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Therapy of spontaneous metastases with an autologous tumor vaccine in a guinea pig model
JOURNAL
OF SURGICAL
Therapy
RESEARCH
30,
409-415 (1981)
of Spontaneous Vaccine
H. C. HOOVER, JR.,
Metastases in a Guinea
with an Autologous Pig Model’
M.D.,* L. C. PETERS, B.S. ,t J. S. BRANDHORST, AND M. G. HANNA, JR., PH.D.t
Tumor
R.N. ,t
*Departments of Surgery and Oncology, Johns Hopkins Medical Institutes, Baltimore, Maryland 21205, and Wancer Metastasis and Treatment Laboratory, NC&Frederick Cancer Research Center, Frederick, Maryland 21701
Presented at the Annual Meeting of the Association for Academic Surgery, Birmingham, Alabama, November 5-8, 1980 Active-specific immunotherapy, which involves activation of host defenses toward antigenic factors distinct for each tumor, is a potentially useful antitumor strategy. Experimental studies have demonstrated the efficacy of autologous tumor cell vaccines supplemented with an adjuvant (Bacillus Calmette-Gutrin, BCG) against intravenously injected tumor cells. In this study, we evaluated the vaccine’s efficacy against a more clinically relevant model, spontaneously occurring metastases. Viable syngeneic hepatocarcinoma cells were injected intradermally into the left shoulder area of inbred guinea pigs. On Day 25, the primary tumor (1.5 cm in diameter) and the first draining node were excised. Animals were randomized into groups that received no further therapy or two, three, or four vaccinations with an autologous preparation of cryopreserved, irradiated tumor cells mixed with live BCG. Vaccinations were started 4 days after surgery and were given weekly on alternate sides of the shoulder and flank regions. Of those animals that received surgery alone, 5% survived, whereas of those that received two, three, or four vaccines, 35% (P = 0.03), 45% (P = 0.004), and 55% (P = 0.002), respectively, were disease free at 200 days. That the effect was systemic and not due to BCG draining into the regionally involved lymph nodes was demonstrated in a repeat experiment in which all vaccinations were given in distant (contralateral) sites with equally effective results. Tumor cells in the lymph nodes beyond those excised were found in animals necropsied on the first day of therapy. Therefore, established distant lymph node metastases were controlled. Human tumors with tumor-specific antigens may respond similarly and should be studied.
INTRODUCTION
In spite of improved surgical procedures, innovative radiotherapy techniques, and many new chemotherapeutic approaches, the common solid malignancies remain major killers. Since it was clearly demonstrated that there are distinct tumor-specific antigens associated with chemically induced tumors of animals [3, 161, the role of the immune system in the control of malignant disease in both experimental animals and humans has been investigated extensively. Most clinical studies reported to date have used nonspecific immune stimulation by 1 Supported by the National Cancer Institute under Contract NOl-CO-75380 with Litton Bionetics, Inc.
agents such as BCG (Bacillus CalmetteGuCrin) or Corynebacterium parvum and the results have been discouraging. A potentially useful antitumor strategy is active specific immunotherapy, which activates the host defenses to antigenic factors distinctive for each tumor in an attempt to quantitatively boost the normal host protective mechanisms. A major impetus to active-specific immunotherapy has been the development and biological characterization of an adequate experimental model that fulfills many of the requirements for studying effective immunotherapy of established metastatic tumors. It has been demonstrated previously that transplanted, syngeneic line 10 (LlO) 409
0022-4804/8l/040409-07$01.00/O Copyright All rights
Q 1981 by Academic Press, Inc. of reproduction in any form reserved.
410
JOURNAL
OF SURGICAL
RESEARCH:
hepatocarcinoma of limited size, established in the skin of inbred guinea pigs (strain 2), regresses, and regional metastases are eliminated after intratumoral injections of an adequate dose of viable BCG [20, 221. During the course of this reaction, systemic, tumor-specific, cellular and humoral immunity develops. Further studies have shown the efficacy of this intratumoral BCG-induced tumor immunity against both regional and systemic malignancies [6, 83. Both in vitro and in viva data suggest that the ultimate effector cells in this form of therapy are cells of the macrophage-histiocyte compartment [2, 191, but the mechanism of induction of these cells is not clearly understood. Although the intratumoral model of immunotherapy has several clinical applications, its major limitations are that many tumors are not accessible for injection or are too large to allow for an adequate response to BCG. Therefore, the ability to achieve systemic tumor immunity by means of a BCG-tumor cell vaccine has been a major advance in this guinea pig immunotherapy model [7]. It has been demonstrated that BCG admixed with tumor cells is effective in inducing the degree of systemic immunity capable of eliminating a limited disseminated tumor burden when the vaccine is carefully controlled for variables such as the number of tumor cells, the ratio of viable BCG organisms to tumor cells, maintenance of nontumorigenic, yet metabolically viable cells, and the vaccination regimen [5, 151. This guinea pig hepatocarcinoma model has been used in recent studies that have focused on questions relevant to clinical situations because it has several features that make it pertinent to the study of human cancer: it is weakly antigenic, syngeneic with its host, and metastasizes spontaneously to draining lymph nodes and eventually visceral organs, following intradermal injection. Whereas most previous experiments in which this model was used evaluated intratumoral or regional therapy or the
VOL. 30, NO. 4, APRIL
1981
efficacy of the tumor cell-BCG vaccine against artificially induced tumor cells that were injected intravenously, the present study tested the vaccine’s efficacy in a more clinically relevant model, that of spontaneously occurring hematogenous and lymphatic metastases from surgically controlled primary tumors. Because drainage of BCG and the subsequent granulomatous reactions in the regional lymph nodes could theoretically influence the results, contralateral immunizations were compared with regional vaccinations. The effects of preexisting tumor immunity and the use of irradiated tumor cells alone as a booster after the standard two vaccinations were also evaluated. MATERIALS
AND METHODS
Animals. Inbred male guinea pigs (SewallWright, strain 2) were obtained from the Animal Production Area, NCI, Frederick Cancer Research Center. They were shown to be histocompatible by skin grafting. They were housed 10 per cage and fed Wayne Guinea Pig Chow and kale daily. All animals weighed 400-500 g at the beginning of the experiments. Tumor. Both induction of the primary hepatocarcinoma by diethylnitrosamine and the antigenic and biologic properties of the derived transplantable ascites tumor (LlO) have been described previously [ 17, 231. Tumor cells were maintained in ascites form by serial passage in the peritoneal cavity of weanling guinea pigs. For the induction of the primary dermal tumors, freshly harvested ascites cells were washed three times in Hanks’ balanced salt solution (HBSS), and lo5 viable cells in 0.1 ml of HBSS were injected intradermally into the left upper dorsal quadrant. Preparation
of BCG-tumor
cell vaccine.
LlO ascites were collected from the peritoneal cavity of donor weanling guinea pigs, washed three times in HBSS and concentrated to 6-8 x lo7 cells/ml. To this suspension an equal volume of chilled HBSS containing 15% DMSO and 10% fetal bovine
HOOVER ET AL.: AUTOLOGOUS TUMOR VACCINE
serum was added slowly. The cells were transferred immediately to l-ml freezer vials (Nunc vial, Vanguard, Red Bank, N. J.) and frozen in a controlled rate biological freezer (Cryo-Med 801) at - l”C/min to -80°C. The cells were then stored in liquid nitrogen. The rationale for this method of freezing has been described in detail elsewhere [ 11, 131. When needed, the cells were rapidly thawed in a 37°C water bath, transferred to a 50-ml centrifuge tube, and diluted slowly over a 30-min period to 50 ml with HBSS, washed, resuspended to 20-30 ml, and exposed to 20,000 rad (500 rad/min) of radiation with a Philips MG-301 X-irradiation unit. The cells were counted, washed, and resuspended to 10R viable cells/ml. Viability, as determined by the trypan blue exclusion test, was greater than 90% for all preparations. To vaccines 1 and 2, an equal volume of BCG preparation (Phipps strain TMC 1029: Trudeau Institute, Saranac, N. Y .) containing lo9 viable organisms/ml was added. In these vaccines, the inoculum contained lo7 irradiated tumor cells and lOa BCG in a 0.2-ml volume. The third and fourth vaccines contained 10’ irradiated tumor cells alone. In Experiment 1 (Fig. l), the first vaccine was given intradermally on the left upper dorsal quadrant adjacent to the surgical excision; the second was
FIG. 1. Surgical model. Protocol for experimental immunotherapy in guinea pigs with dermal tumors in which vaccinations were given to both sides of the body.
411
FIG. 2. Surgical model. Protocol for experimental immunotherapy in guinea pigs with dermal tumors in which vaccinations were given to the side contralateral to the operative site.
given on the right upper dorsal quadrant; and third and fourth were given on the left and right hind quadrants, respectively. In Experiment 2 (Fig. 2), all vaccinations were given contralateral to the operative site. Experimental design. In Experiment 1 (Fig. l), guinea pigs were inoculated intradermally with lo5 LlO cells. Inoculations were given caudal to the accessory axillary lymph nodes. On Day 25, when the mean diameter of the primary tumors was 1.5 cm, both the primary tumor and the adjacent enlarged accessory axillary nodes were excised surgically en bloc. The guinea pigs were randomized into four groups: controls and those receiving two, three, or four vaccinations (the third and fourth vaccines consisting of irradiated tumor cells alone). Five extra animals were killed at the time of the first vaccination to determine the extent of detectable metastases at the beginning of immunotherapy. Vaccinations were first administered 4 days after surgery and then at weekly intervals. The first vaccine was given intradermally on the left upper dorsal quadrant and later vaccinations were alternated between sides. The animals were monitored for survival and necropsied at death or at Day 200. Survival differences among the groups were analyzed statistically by both the Mantel-Haenszel and Cox exact tests.
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survived and, of those receiving two, three, or four vaccinations administered to both sides of the body, 35% (P = 0.03), 45% (P = 0.004), and 55% (P = 0.002), respectively, were disease free at 200 days (Fig. 3). There was no significant statistical difference among those animals receiving two, three, or four vaccinations. Animals in ExFIG. 3. The effect of vaccine on axillary and cer- periment 2, which were given all vaccinavical nodal metastases: percentage cumulative mor- tions at distant contralateral sites, had tality in guinea pigs with regional and disseminated nearly identical survival data (Fig. 4). Necmicrometastases treated with resection of the primary ropsies on control guinea pigs and on those tumor and the first draining lymph node then treated with BCG-tumor cell vaccines to both sides of the that died after treatment revealed widespread lymphatic metastases in all animals. body. Guinea pigs were challenged with lo5 LlO Regional lymph nodes were massively entumor cells administered id. Surgery was performed at 25 days (tumor and first draining node). id vac- larged and were usually the major cause of cinations began at 29 days. death. Pulmonary metastases were often present but rarely were they the cause of In Experiment 2 (Fig. 2), the protocol was death. All surviving animals were killed at exactly that of Experiment 1 except that all 200 days and found to be free of disease. vaccinations were given on the side contraThe immunized survivors in both experilateral to the surgical site. On the day of ments demonstrated tumor immunity by resurgical resection (Day 29, 10 animals with jection of an intradermal challenge with tumors and 10 non-tumor-bearing controls lo6 viable LIO cells on Day 180 following were challenged with IO6 LlO cells intra- surgical resection. Ten untreated control dermally to determine developing tumor im- animals injected similarly developed tumors munity. The challenge sites were measured approximately 1 cm in diameter within twice weekly with vernier calipers. In ad- 1 week. dition, before the experiment was termiAnimals that did not receive vaccinations nated, all surviving animals in the vac- and were challenged with lo6 tumor cells cination groups and 10 non-tumor-bearing on the day of surgery showed a significant controls were challenged intradermally with lo6 LlO cells to determine immunity. RESULTS
The mean diameter of the excised primary tumor was 1.53 2 0.17 cm in Experiment 1 and 1.51 2 0.15 cm in Experiment 2. All accessory axillary lymph nodes removed en bloc with the primary tumor were almost totally replaced by metastatic disease. Metastases were evident in the cervical and axillary lymph nodes beyond the operative site of animals killed on the day of the first vaccination in both experiments. These metastases were obvious only microscopically, as typically only the subcapsular sinuses were full of tumor. In Experiment 1, 5% of animals that received surgery alone
FIG. 4. The effect of vaccine on axillary and cervical nodal metastases: percentage cumulative mortality in guinea pigs with regional and disseminated micrometastases treated with resection of the primary tumor and the first draining lymph node then treated with BCG-tumor cell vaccines limited to the contralateral side from the operative site. Guinea pigs were challenged with lo5 LlO tumor cells administered id. Surgery was performed at 25 days (tumor and first draining node). id vaccinations began at 29 days.
HOOVER ET AL.: AUTOLOGOUS TUMOR VACCINE
413
decrease in the growth rate of the challenge tumor compared with that of normal control animals (Fig. 5). DISCUSSION
Several conclusions are evident from these studies: (i) established lymph node and visceral metastases from a spontaneously metastasizing, surgically controlled primary tumor were eliminated by an autologous tumor cell-BCG vaccine; (ii) the effectiveness was not diminished by administering all vaccinations at a site contralateral to the primary tumor and its draining lymph nodes; (iii) a degree of concomitant immunity did not abrogate the vaccine’s effectiveness; and (iv) one or two additional boosts with irradiated tumor cells alone did not significantly improve the efficacy of the vaccine. Several studies have suggested that intimate contact between BCG and tumor cells is essential for tumor killing [ 1, 21, 221. It has been argued that the primary antitumor effect of BCG therapy could be secondary to the granulomatous reaction of BCG draining into the involved regional lymph nodes. In the present study, Experiment 2, in which the animals received all vaccinations at a distant site from the primary tumor or draining lymph nodes, clearly demonstrated a systemic therapeutic effect. In our experiments, acid-fast organisms or granulomatous reactions in the draining lymph nodes have never occurred when vaccinations were given on the contralateral side. An important factor to evaluate in this model is its relevance to human cancer immunotherapy. The differences between rodent transplantable tumors and autochthonous human tumors are widely recognized. We have attempted to alter the model to the greatest extent possible in terms of clinical relevancy. Because it is estimated that several years are required for the development of a detectable human cancer, the human is presumably exposed to specific tumor antigens for a considerable length
FIG. 5. Tumor growth curves of responses after injection of lo6 LlO cells intradermally to animals with IO5 LlO tumor for 25 days (A) or in control (0) animals that had never had a tumor injection. Standard errors of the means were between 0.03 and 0.1.
of time. There is ample evidence [14, 181 that humans often recognize and respond to these tumor antigens and may have a degree of autogenous immunity to their tumor. The effects of these established immunologic responses on subsequent immunotherapy are unknown. In previous studies in the guinea pig model, therapy was started 6-7 days after intradermal injection of lo6 LIO cells, when the tumor was 1 cm in diameter. Littman et al. [ 121have shown that guinea pigs injected with LlO cells do not develop tumor immunity until the tumor is greater than 1 cm in diameter and has been present in the host for more than 2 weeks. Jessup et al. [9] showed that IO4 LlO cells injected intradermally and permitted to grow for 32 days allowed development of tumor immunity as measured by the ability to reject an intradermal challenge of IO6 LlO tumor cells. The presence of autogenous tumor immunity expressed at the start of therapy may have a significant effect on the outcome. Several investigators have shown that the tumor-specific immune response in tumor-immune animals may be depressed by the development of suppressor T cells [4, 101. These studies suggest that the host’s immune response could enhance tumor growth rather than inhibit it
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and that a degree of concomitant tumor immunity, which is presumably the case in many clinical situations, could be detrimental to active-specific immunotherapy. By administering the low dose of lo5 LlO cells in these studies, the latency of the tumor was increased considerably and allowed us to wait 25 days before tumor resection. The tumor challenge results in the animals (Fig. 5) indicate a significant degree of tumor immunity at the time of surgical resection. Previous studies [9] have demonstrated long-term persistence of this immunity in a similar model. It is encouraging that active-specific immunotherapy was successful in our experiments in spite of a degree of tumor immunity. These findings are similar to those of Jessup et al. [9] who found that intratumoral BCG was capable of mediating regression of both dermal and nodal metastases in the presence of tumor immunity as long as the amount of metastatic tumor in the draining lymph nodes was minimal. It was suggested that tumor burden, not tumor immunity, was the limiting factor to BCG immunotherapy. We came to a similar conclusion with regard to active-specific immunotherapy using an autologous tumor vaccine. The current experiments, although not so designed, are pertinent to the question of tumor burden controllable with an effective vaccination regimen. We know there are variable rates of metastases from even closely size-matched primary tumors such as those in our experiment. Some animals obviously have more systemic disease or handle it less well than others as demonstrated by the variable death rates in the control groups. Random distribution between control and treatment groups should balance this factor overall. However, the early death rates in treated animals were not significantly different from those of controls. This would imply that only animals with a limited residual tumor burden were benefited by the vaccine. This finding is consistent with most studies of immunotherapy and encourages its possible use in patients
whose primary tumor can be successfully managed by resection, but who are at a high risk of having microscopic metastases. Further studies are underway to better quantitate the efficacy of the vaccine in animals with an increased metastatic tumor burden. Of special importance is the possibility of tumor enhancement in animals treated with immunotherapy in the face of an advanced tumor burden. These experiments demonstrated that two vaccinations containing BCG and tumor cells were sufficient to give an antitumor response and that boosts of additional tumor cells administered shortly thereafter were not beneficial in this model. These are important findings if this model has clinical relevance, since obtaining a sufficient number of viable tumor cells for extended vaccinations against some human malignancies would be problematic. As we look toward clinical trials of activespecific immunotherapy, animal models will play an increasingly important role in optimizing therapy and will hopefully lead to an improved clarification of the mechanisms responsible for successful therapy. REFERENCES 1. Baldwin, R. W., and Pimm, M. V. BCG immunotherapy of local subcutaneous growths in post-surgical pulmonary metastases of a transplanted rat epithelioma of spontaneous origin. Int. J. Cancer 12: 420, 1973. 2. Fidler, I. J., Budmen, M. B., and Hanna, M. G., Jr. Characterization of in vitro reactivity by BCGtreated guinea pigs to syngeneic line-10 hepatocarcinoma. Cancer Immunol. Immunother. 1: 179, 1976. 3. Foley, E. J. Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer 13: 835, 1953. 4. Fujimoto, S., Greene, M. I., and Sehon, A. H. Regulation of the immune response to tumor antigens. 1. Immunosuppressor cells in tumorbearing hosts. J. Zmmunol. 116: 791, 1976. 5. Hanna, M. G., Jr., Brandhorst, J. S., and Peters, L. C. Active specific immunotherapy of residual micrometastasis: An evaluation of sources, doses and ratios of BCG with tumor cells. Cancer Zmmunol. Zmmunother. 7: 165, 1979. 6. Hanna, M. G., Jr., and Peters, L. C. Efficacy
HOOVER ET AL.: AUTOLOGOUS TUMOR VACCINE of intralesional BCG therapy in guinea pigs with disseminated tumor. Cancer 36: 1298, 1975. 7. Hanna, M. G., Jr., and Peters, L. C. Immunotherapy of established micrometastases with Baciltumor cell vaccine. Cancer lus Calmette-GuCrin Res. 38: 204, 1978. 8. Hanna, M. G., Jr., Peters, L. C., and Fidler, I. J. The efficacy of BCG-induced tumor immunity in guinea pigs with regional and systemic malignancy. Cancer Immunol. Immunother. 1: 171, 1976. 9. Jessup, J. M., Riggs, C. W., and Hanna, M. G., Jr. Influence of preexisting tumor immunity on Bncillus Calmette-Gutrin immunotherapy of guinea pigs with both regional and disseminated tumor. Cancer Res. 37: 2565, 1977.
10. Kurata, T., and Micksche, M. Correlation of immune response with clinical stage in Lewis lung tumor-bearing mice. Oncology 35: 155, 1978. 11. Leibo, S. P., Farrant, J., Mazur, P., Hanna, M. G., Jr., and Smith, L. H. Effects of freezing on marrow stem cell suspensions: Interactions of cooling and warming rates in the presence of PVP, sucrose, or glycerol. Cryobiology 6: 315, 1970. 12. Littman, B. H., Meltzer, M. S., Cleveland, R. P., Zbar, B., and Rapp, H. J. Tumor-specific, cellmediated immunity in guinea pigs with tumors. J. Natl. Cancer Inst. 51: 1627, 1973. 13. Mazur, P., Leibo, S. P., Farrant, J., Chu, E. H. Y., Hanna, M. G., Jr., and Smith, L. H. Interactions of cooling rate, warming rate and protective additive on the survival of frozen mammalian cells. In G. E. W. Wostenholme and M. O’Connor (Eds.), The Frozen Cell. London: Churchill, 1970. P. 69. 14. Morton, D. L., Eilber, E. R., Malmgrem, R. A., and Wood, W. C. Immunological factors which influence response to immunotherapy in malignant melanoma. Surgery 68: 158, 1970. 15. Peters, L. C., and Hanna, M. G., Jr. Active specific immunotherapy of established metastasis: Effect of cryopreservation procedures on tumor
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cell immunogenicity in guinea pigs. J. Natl. Cancer Inst. 64: 1521, 1980.
16. Prehn, R. T., and Main, J. M. Immunity to methylcholanthrene-induced sarcomas. J. Natl. Cancer Inst. 18: 769, 1957. 17. Rapp, H. J., Churchill, W. H., Jr., Kronman, B. S., Rolley, R. T., Hammond, W. G., and Borsos, T. Antigenicity of a new diethylnitrosamine-induced transplantable guinea pig hepatoma: Pathology and formation of ascites variant. J. Natl. Cancer Inst. 41: 1, 1968. 18. Sjogren, H. O., Hellstrom, I., Bansel, S. C., and Hellstrom, K. E. Suggestive evidence that the “blocking antibodies” of tumor-bearing individuals may be antigen-antibody complexes. Proc. Natl. Acad. Sci. USA 68: 1372, 1971. 19. Snodgrass, M. J., and Hanna. M. G., Jr. Histopathology of tumor regression after intralesional injection of Mycobacterium bovis. Ultrastructural studies of histiocyte-tumor cell interactions. Cancer Res. 33: 701, 1973. 20. Zbar, B., Bernstein, I. D., Bartlett, G. L., Hanna, M. G., Jr., and Rapp, H. J. Immunotherapy of Cancer: Regression of intradermal tumors and prevention of growth of lymph node metastases after intralesional injection of living Mycobacterium bovis (Bacillus Calmette-GuCrin). Inst. 49: 119, 1972.
J. Natl. Cancer
21. Zbar, B., Bernstein, I. D., and Rapp, H. J. Suppression of tumor growth at the site of infection with living Bacillus Calmette-Gutrin. J. Natl. Cancer Inst. 46: 831, 1971. 22. Zbar, B., and Tanaka, T. Immunotherapy of cancer: Regression of tumors after intralesional injection of living Mycobacterium bovis. Science 172: 271, 1971. 23. Zbar, B., Wepsic, H. T., Rapp, H. J., Borsos, T., Kronman, B. S., and Churchill, W. H., Jr. Antigenic specificity of hepatomas induced in strain 2 guinea pigs by diethylnitrosamine. J. Nat/. Cancer Inst. 43: 833, 1969.
OF SURGICAL
Therapy
RESEARCH
30,
409-415 (1981)
of Spontaneous Vaccine
H. C. HOOVER, JR.,
Metastases in a Guinea
with an Autologous Pig Model’
M.D.,* L. C. PETERS, B.S. ,t J. S. BRANDHORST, AND M. G. HANNA, JR., PH.D.t
Tumor
R.N. ,t
*Departments of Surgery and Oncology, Johns Hopkins Medical Institutes, Baltimore, Maryland 21205, and Wancer Metastasis and Treatment Laboratory, NC&Frederick Cancer Research Center, Frederick, Maryland 21701
Presented at the Annual Meeting of the Association for Academic Surgery, Birmingham, Alabama, November 5-8, 1980 Active-specific immunotherapy, which involves activation of host defenses toward antigenic factors distinct for each tumor, is a potentially useful antitumor strategy. Experimental studies have demonstrated the efficacy of autologous tumor cell vaccines supplemented with an adjuvant (Bacillus Calmette-Gutrin, BCG) against intravenously injected tumor cells. In this study, we evaluated the vaccine’s efficacy against a more clinically relevant model, spontaneously occurring metastases. Viable syngeneic hepatocarcinoma cells were injected intradermally into the left shoulder area of inbred guinea pigs. On Day 25, the primary tumor (1.5 cm in diameter) and the first draining node were excised. Animals were randomized into groups that received no further therapy or two, three, or four vaccinations with an autologous preparation of cryopreserved, irradiated tumor cells mixed with live BCG. Vaccinations were started 4 days after surgery and were given weekly on alternate sides of the shoulder and flank regions. Of those animals that received surgery alone, 5% survived, whereas of those that received two, three, or four vaccines, 35% (P = 0.03), 45% (P = 0.004), and 55% (P = 0.002), respectively, were disease free at 200 days. That the effect was systemic and not due to BCG draining into the regionally involved lymph nodes was demonstrated in a repeat experiment in which all vaccinations were given in distant (contralateral) sites with equally effective results. Tumor cells in the lymph nodes beyond those excised were found in animals necropsied on the first day of therapy. Therefore, established distant lymph node metastases were controlled. Human tumors with tumor-specific antigens may respond similarly and should be studied.
INTRODUCTION
In spite of improved surgical procedures, innovative radiotherapy techniques, and many new chemotherapeutic approaches, the common solid malignancies remain major killers. Since it was clearly demonstrated that there are distinct tumor-specific antigens associated with chemically induced tumors of animals [3, 161, the role of the immune system in the control of malignant disease in both experimental animals and humans has been investigated extensively. Most clinical studies reported to date have used nonspecific immune stimulation by 1 Supported by the National Cancer Institute under Contract NOl-CO-75380 with Litton Bionetics, Inc.
agents such as BCG (Bacillus CalmetteGuCrin) or Corynebacterium parvum and the results have been discouraging. A potentially useful antitumor strategy is active specific immunotherapy, which activates the host defenses to antigenic factors distinctive for each tumor in an attempt to quantitatively boost the normal host protective mechanisms. A major impetus to active-specific immunotherapy has been the development and biological characterization of an adequate experimental model that fulfills many of the requirements for studying effective immunotherapy of established metastatic tumors. It has been demonstrated previously that transplanted, syngeneic line 10 (LlO) 409
0022-4804/8l/040409-07$01.00/O Copyright All rights
Q 1981 by Academic Press, Inc. of reproduction in any form reserved.
410
JOURNAL
OF SURGICAL
RESEARCH:
hepatocarcinoma of limited size, established in the skin of inbred guinea pigs (strain 2), regresses, and regional metastases are eliminated after intratumoral injections of an adequate dose of viable BCG [20, 221. During the course of this reaction, systemic, tumor-specific, cellular and humoral immunity develops. Further studies have shown the efficacy of this intratumoral BCG-induced tumor immunity against both regional and systemic malignancies [6, 83. Both in vitro and in viva data suggest that the ultimate effector cells in this form of therapy are cells of the macrophage-histiocyte compartment [2, 191, but the mechanism of induction of these cells is not clearly understood. Although the intratumoral model of immunotherapy has several clinical applications, its major limitations are that many tumors are not accessible for injection or are too large to allow for an adequate response to BCG. Therefore, the ability to achieve systemic tumor immunity by means of a BCG-tumor cell vaccine has been a major advance in this guinea pig immunotherapy model [7]. It has been demonstrated that BCG admixed with tumor cells is effective in inducing the degree of systemic immunity capable of eliminating a limited disseminated tumor burden when the vaccine is carefully controlled for variables such as the number of tumor cells, the ratio of viable BCG organisms to tumor cells, maintenance of nontumorigenic, yet metabolically viable cells, and the vaccination regimen [5, 151. This guinea pig hepatocarcinoma model has been used in recent studies that have focused on questions relevant to clinical situations because it has several features that make it pertinent to the study of human cancer: it is weakly antigenic, syngeneic with its host, and metastasizes spontaneously to draining lymph nodes and eventually visceral organs, following intradermal injection. Whereas most previous experiments in which this model was used evaluated intratumoral or regional therapy or the
VOL. 30, NO. 4, APRIL
1981
efficacy of the tumor cell-BCG vaccine against artificially induced tumor cells that were injected intravenously, the present study tested the vaccine’s efficacy in a more clinically relevant model, that of spontaneously occurring hematogenous and lymphatic metastases from surgically controlled primary tumors. Because drainage of BCG and the subsequent granulomatous reactions in the regional lymph nodes could theoretically influence the results, contralateral immunizations were compared with regional vaccinations. The effects of preexisting tumor immunity and the use of irradiated tumor cells alone as a booster after the standard two vaccinations were also evaluated. MATERIALS
AND METHODS
Animals. Inbred male guinea pigs (SewallWright, strain 2) were obtained from the Animal Production Area, NCI, Frederick Cancer Research Center. They were shown to be histocompatible by skin grafting. They were housed 10 per cage and fed Wayne Guinea Pig Chow and kale daily. All animals weighed 400-500 g at the beginning of the experiments. Tumor. Both induction of the primary hepatocarcinoma by diethylnitrosamine and the antigenic and biologic properties of the derived transplantable ascites tumor (LlO) have been described previously [ 17, 231. Tumor cells were maintained in ascites form by serial passage in the peritoneal cavity of weanling guinea pigs. For the induction of the primary dermal tumors, freshly harvested ascites cells were washed three times in Hanks’ balanced salt solution (HBSS), and lo5 viable cells in 0.1 ml of HBSS were injected intradermally into the left upper dorsal quadrant. Preparation
of BCG-tumor
cell vaccine.
LlO ascites were collected from the peritoneal cavity of donor weanling guinea pigs, washed three times in HBSS and concentrated to 6-8 x lo7 cells/ml. To this suspension an equal volume of chilled HBSS containing 15% DMSO and 10% fetal bovine
HOOVER ET AL.: AUTOLOGOUS TUMOR VACCINE
serum was added slowly. The cells were transferred immediately to l-ml freezer vials (Nunc vial, Vanguard, Red Bank, N. J.) and frozen in a controlled rate biological freezer (Cryo-Med 801) at - l”C/min to -80°C. The cells were then stored in liquid nitrogen. The rationale for this method of freezing has been described in detail elsewhere [ 11, 131. When needed, the cells were rapidly thawed in a 37°C water bath, transferred to a 50-ml centrifuge tube, and diluted slowly over a 30-min period to 50 ml with HBSS, washed, resuspended to 20-30 ml, and exposed to 20,000 rad (500 rad/min) of radiation with a Philips MG-301 X-irradiation unit. The cells were counted, washed, and resuspended to 10R viable cells/ml. Viability, as determined by the trypan blue exclusion test, was greater than 90% for all preparations. To vaccines 1 and 2, an equal volume of BCG preparation (Phipps strain TMC 1029: Trudeau Institute, Saranac, N. Y .) containing lo9 viable organisms/ml was added. In these vaccines, the inoculum contained lo7 irradiated tumor cells and lOa BCG in a 0.2-ml volume. The third and fourth vaccines contained 10’ irradiated tumor cells alone. In Experiment 1 (Fig. l), the first vaccine was given intradermally on the left upper dorsal quadrant adjacent to the surgical excision; the second was
FIG. 1. Surgical model. Protocol for experimental immunotherapy in guinea pigs with dermal tumors in which vaccinations were given to both sides of the body.
411
FIG. 2. Surgical model. Protocol for experimental immunotherapy in guinea pigs with dermal tumors in which vaccinations were given to the side contralateral to the operative site.
given on the right upper dorsal quadrant; and third and fourth were given on the left and right hind quadrants, respectively. In Experiment 2 (Fig. 2), all vaccinations were given contralateral to the operative site. Experimental design. In Experiment 1 (Fig. l), guinea pigs were inoculated intradermally with lo5 LlO cells. Inoculations were given caudal to the accessory axillary lymph nodes. On Day 25, when the mean diameter of the primary tumors was 1.5 cm, both the primary tumor and the adjacent enlarged accessory axillary nodes were excised surgically en bloc. The guinea pigs were randomized into four groups: controls and those receiving two, three, or four vaccinations (the third and fourth vaccines consisting of irradiated tumor cells alone). Five extra animals were killed at the time of the first vaccination to determine the extent of detectable metastases at the beginning of immunotherapy. Vaccinations were first administered 4 days after surgery and then at weekly intervals. The first vaccine was given intradermally on the left upper dorsal quadrant and later vaccinations were alternated between sides. The animals were monitored for survival and necropsied at death or at Day 200. Survival differences among the groups were analyzed statistically by both the Mantel-Haenszel and Cox exact tests.
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survived and, of those receiving two, three, or four vaccinations administered to both sides of the body, 35% (P = 0.03), 45% (P = 0.004), and 55% (P = 0.002), respectively, were disease free at 200 days (Fig. 3). There was no significant statistical difference among those animals receiving two, three, or four vaccinations. Animals in ExFIG. 3. The effect of vaccine on axillary and cer- periment 2, which were given all vaccinavical nodal metastases: percentage cumulative mor- tions at distant contralateral sites, had tality in guinea pigs with regional and disseminated nearly identical survival data (Fig. 4). Necmicrometastases treated with resection of the primary ropsies on control guinea pigs and on those tumor and the first draining lymph node then treated with BCG-tumor cell vaccines to both sides of the that died after treatment revealed widespread lymphatic metastases in all animals. body. Guinea pigs were challenged with lo5 LlO Regional lymph nodes were massively entumor cells administered id. Surgery was performed at 25 days (tumor and first draining node). id vac- larged and were usually the major cause of cinations began at 29 days. death. Pulmonary metastases were often present but rarely were they the cause of In Experiment 2 (Fig. 2), the protocol was death. All surviving animals were killed at exactly that of Experiment 1 except that all 200 days and found to be free of disease. vaccinations were given on the side contraThe immunized survivors in both experilateral to the surgical site. On the day of ments demonstrated tumor immunity by resurgical resection (Day 29, 10 animals with jection of an intradermal challenge with tumors and 10 non-tumor-bearing controls lo6 viable LIO cells on Day 180 following were challenged with IO6 LlO cells intra- surgical resection. Ten untreated control dermally to determine developing tumor im- animals injected similarly developed tumors munity. The challenge sites were measured approximately 1 cm in diameter within twice weekly with vernier calipers. In ad- 1 week. dition, before the experiment was termiAnimals that did not receive vaccinations nated, all surviving animals in the vac- and were challenged with lo6 tumor cells cination groups and 10 non-tumor-bearing on the day of surgery showed a significant controls were challenged intradermally with lo6 LlO cells to determine immunity. RESULTS
The mean diameter of the excised primary tumor was 1.53 2 0.17 cm in Experiment 1 and 1.51 2 0.15 cm in Experiment 2. All accessory axillary lymph nodes removed en bloc with the primary tumor were almost totally replaced by metastatic disease. Metastases were evident in the cervical and axillary lymph nodes beyond the operative site of animals killed on the day of the first vaccination in both experiments. These metastases were obvious only microscopically, as typically only the subcapsular sinuses were full of tumor. In Experiment 1, 5% of animals that received surgery alone
FIG. 4. The effect of vaccine on axillary and cervical nodal metastases: percentage cumulative mortality in guinea pigs with regional and disseminated micrometastases treated with resection of the primary tumor and the first draining lymph node then treated with BCG-tumor cell vaccines limited to the contralateral side from the operative site. Guinea pigs were challenged with lo5 LlO tumor cells administered id. Surgery was performed at 25 days (tumor and first draining node). id vaccinations began at 29 days.
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decrease in the growth rate of the challenge tumor compared with that of normal control animals (Fig. 5). DISCUSSION
Several conclusions are evident from these studies: (i) established lymph node and visceral metastases from a spontaneously metastasizing, surgically controlled primary tumor were eliminated by an autologous tumor cell-BCG vaccine; (ii) the effectiveness was not diminished by administering all vaccinations at a site contralateral to the primary tumor and its draining lymph nodes; (iii) a degree of concomitant immunity did not abrogate the vaccine’s effectiveness; and (iv) one or two additional boosts with irradiated tumor cells alone did not significantly improve the efficacy of the vaccine. Several studies have suggested that intimate contact between BCG and tumor cells is essential for tumor killing [ 1, 21, 221. It has been argued that the primary antitumor effect of BCG therapy could be secondary to the granulomatous reaction of BCG draining into the involved regional lymph nodes. In the present study, Experiment 2, in which the animals received all vaccinations at a distant site from the primary tumor or draining lymph nodes, clearly demonstrated a systemic therapeutic effect. In our experiments, acid-fast organisms or granulomatous reactions in the draining lymph nodes have never occurred when vaccinations were given on the contralateral side. An important factor to evaluate in this model is its relevance to human cancer immunotherapy. The differences between rodent transplantable tumors and autochthonous human tumors are widely recognized. We have attempted to alter the model to the greatest extent possible in terms of clinical relevancy. Because it is estimated that several years are required for the development of a detectable human cancer, the human is presumably exposed to specific tumor antigens for a considerable length
FIG. 5. Tumor growth curves of responses after injection of lo6 LlO cells intradermally to animals with IO5 LlO tumor for 25 days (A) or in control (0) animals that had never had a tumor injection. Standard errors of the means were between 0.03 and 0.1.
of time. There is ample evidence [14, 181 that humans often recognize and respond to these tumor antigens and may have a degree of autogenous immunity to their tumor. The effects of these established immunologic responses on subsequent immunotherapy are unknown. In previous studies in the guinea pig model, therapy was started 6-7 days after intradermal injection of lo6 LIO cells, when the tumor was 1 cm in diameter. Littman et al. [ 121have shown that guinea pigs injected with LlO cells do not develop tumor immunity until the tumor is greater than 1 cm in diameter and has been present in the host for more than 2 weeks. Jessup et al. [9] showed that IO4 LlO cells injected intradermally and permitted to grow for 32 days allowed development of tumor immunity as measured by the ability to reject an intradermal challenge of IO6 LlO tumor cells. The presence of autogenous tumor immunity expressed at the start of therapy may have a significant effect on the outcome. Several investigators have shown that the tumor-specific immune response in tumor-immune animals may be depressed by the development of suppressor T cells [4, 101. These studies suggest that the host’s immune response could enhance tumor growth rather than inhibit it
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and that a degree of concomitant tumor immunity, which is presumably the case in many clinical situations, could be detrimental to active-specific immunotherapy. By administering the low dose of lo5 LlO cells in these studies, the latency of the tumor was increased considerably and allowed us to wait 25 days before tumor resection. The tumor challenge results in the animals (Fig. 5) indicate a significant degree of tumor immunity at the time of surgical resection. Previous studies [9] have demonstrated long-term persistence of this immunity in a similar model. It is encouraging that active-specific immunotherapy was successful in our experiments in spite of a degree of tumor immunity. These findings are similar to those of Jessup et al. [9] who found that intratumoral BCG was capable of mediating regression of both dermal and nodal metastases in the presence of tumor immunity as long as the amount of metastatic tumor in the draining lymph nodes was minimal. It was suggested that tumor burden, not tumor immunity, was the limiting factor to BCG immunotherapy. We came to a similar conclusion with regard to active-specific immunotherapy using an autologous tumor vaccine. The current experiments, although not so designed, are pertinent to the question of tumor burden controllable with an effective vaccination regimen. We know there are variable rates of metastases from even closely size-matched primary tumors such as those in our experiment. Some animals obviously have more systemic disease or handle it less well than others as demonstrated by the variable death rates in the control groups. Random distribution between control and treatment groups should balance this factor overall. However, the early death rates in treated animals were not significantly different from those of controls. This would imply that only animals with a limited residual tumor burden were benefited by the vaccine. This finding is consistent with most studies of immunotherapy and encourages its possible use in patients
whose primary tumor can be successfully managed by resection, but who are at a high risk of having microscopic metastases. Further studies are underway to better quantitate the efficacy of the vaccine in animals with an increased metastatic tumor burden. Of special importance is the possibility of tumor enhancement in animals treated with immunotherapy in the face of an advanced tumor burden. These experiments demonstrated that two vaccinations containing BCG and tumor cells were sufficient to give an antitumor response and that boosts of additional tumor cells administered shortly thereafter were not beneficial in this model. These are important findings if this model has clinical relevance, since obtaining a sufficient number of viable tumor cells for extended vaccinations against some human malignancies would be problematic. As we look toward clinical trials of activespecific immunotherapy, animal models will play an increasingly important role in optimizing therapy and will hopefully lead to an improved clarification of the mechanisms responsible for successful therapy. REFERENCES 1. Baldwin, R. W., and Pimm, M. V. BCG immunotherapy of local subcutaneous growths in post-surgical pulmonary metastases of a transplanted rat epithelioma of spontaneous origin. Int. J. Cancer 12: 420, 1973. 2. Fidler, I. J., Budmen, M. B., and Hanna, M. G., Jr. Characterization of in vitro reactivity by BCGtreated guinea pigs to syngeneic line-10 hepatocarcinoma. Cancer Immunol. Immunother. 1: 179, 1976. 3. Foley, E. J. Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer 13: 835, 1953. 4. Fujimoto, S., Greene, M. I., and Sehon, A. H. Regulation of the immune response to tumor antigens. 1. Immunosuppressor cells in tumorbearing hosts. J. Zmmunol. 116: 791, 1976. 5. Hanna, M. G., Jr., Brandhorst, J. S., and Peters, L. C. Active specific immunotherapy of residual micrometastasis: An evaluation of sources, doses and ratios of BCG with tumor cells. Cancer Zmmunol. Zmmunother. 7: 165, 1979. 6. Hanna, M. G., Jr., and Peters, L. C. Efficacy
HOOVER ET AL.: AUTOLOGOUS TUMOR VACCINE of intralesional BCG therapy in guinea pigs with disseminated tumor. Cancer 36: 1298, 1975. 7. Hanna, M. G., Jr., and Peters, L. C. Immunotherapy of established micrometastases with Baciltumor cell vaccine. Cancer lus Calmette-GuCrin Res. 38: 204, 1978. 8. Hanna, M. G., Jr., Peters, L. C., and Fidler, I. J. The efficacy of BCG-induced tumor immunity in guinea pigs with regional and systemic malignancy. Cancer Immunol. Immunother. 1: 171, 1976. 9. Jessup, J. M., Riggs, C. W., and Hanna, M. G., Jr. Influence of preexisting tumor immunity on Bncillus Calmette-Gutrin immunotherapy of guinea pigs with both regional and disseminated tumor. Cancer Res. 37: 2565, 1977.
10. Kurata, T., and Micksche, M. Correlation of immune response with clinical stage in Lewis lung tumor-bearing mice. Oncology 35: 155, 1978. 11. Leibo, S. P., Farrant, J., Mazur, P., Hanna, M. G., Jr., and Smith, L. H. Effects of freezing on marrow stem cell suspensions: Interactions of cooling and warming rates in the presence of PVP, sucrose, or glycerol. Cryobiology 6: 315, 1970. 12. Littman, B. H., Meltzer, M. S., Cleveland, R. P., Zbar, B., and Rapp, H. J. Tumor-specific, cellmediated immunity in guinea pigs with tumors. J. Natl. Cancer Inst. 51: 1627, 1973. 13. Mazur, P., Leibo, S. P., Farrant, J., Chu, E. H. Y., Hanna, M. G., Jr., and Smith, L. H. Interactions of cooling rate, warming rate and protective additive on the survival of frozen mammalian cells. In G. E. W. Wostenholme and M. O’Connor (Eds.), The Frozen Cell. London: Churchill, 1970. P. 69. 14. Morton, D. L., Eilber, E. R., Malmgrem, R. A., and Wood, W. C. Immunological factors which influence response to immunotherapy in malignant melanoma. Surgery 68: 158, 1970. 15. Peters, L. C., and Hanna, M. G., Jr. Active specific immunotherapy of established metastasis: Effect of cryopreservation procedures on tumor
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16. Prehn, R. T., and Main, J. M. Immunity to methylcholanthrene-induced sarcomas. J. Natl. Cancer Inst. 18: 769, 1957. 17. Rapp, H. J., Churchill, W. H., Jr., Kronman, B. S., Rolley, R. T., Hammond, W. G., and Borsos, T. Antigenicity of a new diethylnitrosamine-induced transplantable guinea pig hepatoma: Pathology and formation of ascites variant. J. Natl. Cancer Inst. 41: 1, 1968. 18. Sjogren, H. O., Hellstrom, I., Bansel, S. C., and Hellstrom, K. E. Suggestive evidence that the “blocking antibodies” of tumor-bearing individuals may be antigen-antibody complexes. Proc. Natl. Acad. Sci. USA 68: 1372, 1971. 19. Snodgrass, M. J., and Hanna. M. G., Jr. Histopathology of tumor regression after intralesional injection of Mycobacterium bovis. Ultrastructural studies of histiocyte-tumor cell interactions. Cancer Res. 33: 701, 1973. 20. Zbar, B., Bernstein, I. D., Bartlett, G. L., Hanna, M. G., Jr., and Rapp, H. J. Immunotherapy of Cancer: Regression of intradermal tumors and prevention of growth of lymph node metastases after intralesional injection of living Mycobacterium bovis (Bacillus Calmette-GuCrin). Inst. 49: 119, 1972.
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21. Zbar, B., Bernstein, I. D., and Rapp, H. J. Suppression of tumor growth at the site of infection with living Bacillus Calmette-Gutrin. J. Natl. Cancer Inst. 46: 831, 1971. 22. Zbar, B., and Tanaka, T. Immunotherapy of cancer: Regression of tumors after intralesional injection of living Mycobacterium bovis. Science 172: 271, 1971. 23. Zbar, B., Wepsic, H. T., Rapp, H. J., Borsos, T., Kronman, B. S., and Churchill, W. H., Jr. Antigenic specificity of hepatomas induced in strain 2 guinea pigs by diethylnitrosamine. J. Nat/. Cancer Inst. 43: 833, 1969.