, for which the paired Student’s t test was used.

Results

Details of histologic diagnosis and numbers of patients can be seen in Table 1, and results of serologic investi- gation of hepatitis B antigens, antibodies to EBV, VCA, and immunoglobulin levels, in Table 2. Patients with tu-

TABLE 2. Seroloeic Values

No. of Condition patients

Ameloblastoma 19 Matched controls 15 All controls 30

Squamous cell carcinoma 9 Matched controls 9 All controls 30

Antibodies to EBV YCA Total immunoglobulins 1U/ml

k G (arithmetic mean values) (Geometric IgA IgM mean titers) (Patients +ve) (Patients +ve) IgG IgA IgM

24 3 0 216 123 264 49 <0.05 0 0 208 NS 117NS 254NS 33 NS 0 0 201 NS 130 NS 324 NS

19 1 0 248 148 226 47 NS 0 0 231 NS 172 NS 254NS 33 NS 0 0 201 <0.05 130 NS 324 NS

All tumors 48 8 (0) 21 4 0 240 150 257 All controls 30 33 <0.01 0 0 201 NS 130 NS 324 NS

Ig: Immunoglobulin; EBV Epstein-Barr virus; VCA: EBV capsid an- tigens; HBeAg: Hepatitis B ‘e’ antigen; HBsAg: Hepatitis B surface an-

tigen; +ve: positive; NS: not significant.

1152 CANCER March 15 1987 VOl. 59

TABLE 3. Relationship Between Preoperative and Postoperative Levels of Immunoglobulin and Antibodies to Emtein-Barr Virus

Preoperative Postoperative

Mean immunoglobulin (IU/ml)t Mean immunoglobulins (IU/ml)t

No. of Mean* VCA Mean* VCA patients IgG IgA IgM IgG IsG IgA IgM IgG

15 242 145 332 16.9 267 173 366 27.3 Significance level between pre and postoperative means NS NS NS P = < 0.05

Ig: Immunoglobin; VCA Epstein-Barr virus capsid antigens; NS: not significant.

mors, and those with ameloblastomas demonstrated sig- nificantly lower mean IgG levels compared with control patients, but no significant differences were detected with regard to total immunoglobulin levels. Of eight HBsAg positive patients, seven were tumor patients and only one a control. Postoperative immunoglobulin levels are shown in Table 3, and the relationship between HBsAg, anti- bodies to EBV, VCA and total immunoglobulins in Table 4. Mean EBV, IgG levels were significantly increased 2 weeks after operation in tumor patients, as were total im- munoglobulin levels, although this was not statistically significant. Tumor patients positive for HBsAg demon- strated a significantly lower level of EBV, IgG with a four- fold difference in mean titer than those negative for HBsAg.

Discussion

Results in this study provided no evidence that EBV plays any part in the etiology of ameloblastoma, or the spectrum of tumors as a whole. Antibody titers consistent with Burkitt's lymphoma were not encountered in any patient in this study. Nevertheless, there was also no ev- idence that ruled out the possibility that EBV contributed in some way, particularly as this was an indirect, serologic study. Numbers of patients with specific tumors other than ameloblastoma were small, the largest group being those with squamous cell carcinoma. Surprisingly, there was evidence that titers of IgG antibody to EBV, VCA were

TABLE 4. Effect of HBsAg Status (Tumor Patients Onlv)

Total immuno- Geometric globulin levels mean titers (arithmetic for antibody means) (IU/ml)

No. of to EBV VCA patients (IgG) IgG IgA IgM

HBsAg +ve 8 9.2 245 132 273 HBsAg -ve 40 36.6 244 138 264 Statistical significance P=<O.Ol NS NS NS

HBsAg: Hepatitis B surface antigens; EBV VCA: Epstein-Barr virus capsid antigens; +ve: positive; -ve: negative; NS: not significant.

* Geometric mean titers. t Arithmetic mean titers.

significantly reduced in both ameloblastoma patients, and the tumor group as a whole, compared with the control group (Table 2). This was in contrast to total immuno- globulin levels, where no significant difference was de- tected between tumor and control patients.

There is evidence that the presence of IgA antibodies to EBV, VCA is particularly important in establishing an etiologic link between EBV and NPC. In this study, only four patients of 8 I showed evidence of this antibody, al- though all four were tumor patients; three with amelo- blastoma and one with squamous carcinoma (Table 2).

EBV antibody titers, both in ameloblastoma patients and the whole tumor group and controls, indicate wide- spread exposure of the Nigerian population to EBV. Pa- tients in this study were almost all seropositive. However, titers were of a low level, and explanations of this include the adverse conditions of storage before testing; 3 1 % of the sera were to some extent hemolyzed upon receipt at the laboratory.

Titers of IgG antibody to EBV, VCA obtained in this study possibly indicate that the tumors, and particularly ameloblastoma, in some way cause a specific decrease in antibody production to EBV. Because EBV infects cells of the immune system, there are many potential com- plexities in the serologic response, due to suppression of various areas of the immune system." Perhaps tumor load has a suppressive effect on antibody synthesis, or on the ability of B cells to synthesise the viral antigens needed to maintain antibody stimulation.

In this study, tumor patients demonstrating HBsAg also had significantly lower levels of EBV, VCA, and IgG, compared with tumor patients who were HBsAg negative. Total immunoglobulins were, however, not significantly different. Explanations of this include immunodeficiency, possibly specifically relating to EBV. It was striking that of nine patients demonstrating HBsAg, eight were tumor patients, and only one a control.

Two weeks postoperatively, total IgG, IgA, and IgM levels were greater than preoperatively in the 15 patients tested at this interval; although this was not statistically significant. However, EBV, VCA, and IgG levels, were

No. 6 EPSTEIN-BARR VIRUS AND JAW TUMORS - Shepherd et a[. 1153

just significantly higher. The raised immunoglobulin levels following surgery may reflect a better dietary status during hopsitalization, or a response to the removal of tumor load. The latter might release the patient from the inherent immunosuppressed state associated with tumors, allowing the immune system to respond in a more normal manner.

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