ANTIBODY TO EPSTEIN-BARR VIRUS IN PATIENTS WITH CARCINOMA OF THE NASOPHARYNX

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1 Keio J. Med. 26: 79-90, 1977 ANTIBODY TO EPSTEIN-BARR VIRUS IN PATIENTS WITH CARCINOMA OF THE NASOPHARYNX YUKIO INUYAMA*, KAZUYUKI ASAOKA*, YASUO NAKAJIMA*, MASATOSHI HORIUCHI*, KEI TAKASAKI** and RAISUKE OZU** *Department of Oto-rhino -laryngology, School of Medicine, Keio University, Tokyo, Japan ** Department of Oto-rhino-laryngology, Saiseikai Central Hospital of Tokyo, Tokyo, Japan (Received for publication May 4, 1977) ABSTRACT The antibody against viral capsid antigen (VCA) of Epstein-Barr virus (EB virus) has been studied in 59 patients with malignant tumor of the nasopharynx, in 117 patients with tumor of the other sites of the head and neck, and in 263 patients with nonmalignant disease. The anti-vca titer was determined by Kawamura's modification of Henle's indirect immunofluorescence technic. The geometric mean titer was 1:794 and the antibody positive rate (? 1:640) 82% in patients with nasopharyngeal tumor, while being 1:190 and 37% respectively in those with tumor of the other sites of the head and neck. In 263 patients with nonmalignancy serving as controls, the values were respectively 1:120 and 20%. The anti-vca titer in patients with naso pharyngeal carcinoma was significantly higher than that in controls by ridit analysis. The anti-vca titer was repeatedly determined in patients with naso pharyngeal carcinoma at different points of the clinical course. As the result of the present study, it was shown that in cases of recur rence or metastasis there was a remarkable elevation of the anti-vca titer and in cases of short and long term remission there was a decrease in the titer. We concluded that the anti-vca titers might serve as a useful clinical parameter for prognosis of the nasopharyngeal carcinoma. INTRODUCTION Many studies on oncogenic virus have been made in experimental animals since the beginning of this century. It is not an overstatement, however that 79

2 80 Yukio Inuyama et al virological studies on human cancer make up a history of successive failures. Epstein and Barr1 first discovered herpes-type virus (EB virus) in cultured cells of Burkitt's lymphoma in In 1966, Old et al.2 found the elevated anti EBV antibody titers in patients with nasopharyngeal carcinoma. Recently, other investigators3,4 have also reported on the high anti-ebv antibody titers in pa itents with Burkitt's lymphoma, nasopharyngeal carcinoma or infectious mono nucleosis. Despite of the facts mentioned above, it has not been made clear whether EB virus is the carcinogenic virus or only the passenger virus. The antibody against viral capsid antigen (VCA) of EB virus has been determined in 59 patients with malignant tumor of the nasopharynx seen in our department from September 1972 through March MATERIALS AND METHODS Serum was collected from 59 patients with malignant tumor of the naso pharynx. Among 59 cases, 51 carcinomas and 8 malignant lymphomas were found. (Table 1) All patients were treated with Linac. Serum was also obtained from 117 patients with malignant tumor of the other sites in the head and neck, and 263 control patients with non-malignant disease. The anti-vca titer was determined by Kawamura's modification4 of Henle's indirect immunofluorescence technic. P3HR-1 cells derived from P3 (Jijoye) strain of Burkitt's lymphoma cultured cells were employed as the target cell. In this study, the titer of 1:640 or higher was regarded as positive. Histological Table 1 classification RESULTS The anti-vca antibody titer in patients with malignant tumor of the h ead

3 Nasopharyngeal carcinoma and Epstein-Barr virus 81 and neck is shown in Table 2. The anti-vca titer in patients with naso pharyngeal carcinoma was generally high. The geometric mean titer was 1:794 and the positivity rate was 82% in them. (Fig. 1) On the contrary, the anti-vca titer in patients with malignant tumor of some other site in the head and neck was lower than that in cases of naso pharyngeal carcinoma. The geometric mean titer was 1:190 and the positivity rate was 37%. The anti-vca titer in control patients with nonmalignant disease is shown in Table 3. The anti-vca titer of the controls was also low. The geometric mean titer was 1:120 and the positive rate was 20% in them. The anti-vca titer in patients with nasopharyngeal carcinoma was significantly higher than that in the controls by ridit analysis. (Fig. 2, 90) The difference in titer between non-nasopharyngeal tumors of the head and neck and non-malignant diseases was also significant by ridit analysis. The distribution of anti-vca titer positive rate among patients with naso pharyngeal carcinoma, patients with non-nasopharyngeal tumor and patients with non-malignant disease is shown in Fig. 4. The anti-vca titer at the time when Table 2 Anti-VCA antibody titers in patients with malignant tumors of the head and neck

4 82 Yukio Inuyama et al Fig. 1 Anti-EB virus-positive rate in sera from patients with nasopharyngeal carcinoma, tumor of the other sites or nonmalignant disease. Table 3 Anti-VCA antibody titers in patients with nonmalignant diseases

5 Nasopharyngeal carcinoma and Epstein-Barr virus 83 Fig. 2 Ridit analysis of anti-vca titers in patients with the head and neck tumor. the positivity rate reached its peak was 1:2560 or higher in nasopharyngeal carcinoma and 1:160 in other diseases. Table 4 shows the anti-vca titers determined in patients with nasopharyn geal carcinoma at different points of the clinical course. The geometric mean titer before treatment was 1 :662 and the positivity rate was 82% in these pa tients. The anti-vca titer after radiotherapy seemed higher than that before treatment in them. Patients after radiotherapy had the geometric mean titer of

6 84 Yukio Inuyama et al Fig. 3 Ridit analysis of anti-vca titers in patients with nonmalignant disease. Table 4 Clinical course and anti-vca antibody titer

7 Nasopharyngeal carcinoma and Epstein-Barr virus 85 Fig. 4 Distribution of anti-vca titers among patients with nasopharyngeal car cinoma, patients with tumor of the other sites of the head neck and patients with nonmalignant disease. 1:1278 and the positivity rate of 90%. The anti-vca titer of patients in short term remission (less than three years) seemed a little lower than that after treatment. Patients in short term remission had the geometric mean titer of 1:765 and the positivity rate of 82%. When recurrence or metastasis of malig nancy occurred, the anti-vca titer was elevated to 1:1808 in terms of the geo metric mean and the positivity rate to 100%. The high anti-vca titer was also presented by patients in the last stage. On the contrary, the anti-vca titer of patients in long term remission (more than three years) decreased to 1:451 in terms of the geometric mean and the positivity rate to 62%. DISCUSSION The elevation of anti-ebv titer in patients with nasopharyngeal carcinoma had been referred to in the previous reports (Table 5) It was also shown in

8 86 Yukio Inuyama et al Table 5 Anti-VCA antibody titers in patients with nasopharyngeal carcinoma the present study that the anti-vca titer in patients with nasopharyngeal car cinoma was significantly higher than in patients with non-nasopharyngeal tumor or nonmalignant disease. As to the role of EB virus, there are two possibilities. The first possibility is that EB virus might be carcinogenic, and the second one is that EB virus might be only a passenger virus. It is the task for the future to determine which of the hypotheses is right. Lynn et al.9 reported that the titer was not significantly correlated with the size of the primary tumor, while there statistically was a high degree of cor relation between the titer and metastasis to the neck. As to the relationship between the histopathologic findings and the anti-vca titer, Sako et al.x4 reported that there was no definite relation between the two. According to Sawaki's report,r3 however, the antibody titer was positive in 80% of patients with lymphoepithelioma, and in 33% (well-differentiated squamous cell carcinoma) to 47% (undifferentiated carcinoma, transitional cell carcinoma) of patients with some other type of tumor. Henderson" reported that, while elevation of the anti-ebv titer was not observed in nasopharyngeal tumors other than carcinoma, it was noted in epider moid carcinoma of the hypopharynx or the nasal cavity. According to our results, however, the geometric mean titer was 1:163 in 53 cases of maxillary carcinoma,

9 Nasopharyngeal carcinoma and Epstein-Barr virus 87 1:214 in 14 cases of laryngeal carcinoma and 1:219 in 13 cases of lingual car cinoma. These titers were much lower than that in nasopharyngeal carcinoma. Concerning the relationship between the anti-vca titer and the clinical course, although the titer was elevated after radiotherapy in our trial, Lynn et al.10 reported that the geometric mean value for the anti-vca titer was rela tively low after radiotherapy. In cases of Burkitt's lymphoma, Einhorn et al.1u observed a rise in anti-vca titer after radiotherapy and suggested that irra diation, might activate VCA synthesis in Burkitt's lymphoma cells. In our opin ion, elevation of the anti-vca titer after irradiation in patients with naso pharyngeal carcinoma can be interpreted in two ways. The first possibility is that the antigen stimulation due to destruction of cancer cells after radiotherapy causes the elevation of the titer. According to Wara's report,'6 eleven patients with nasopharyngeal carcinoma treated by standard radiation therapy were found to have depressed cell mediated immunity. In addition, all these patients had depressed total lymphocytes count and eight of eleven patients had depressed T-cell rosettes after radiotherapy. Tuberculin reactivity skin test was carried out in our 33 patients with malig nant tumor of the nasopharynx. Tuberculin reactivity had a tendency to decrease after radiotherapy. These findings suggest the second possibility that B-cells might produce more antibodies as a sequel of relief from the suppression of T-cells. The relatively low anti-vca titer of our patients in short and long term remission was compatible with the results of Lynn, Henderson and Goldman, while Sako reported that out of eleven patients who had previously been treated and in whom no active disease was clinically present in a follow-up period rang ing from three to eleven years, seven still had elevated titers. When recurrence of malignancy occurrs, the anti-vca titer is generally elevated. Lynn et al. reported that the anti-vca titer rose to 1:424 in terms of the geometric mean and the positivity rate 56.8% in 37 patients with recurrences, while Sako et al. stated that out of twelve patients with active disease, ten had elevated titers. It is considered that persistence of VCA, that of cancer cells themselves and depression of the cell mediated immunity might affect elevation of the anti-vca titer. From the results mentioned above, it was clarified that in cases of recur rence or metastasis there was a remarkable elevation of the anti-vca titer and in cases of short and long term remission there was a decrease in the titer.

10 88 Yukio Inuyama et al SUMMARY AND CONCLUSIONS 1. The anti-vca titer in patients with nasopharyngeal carcinoma was significantly higher than that in controls by ridit analysis. 2. The anti-vca titer in patients after radiotherapy tended to be higher than that before treatment. 3. Patients with recurrence or metastasis and in the last stage had the high anti-vca titer. 4. Patients in short and long term remission had the relatively low anti VCA titer. We concluded that the anti-vca titer might be applied clinically as a useful parameter for prognosis of the nasopharyngeal carcinoma. ACKNOWLEDGEMENT The authors are greatly indeted to Drs. A. Kawamura, Jr., T. Hirayama, T. Urano and H. Hara for their invaluable instruction and advices, and also to Miss S. Taniguchi for her technical assistance. This study was partly supported by research grant from the Ministry of Education. REFERENCES 1. Epstein, M. A., Achong, B. G. and Barr, Y. M.: Virus particles in cultured lym phoblasts lymphoma. Lancet 1: , Old, L. J., Boyse, E. A., Oettgen, H. F., de Harven, E., Geering, G., Williamson, B. and Clifford, P.: Precipitating antibody in human serum to an antigen pres ent in cultured Burkitt's lymphoma cells. Proc. Nat. Acad. Sci., 56: , Henle, G., Henle, W. and Diehl, V.: Relation of Burkitt's tumor-associated Herpes type virus to infections mononucleosis. Proc. Nat. Acad. Sci. 59: , Kawamura, A. Jr., Takada, M., Gotoh, A., Hamajima, K., Sanpe, T., Murata, M., Ito, Y., Takahashi, T., Yoshida, T., Hirayama, T., Tu, S., Liu, C., Yang, C. and Wang, C.: Seroepidemiological studies on nasopharyngeal carcinoma by fluo rescent antibody techniques with cultured Burkitt's lymphoma cells. Gann 61: 55-71, Henle, W., Henle, G., Ho, H.C., Burtin, P., Cachin, Y., Clifford, P., de Schryver, A., de The G., Diehl, V. and Klein, G.: Antibodies to Epstein-Barrvirus in naso pharyngeal carcinoma, other head and neck neoplasms, and control groups. J. Nat. Cancer Inst. 44: , Hosokawa, T.: Anti-EB virus titer in sera of patients with rhinological carcinoma. (in Japanese). Jap. Jour. Otol. 73: , Goldman, J. M., Goodman, M. L. and Miller, D.: Antibody to Epstein-Barr virus in American patients with carcinoma of the nasopharynx. J.A.M.A. 216: , Awataguchi, S. et al,: Studies on the relationship between nasopharyngeal cancer and specific anti-v and anti-n induced by EB virus. (in Japanese). Otologia Tokyo 45: , 1973

11 Nasopharyngeal carcinoma and Epstein-Barr virus Lynn, T. C., Tu, S. M., Hirayama, T. and Kawamura, A. Jr.: Nasopharyngeal carcinoma and Epstein-Barr virus. I. Factors related to the anti-vca antibody. Japan J. Exp. Med. 43: Lynn, T. C., Tu, S. M., Hirayama, T. and Kawamura, A. Jr.: Nasopharyngeal carcinoma and Epstein-Barr virus. II Clinical course and the anti-vca antibody. Japan J. Exp. Med. 43: Henderson, B. E., Louie, E., Bogdanoff, E., Henle, W., Alena, B. and Henle, G.: Antibodies to herpes group viruses in patients with nasopharyngeal and other head and neck cancers. Cancer Research 34: , Sakai, S.: Maxillary cancer. (in Japanese). Kaneharashuppan, Sawaki, S., Sugano, H., Hirayama, T., Kawamura, A. Jr. nd Tachibana, T.: Histopathological and immunological studies of nasopharyngel carcinoma. Chinese J. Microbiology. 8: 73-81, Sako, K., Minowada, J. and Marchetta, F. C.: Epstein-Barr virus antibodies in patients with carcinoma of the nasopharynx and carcinoma of other sites in the head and neck. Am, J. Surg. 130: , Einhorn, N., Henle, G., Henle, W., Klein, G. and Clifford, P.: Effect of local radiotherapy on the antibody levels against EBV-induced early and capsid anti gens (EA and VCA) in patients with certain malignant tumors. Int. J. Cancer. 9 : , Wara, W. M., Phillips, T. L., Wara, D. W., Ammann, A. J. and Smith, V.: Im munosuppression following radiation therapy for carcinoma of the nasopharynx. Am. J. Roent. 123: , 1975