In Vivo and in Vitro Studies of Immunotherapy of Nasopharyngeal Carcinoma with Transfer Factor I

[CANCER RESEARCH 36, 720-723, February 1976]

Gerald J. Goldenberg 2 and Lorne J. Brandes :~

Department of Medicine, University of Manitoba, and The Manitoba Institute of Cell Biology, Winnipeg, Manitoba R3E OV9, Canada

Summary kines from those cells might have the potential to program and/or recruit uncommitted lymphocytes to react against Epstein-Barr virus, the apparent cause of infectious mono- EBV and/or viral antigens. This report describes a patient nucleosis, may also be an etiological agent in nasopharyn- with NPC refractory to conventional therapy, who was geal carcinoma and Burkitt's lymphoma. Lymphocytes from treated with TF derived from normal adult donors with a normal individuals with anti-Epstein-Barr virus antibody ac- past history of IM. The findings are discussed in relationship tivity may be sensitized to Epstein-Barr virus and contain to in vitro studies in which transfer of CMI against NPC was transfer factor with the potential to program and/or recruit demonstrated using TF from donors with EBV antibody other lymphocytes to react against the virus and/or viral activity (2). antigens. A patient with nasopharyngeal carcinoma refractory to conventional therapy was treated with transfer factor obtained from normal, young adults with a previous history Case Report of infectious mononucleosis. Following immunotherapy, apparent slowing of tumor growth was observed, which was A 31-year-old Chin, ; male from Hong Kong presented in associated with intense lymphocytic infiltration of the tumor November 1970 with right ear deafness and an enlarged and reconstitution of delayed cutaneous hypersensitivity lymph node in the right upper neck. On physical examina- reactions to microbial recall antigens. A double-blind ran- tion, a soft tissue mass was noted in the nasopharynx. No domized clinical trial has been initiated to determine cranial nerve involvement was noted. A skull film showed a whether transfer factor immunotherapy is a useful adjunct soft tissue mass in the right nasopharynx. On December 1, to radiotherapy in the primary treatment of patients with 1970, a biopsy of the nasopharyngeal tumor revealed infil- nasopharyngeal carcinoma. If successful, a similar trial trating epidermoid carcinoma Grade 3 (Fig. 1A). The patient might be considered for African patients with Burkitt's lym- was treated with radiotherapy (6000 rads) to the nasophar- phoma. ynx and upper cervical region (Chart 1), resulting in hearing improvement and complete tumor regression. In July 1971, hearing loss reappeared, along with a naso- Introduction pharyngeal mass. On August 31, 1971, repeat biopsy showed infiltrating epidermoid carcinoma Grade 3 similar in Recently, TF 4 has been applied to the immunotherapy of appearance to that of the original tumor (Fig. 1B). Skin tests patients with malignant disease, with some reports of tumor of delayed cutaneous hypersensitivity demonstrated a nega- regression (1,9, 13, 15, 19, 21,25, 26). A major problem has tive reaction to PPD (5 units) and Trichophyton rubrum and been the identification of suitable donors of tumor-specific a weekly positive reaction (5 to 10 mm) to Candida albicans TF. The approach used here, and described previously (9), and mumps (Chart 1). The patient had been PPD-positive in is based on the premise that individuals exposed to IM 1960 on entrance to University and also on a repeat test in might serve as ideal tumor-specific TF donors in patients 1964. with either NPC or BL. The following assumptions are On September 9, 1971, combination chemotherapy was made: EBV is an etiological factor in all 3 diseases, IM, NPC, instituted, following which rapid tumor growth was ob- and BL; lymphocytes from patients with a past history of IM served. Antibody against VCA was present at a titer of 1/512 may be sensitized to EBV, and finally, TF or other lympho- (Chart 1). On October 5, 1971, as much tumor as possible was excised from the nasopharynx. The histological appearance of the tumor was unchanged; lymphocytic infiltration Presented at the symposium "Immunological Control of Virus-associated was not evident. Tumors in Man: Prospects and Problems," April 7 to 9, 1975, Bethesda, Md. On October 7, 1971, immunotherapy with crude buffy This work was supported by a grant from the National Cancer Institute of Canada. coat extract from patients with a history of infectious mono- 2 Presenter. Clinical Research Associate of the National Cancer Institute of nucleosis was initiated. Approximately 5 to 10 ml of crude Canada. extract were injected i.m. weekly. Biopsy of the nasophar- :~ Centennial Fellow of the Medical Research Council of Canada. 4 The abbreviations used are: TF, transfer factor; IM, infectious mononu- ynx on November 3, 1971, showed persistance of carci- cleosis; NPC, nasopharyngeal carcinoma; BL, Burkitt's lymphoma; EBV, noma; however, a striking round-cell infiltration of the tu- Epstein-Barr virus; CMI, cell-mediated immunity; PPD, purified-protein deriv- ative; VCA, viral capsid antigen; PLMI, peripheral leukocyte migration inhibi- mor was noted (Fig. 1C). The anti-VCA titer remained ele- tion. vated at 1/512. Tests of delayed hypersensitivity now

720 CANCER RESEARCH VOL. 36

ANTI-512- I o~-~ ~~.~fL..~_Fz/~ mained stable at 1/256 to 1/512 (Chart 1). In view of the VCA 2~.J ", obvious tumor relapse, chemotherapy with bleomycin was started July 8, and a total dose of 285 units was adminis- tered up to August 18, 1972. Immunotherapy with dialyzable

D.H. SKIN PPDI R,,~ ..... 1-t t 9 , TF was continued once weekly. The clinical status remained TESTS I .... -. Cond,do + { i relatively stable. The patient received further radiotherapy Moml)~ r Jr- , -" I and once-weekly TF in Hong Kong in August 1972. His LYMPH INFILTN ~ 9 9 9 condition was stable temporarily but then began to deterio- OF TUMOUR ATION CHEMOTHERAPY ,MMUNOTHERAPY rate; TF was discontinued and he died on March 12, 1974. At autopsy, no residual tumor was detected in the nasophar- ~ ~ 7 .ALYZ, T F 'oJ J I F 'MIA'MI J' J IAIS IO'NI DIJ" F'MI A~MI J' J'A' ynx, but there was tumor invasion of the meninges, poste- 1971 ~ 1972 rior cerebral fossa, and right cerebellar hemisphere. No Chart 1-i-A-time course of antibody levels to VCA, tests of delayed cuta- distant metastases were noted. neous hypersensitivity (DH) to PPD, T. rubrum, C. albicans, and mumps, and the presence or absence of lymphocytic infiltration of the tumor (Lymph Infiltn. of Tumour) in relation to the type and duration of therapy. Antibody to Materials and Methods VCA was determined by the indirect immunofluorescent antibody technique, using acetone-fixed cells of the HR1-K subline of BL as described by Henle et al. (11). BLEO., bleomycin; DIALYZ., dialyzable. Selection of TF donors. Normal, young adults, who had a history of IM supported by positive serology (Paul Bunnell showed a positive response (>10 mm) to PPD and T. rub- or Mono spot test) 3 months to 6 years previously, were rum, as well as Candida and mumps (Chart 1). On Decem- recruited from the nursing and medical staff to serve as TF ber 17, 1971, the anti-VCA antibody titer had dropped to donors. All individuals with a past history of hepatitis, ma- 1/64. Biopsy of the tumor in January 1972 showed persist- laria, or syphilis were excluded; donors were screened for ence of the round-cell infiltration. The patient reported im- hepatitis-associated antigen, and positive reactors were re- proved hearing acuity in the right ear. jected. On February 16, blood appeared in the right ear canal, a Preparation of TF. Crude buffy coat extract and dialyza- mass was noted in the nasopharynx, and immunotherapy ble TF were prepared as described previously (2). This tech- with crude buffy coat extract was discontinued. A biopsy of nique of preparing TF is more rapid than the prolonged the tumor showed epidermoid carcinoma Grade 3 similar to dialysis method of Lawrence (16-18), and the reconstitution the original biopsy; there was little or no round-cell infiltra- of delayed cutaneous hypersensitivity to PPD and Trichophy- tion (Fig. 1D). An elevation of anti-VCA antibody titer to ton in this case may attest to the biological activity of the 1/256 was noted. Skin tests demonstrated positive re- preparation. Dialyzable TF, unlike the crude buffy coat ex- sponses to Candida and mumps but showed loss of reactiv- tract, produced little pain on i.m. injection. ity to PPD and T. rubrum (Chart 1). Crude buffy coat extract from IM donors might be ex- Discussion pected to contain viable EBV and/or viral antigens that could elicit malignant transformation of other recipient cells Evidence exists that EBV may be an etiological agent in and/or induce immune paralysis by virtue of antigen excess. NPC (5, 7, 11, 20, 23). Chinese and East African patients For this reason, subsequent immunotherapy was performed with NPC had high antibody titers to VCA relative to control with dialyzable TF, which eliminates molecules greater than patients with other head and neck neoplasms (3, 12). Anti- 10,000 in molecular weight (16-18), thus excluding whole bodies to membrane-associated antigen and early antigen virus and, possibly, viral antigen. were also preferentially elevated in patients with NPC, com- On March 6, 1972, immunotherapy with dialyzable TF was pared with controls with lymphoproliferative neoplasms, initiated in which approximately 109 lymphocyte equivalents carcinomas, or sarcomas (3, 4). The association of elevated in 4 to 6 ml were given as a single i.m. weekly injection. On titers with advanced disease does not differentiate between March 18, 1972, positive cutaneous reactions to PPD and T. a passenger virus and a causal agent since, in either case, rubrum had been restored (Chart 1). On March 27, 1972, viral proliferation with antibody formation could accompany nasopharyngeal biopsy showed epidermoid carcinoma with tumor growth (10, 12). pronounced round-cell infiltration (Fig. 1E). Examination of Using an anticomplementary fluorescent staining method the nasopharynx on May 9, 1972, showed no obvious tumor to examine biopsy sections of NPC, zur Hausen (28) demon- mass. However, biopsy disclosed residual epidermoid carci- strated a clearly positive fluorescence against the epithelial noma once again with heavy round-cell infiltration (Fig. 1F). cell component of NPC tissue, using EBV antibody-positive The patient remained well until the end of June 1972, sera from normal donors. The fluorescence depended on when he became symptomatic with evidence of involvement the concurrent presence in the test sera of antibodies of the right cranial nerves V, IX, X, XI, and Xll. An X-ray against membrane-associated antigen. In addition, in hy- demonstrated a large soft tissue mass on the right side of bridization studies, zur Hausen and Schulte-Holthausen the nasopharynx and destruction of the base of the skull. A (29) detected evidence for the presence of the EBV genome biopsy showed infiltrating epidermoid carcinoma with little in the epithelial cell component. plasma cell or lymphocytic reaction. Skin tests revealed a Gerber and Lucas (8), using thymidine incorporation as loss of reactivity to PPD and mumps but positive responses an index of CMI against EBV demonstrated reactivity only in to T. rubrurn and Candida. The anti-VCA antibody titer re- EBV-seropositive subjects. Fimmel et al. (6), using PLMI,

FEBRUARY 1976 721

also demonstrated CMI to EBV in normal individuals with reviewing the numerous biopsy specimens; Catherine Anderson and Dr. J. M. Bowman for their assistance in preparation of buffy coat extract; Dr. J. C. anti-VCA antibody activity. Evidence that the reactivity was Wilt, Rosanna Peeling, Dr. A. M. Wallbank, and Tom Sinclair for determina- directed against the EBV genome, rather than membrane- tion of anti-VCA antibody titers; Dr. L. G. Israels for his interest, encourage- associated antigen on BL cell lines, was that only cell ex- ment, and assistance in organizing the prospective trial of TF immunotherapy of NPC; and Dorothy Faulkner for typing the manuscript. tracts from EBV-producer cell lines but not that from the nonproducing Raji cell line provided a suitable source of antigen. References We reported previously (2) that the PLMI test has been adapted both to measure CMI against an antigen extract of 1. Brandes, L. J., Galton, D. A. G., and Wiltshaw, E. New Approach to the Immunotherapy of Melanoma. Lancet, 2: 293-295, 1971. NPC and as an assay for TF in normal individuals and 2. Brandes, L. J., and Goldenberg, G. J. In Vitro Transfer of Cellular patients with NPC. The study did not resolve whether the Immunity against Nasopharyngeal Carcinoma Using Transfer Factor from Donors with Epstein-Barr Virus Antibody Activity. Cancer Res., 34: PLMI reactivity was directed against EBV genome or viral- 3095-3101,1974. associated antigens such as early antigen or membrane- 3. de Schryver, A., Friberg, S., Jr., Klein G., Henle, W., Henle, G., de-The, associated antigen. TF from EBV-positive donor leukocytes G., Clifford, P., and Ho, H. C. Epstein-Barr Virus-associated Antibody Patterns in Carcinoma of the Post-Nasal Space. Clin. Exptl. Immunol., 5: converted the teukocytes in 7 of 9 EBV-negative normal 443-459, 1969. subjects to a reactive state in vitro. TF also conveyed similar 4. de Schryver, A., Klein, G., Henle, G., Henle, W., Cameron, H. M., Santes- reactivity upon the leukocytes of 2 of 4 patients with NPC in son, L., and Clifford, P. EB-Virus Associated Serology in Malignant Disease: Antibody Levels to Viral Capsid Antigens (VCA), Membrane relapse. Antigen (MA) and Early Antigens (EA) in Patients with Various Neoplastic In this report, which preceded the above in vitro study, TF Conditions. Intern. J. Cancer, 9: 353-365, 1972. 5. Evans, A. S., Niederman, J. C., and McCollum, R. W. Seroepidemiologic immunotherapy of a patient with NPC, on 2 occasions was Studies of Infectious Mononucleosis with EB Virus. New Engl. J. Med., associated with apparent slowing of tumor growth coinci- 279: 1121-1127, 1968. dent with marked lymphocytic inf;Itration of the tumor and 6. Fimmel, P. J., Lai, P. K., Keast, D., Alpers, M. P., and MacKay-Scollay, E. M. In Vitro Cellular Immunity to Epstein-Barr Virus in Normal Human reconstitution of delayed cutaneous hypersensitivity to PPD Subjects. Intern. J. Cancer, 13: 599-605, 1974. and Trichophyton. It is possible that sampling error could 7. Gerber, P., Hamre, D., Moy, R. A., and Rosenblum, E. N. Infectious influence the histological findings, or the observed changes Mononucleosis: Complement-fixing Antibodies to Herpes-like Virus As- sociated with Burkitt's Lymphoma. Science, 161: 173-175, 1968. might be interpreted as a biological variation of NPC, since 8. Gerber, P., and Lucas, S. J. In Vitro Stimulation of Human Lymphocytes many tumors of the nasopharynx have a heavy lymphoid by Epstein-Barr Virus. Cellular Immunol., 5: 318-324, 1972. 9. Goldenberg, G. J., and Brandes, L. J. Immunotherapy of Nasopharyngeal component (10, 22, 24, 27). Several morphological studies Carcinoma with Transfer Factor from Donors with Previous Infectious support the concept that so-called lymphoepithelioma is Mononucleosis. Clin. Res., 20: 947, 1972. not a separate entity but merely a variant of undifferentiated 10. Goldman, J. M., Goodman, M. L., and Miller, D. Antibody to Epstein-Barr Virus in American Patients with Carcinoma of the Nasopharynx. J. Am. carcinoma and that lymphocytes are not an integral part of Med. Assoc., 216: 1618-1622, 1971. the tumor but represent a reactive infiltrate (22, 24, 27). 11. Henle, G., Henle, W., and Diehl, V. Relation of Burkitt's Tumor-associ- Following immunotherapy, not only was there a marked ated Herpes-type Virus to Infectious Mononucleosis. Proc. Natl. Acad. Sci. U.S., 59: 94-101,1968. round-cell infiltration of the tumor, but tumor cell degenera- 12. Henle, W., Henle, G., Ho, H. C., Burton, P., Cachin, Y., Clifford, P., de tion was also observed. Schryver, A., de-The, G., Diehl, V., and Klein, G. Antibodies to Epstein- Barr Virus in Nasopharyngeal Carcinoma, Other Head and Neck Neo- It is conceivable that TF immunotherapy had no effect and plasms, and Control Groups. J. Natl. Cancer Inst., 44: 225-231,1970. the clinical and pathological changes observed were those 13. Hitzig, W. H., and Grob, P. J. Therapeutic Uses of Transfer Factor. Progr. of the natural course of the disease. Patients with NPC may Clin. Immunol., 2: 60-100, 1974. 14. Ho, J. H. C. Nasopharyngeal Carcinoma in Hong Kong. Union Intern. survive up to 10 years with active disease without therapy Contre Cancer Monograph Ser., 1: 58-63, 1967. (14). However, the apparent slowing of tumor growth and 15. Kirkpatrick, C. H., and Gallin, J. I. Treatment of Infectious and Neoplastic the associated finding of lymphocytic infiltration of the tu- Diseases with Transfer Factor. Oncology, 29: 46-73, 1974. 16. Lawrence, H. S. Transfer Factor. Advan. Immunol., 11: 195-266, 1969. mor and reconstitution of delayed hypersensitivity reactions 17. Lawrence, H. S. Transfer Factor and Cellular Immune Deficiency Dis- suggest a favorable response to TF immunotherapy. ease. New Engl. J. Med., 283: 411-419, 1970. The literature regarding TF immunotherapy is replete with 18. Lawrence, H. S. In: J. Uhr, and M. Landy (eds.), Immunologic Interven- tion, pp. 20-27. New York: Academic Press, Inc., 1971. anecdotal case reports such as the one reported here (13, 19. Levin, A. S., Spitler, L. E., Wybran, J., Fudenberg, H. H., Hellstrom, I., 15). A need exists for controlled clinical trials to determine and Hellstrom, K. E. Treatment of Osteogenic Sarcoma with Tumor Specific Transfer Factor. Clin. Res., 20: 568, 1972. whether TF immunotherapy of any disease is beneficial. As 20. Niederman, J. C., McCollum, R. W., Henle, G., and Henle, W. Infectious a result of our limited clinical experience with TF immuno- Mononucleosis. Clinical Manifestations in Relation to EB Virus Antibod- therapy and our in vitro studies (2), a randomized, double- ies. J. Am. Med. Assoc., 203: 205-209, 1968. 21. Oettgen, H., Old, L., Farrow, J., Valentine, F., Lawrence, H. S., and blind clinical trial is currently underway in Hong Kong, in Thomas, L. Effects of Transfer Factor in Cancer Patients. J. Clin. Invest., collaboration with Dr. J. H. C. Ho, in which patients with 50: 71a, 1971. NPC will be treated with conventional radiotherapy, and 22. Shanmugaratnam, K., and Muir, C. S. Nasopharyngeal Carcinoma. Ori- gin and Structure. Union Intern. Contre Cancer Monograph Ser., 1: 153- one-half of the group will also receive dialyzable TF from 162, 1967. normal adults with a history of IM and/or with an anti-VCA 23. Smith, R. T., and Bausher, J. C. Epstein-Barr Virus Infection in Relation to Infectious Mononucleosis and Burkitt's Lymphoma. Ann. Rev. Med., antibody titer of 1/64 or greater. If successful, a similar trial 23: 39-56, 1972. of TF as an adjunct to chemotherapy might be considered in 24. Svoboda, D. J., Kirchner, F. R., and Shanmugaratnam, K. The Fine African patients with BL. Structure of Nasopharyngeal Carcinomas. Union Intern. Contre Cancer Monograph Ser., 1: 163-171, 1967. 25. Thompson, R. B. Lymphocyte Transfer Factor. Rev. European Etudes Acknowledgments Clin. Biol., 16: 201-204, 1971. 26. Thompson, R. B., and Mathe, G. Adoptive Immunotherapy in Malignant We thank Dr. G. A. Brown for referring the patient, for his keen interest Disease. Transplant. Rev., 9: 54-72, 1972. and active cooperation in clinical management; Dr. G. R. Hogg for carefully 27. Yeh, S. Histology of Nasopharyngeal Cancer. Union Intern. Contre Can-

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cer Monograph Ser., 1: 147-152, 1967. Virus Genomes in Human Tumor Cells by Nucleic Acid Hybridization. In: 28. zur Hausen, H. Epstein-Barr Virus in Human Tumor Cells 9Intern. Rev. P. M. Biggs, G. de-The, and L. N. Payne (eds.), Oncogenesis and Herpes- Exptl. Pathol., 21: 233-258, 1972. viruses, IARC Publication No. 2, pp. 321-325, Lyon, France: Interna- 29. zur Hausen, H., and Schulte-Holthausen, H. Detection of Epstein-Barr tional Agency for Research on Cancer, 1972.

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Fig. 1. Photomicrographs of serial nasopharyngealtumor biopsies. Except as stated, H & E, • 125. A, December 1, 1970. The initial biopsy on which a diagnosis of infiltrating epidermoid carcinoma Grade 3 was made. The predominant feature is undifferentiated carcinoma showing little evidence of lymphocytic infiltration. B, August 31,1971. Tumor recurrence 7 months after radiotherapy, showing infiltrating epidermoid carcinoma Grade 3 similar to the original tumor. C, November 3, 1971. Nasopharyngeal tumor biopsy approximately 1 month after starting immunotherapy with crude buffy coat extract, showing marked round-cell infiltration of the tumor. D, February 22, 1972. Tumor progression concomitant with loss of delayed cutaneous hypersensitivity reactions to PPD and T. rubrum. The biopsy reveals epidermoid carcinoma with little evidence of lymphocytic infiltration and resembles the histological appearance of the original tumor (A). E, March 27, 1972. Nasopharyngeal tumor biopsy 3 weeks after starting immunotherapy with dialyzable TF showing reappearance of round-cell infiltration of the tumor; positive delayed hypersensitivity reactions to PPD and T. rubrum were obtained approximately 1 week before and 2 weeks after the biopsy. F, May 9, 1972. Nasopharyngeal tumor biopsy approximately 2 months after starting immunotherapy with dialyzable TF; nests of degenerating tumor cells are encircled by an intense lymphocytic and plasma cellular infiltrate. • 200.

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Gerald J. Goldenberg and Lorne J. Brandes

Cancer Res 1976;36:720-723.

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