- Email: [email protected]
Immunotherapeutic Effect of Cryosurgical Tumor Necrosis
Symposium on Cryosurgery
Immunotherapeutic Effect of Cryosurgical Tumor Necrosis H. Bryan Neel III, M.D., Ph.D.*
Cryosurgery has been applied clinically for both cure and palliation of benign and malignant neoplasms. Detailed studies of temporal and physical factors have provided the information necessary to ensure a reasonable chance of therapeutic success. 4 •5 Cryosurgery not only has a local tumoricidal effect but also has systemic immunologic antitumor effects. Of particular interest has been the observation that destruction of tumor by cryosurgery leads to increased tumor-specific transplantation immunity. Tumor-specific transplantation immunity to tumors can be induced in animals by a variety of means, and early studies along these lines constitute one of the most important advances in cancer biology. Basic to this achievement was the development and ready availability of highly inbred, syngeneic strains of rodents. Now there is evidence that many human tumors contain tumorspecific antigens that are accessible for immunologic recognition and capable of inducing responses that are cytotoxic to tumor cells. 10 This has led to a resurgence of interest and investigation of immunotherapy for cancer and has provided the impetus to study, in the laboratory, the effects of tumor destruction by various means, including cryosurgery, on tumor-specific transplantation immunity and related immunologic parameters. Immunotherapy has the theoretical capacity of destroying all tumor cells, provided that the tumor burden is minimal-such as after a "curative" surgical procedure in which the primary tumor is completely excised yet tumor cells remain in circulation or in the form of clinically undetectable microscopic metastases. Modest or minimal toxicity is associated with immunotherapy in contrast to chemotherapy, which, in addition, may impair cell-mediated immunity directed against tumor. Unfortunately, immunotherapy may facilitate or enhance tumor *Consultant, Department of Otorhinolaryngology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota; Associate Professor, Mayo Medical School, Rochester, Minnesota Veterinary Clinics of North America: Small Animal Practice-Val. 10, No.4, November 1980
763
764
H.
BRYAN NEEL
III
growth, a hazard that must be considered, particularly if specific immunization is to be used. Therefore, cryosurgery is an attractive form of cancer treatment because it is highly successful in selected patients and it has a nontoxic immunotherapeutic effect. Necrosis of tumor 'in the host by cryosurgery or other methods, alone or in combination with excision, is unlikely to result in facilitation of tumor growth and will have a protective effect.
HISTORICAL PERSPECTIVE Little is known about the relationship between the nature and duration of the host's exposure to tumor antigen and the subsequent effectiveness of the host's immune response. Strauss et al. were the first to propose that destruction of tumor in the host augmented that host's resistance to tumor. 13 They found that partial electrocoagulation of human rectal tumors was followed by regression and disappearance of residual tumor in some cases. From this observation, they inferred that both local and systemic effects were induced by prolonged absorption of dead tumor tissue, including tumor-specific antigens presumably. Immunologic mechanisms in explanation of these observations in man were adduced from tumor challenge data in rabbits after electrocoagulation of Brown-Pearce tumors. Unfortunately, this host is not syngeneic and the tumor was not of recent origin, and furthermore immunogenicity of the tumor was not demonstrated; these are recurrent deficiencies in many studies of tumor immunobiology even today. However, these observations were confirmed by Sobel and Patterson, who found that electrocoagulation of tumor in a syngeneic murine tumor-bearing host produced greater tumor-specific transplantation immunity than that observed after amputation (excision) of tumor. 12 Others have since provided further confirmation. Moore et al. reported that cryosurgical treatment of several different human tumors induced an immune response that was apparently directed against the tumors. 3 Soanes et al. observed roentgenographic regression of pulmonary metastases in patients with prostatic carcinoma after cryosurgery of the primary tumor.U Addressing the issue directly in the laboratory, Neel and associates showed that tumor-specific transplantation immunity after challenge with the same tumor was greater after cryosurgery than it was after simple excision of the primary tumor. 5
RECENT STUDIES Several studies have been done to determine the relative effectiveness of cryosurgery and other methods of tumor necrosis in situ. The
765
IMMUNOTHERAPEUTIC EFFECT OF CRYOSURGICAL TUMOR NECROSIS
materials, methods, and experimental design have been described. 5 - 7 •9 Briefly, inbred strains of mice and tumors with demonstrable immunogenicity were used. Viral and chemically induced tumors were maintained by serial transplantation in their strains of origin. Appropriate dilutions of single-cell suspensions of tumor were prepared and used for transplantation or challenge of treated mice. In the most recent studies, tumors were treated in one of the following ways: (1) completely excised, (2) frozen through the intact overlying skin, (3) frozen through the intact overlying skin and excised four hours after cryosurgery, (4) frozen through the intact overlying skin and excised 24 hours after cryosurgery, and (5) infarcted by ligation of the tumor. All groups were challenged 7 to 21 days after treatment of the primary tumor. Groups to be compared were all inoculated on the same day with appropriate dilutions of single-cell suspensions of tumor. The cumulative incidence of palpable tumor at the challenge site was recorded every three or four days. From the long and short diameters, individual tumor volumes were calculated from the formula V = 0.4 ab2 • 1 The significance of the differences when mean tumor volumes in the various groups were compared was tested with Student's t test.
Figure 1. Cumulative tumor incidences and mean tumor volumes after challenge with 104 viable tumor cells 21 days after treatment in C57BL/ 6J-sarcoma system. (From Nee!, H. B., Ill: Tumor immunology: An overview. In Ballenger, J. (ed.): Diseases of the Nose, Throat, and Ears. Edition 12. Philadelphia, Lea & Febiger, 1977, p. 529; reprinted with permission.)
BOO
o--o Control
.....__
......__..... Excised
E: ~
..--.Ligated
.., 700
~
~ ~
.....
C)
§ .....
600
- . . . Frozen
o--o
Coagulated
500 400
§ 300
~
200 100 8 1
30
766
H.
BRYAN NEEL
III
The relative strength of immunity can be interpreted from the percentage of mice in which tumor develops from the challenge inoculum, the latent period from the time of challenge and the appearance of tumor, and the mean sequential measurements of volume at the challenge site in those animals in which tumors develop at the challenge site. That information is shown in Figure 1, which represents a typical experiment. Tumors appeared early and enlarged rapidly in control groups (mice not previously exposed to tumor) and after excision. Either no tumor developed or the appearance of the tumor at the challenge site was less frequent after cryosurgery, ligation, and electrocoagulation of the primary tumor. There was a longer latent period before the tumor appeared in mice in which tumors had been frozen, ligated, or coagulated than in the mice in which tumors had been excised and in the control mice. Mean tumor volumes after ligation, electrocoagulation, or cryosurgery were significantly less than those observed in mice that had had tumor excised and in controls (P<0.05). Of the three methods of producing necrosis, electrocoagulation seemed to induce the least immunity, particularly as reflected in mean tumor volumes. This pattern of differential immunity was generally consistent after challenge with the various doses of tumor inocula in all the tumor-host systems that were studied.
1400
Figure 2. Cumulative tumor incidences and mean tumor volumes after challenge with 5 x 105 viable tumor cells 21 days after treatment in C57BLI 6J-sarcoma system. Frz-exc 4 h = frozen-excision 4 hours; Frz-exc 24 h = frozen-excision 24 hours. (From Nee!, H. B., III, and Ritts, R. E., Jr.: Immunotherapeutic effect of tumor necrosis after cryosurgery, electrocoagulation, and ligation. ]. Surg. Oncol., 11:45, 1979; reprinted with permission of Alan R. Liss, Inc.)
o--o Control .............., Excised
x-
Frz-exc 4 h
o----¢ Frz-exc 24 h
1000
....
0
--Frozen
600
§
.....
200 5
10
15
20
Days after challenge
25
IMMUNOTHERAPEUTIC EFFECT OF CRYOSURGICAL TUMOR NECROSIS
767
The differential immunity was somewhat less when the primary tumors were excised 4 or 24 hours after cryosurgical necrosis. Immunity was present but it was less than that seen when the necrotic tumor remained in the host and similar in degree to that which followed simple excision without cryosurgery (Fig. 2). 9 Both the cellular and the humoral arms of the immune mechanism are augmented by cryosurgery, as was shown by Faraci and associates in an elegant series of experiments that were performed in vitro. 2 They assessed differential cellular and humoral immunity after cryosurgery and excision, using an immunogenic methylcholanthrene-induced fibrosarcoma in C57BL mice. Lymphocyte-mediated cytotoxicity against tumor cells was markedly greater when cells from the cryosurgically treated mice were used as compared with cells from the excision (amputation) and control groups (Fig. 3). This immunity, which was markedly augmented for more than 14 days after cryosurgery, was shown to be specifically directed against the tumor because lymphocytes from cryosurgically treated tumor-bearing and normal mice were not cytotoxic to another type of tumor. Serum-mediated (humoral) cytotoxicity tests were also done. Serum containing complement-dependent antibodies from cryosurgically treated mice was extremely cytotoxic against tumor cells in vitro for the -
Fl Lymphocytes
----- A Lymphocytes TB Lymphocytes
03
- - FN Lymphocytes
u.J <..)
Ci ~
02
~ X
0
>0 >-
><..)
01
''
OL-------------------~~~------------~--~~~-
7
14
TIME FOLLOWING TREATMENT (days)
Figure 3. Results of lymphocyte-mediated cytotoxicity in frozen-immune (FI), amputation/excision (A), untreated tumor-bearing (TB), and frozen normal (FN) mice. (From Faraci, R. P., Bagley, D. H., Marrone, ]., et al.: In vitro demonstration of cryosurgical augmentation of tumor immunity. Surgery, 77:433, 1975; reprinted with permission of C. V. Mosbv Companv.)
768
H.
BRYAN NEEL
III
0.7
06
05
--
04
03
·~
····-I------FI A TB FN
02
I
.. -~
-----------1
Sere Sera Sera Sera
01
Figure 4. Results of serum-mediated cytotoxicity in frozen-immune (Fl), amputation/excision (A), untreated tumor-bearing (TB), and frozen normal (FN) mice. (From Faraci, R. P., Bagley, D. H., Marrone,]., et al.: In vitro demonstration of cryosurgical augmentation of tumor immunity. Surgery, 77:433, 197 5; reprinted with permission of C. V. Mosby Company.)
---~
--1. 0~~--~~--=----------------~---------------~ 14 21 7
TIME FOLLOWING TREATMENT (doy•l
21-day duration of the experiments (Fig. 4). 2 Serum from cryosurgically treated normal mice (no exposure to tumor) was not cytotoxic and therefore did not contain any antibodies directed against the tumor. These experiments in mice show that cell-mediated cytotoxicity and humoral cytotoxicity are augmented by cryosurgery. Neel and Ritts designed experiments to assess the relative immunotherapeutic effects of adoptive transfer of either viable lymphoid spleen cells or serum from normal and tumor-bearing mice. 8 In one of these experiments it was shown that the protective effect against challenge with the same tumor after "immune" serum or cells had been given was similar to that seen in mice after tumors had been destroyed cryosurgically. Cells and serum from normal animals did not convey a protective effect. In other words, a tumor-specific protective effect was evoked by augmentation of immunity, and the protection seen after cryosurgery of tumor was equivalent to that which could be transferred passively to normal animals by either immune lymphoid cells or serum.
SUMMARY It is clear, then, that cryosurgery augments immunity that is specifically directed against the tumor in several murine tumor systems. Both cell-mediated and humoral immunity are involved. The degree of augmentation of immunity is greater than that following a period of
IMMUNOTHERAPEUTIC EFFECT OF CRYOSURGICAL TUMOR NECROSIS
769
tumor growth and cold-knife excision and that following electrocoagulation of tumor. The degree of tumor-specific immunity after cryosurgery is not sufficient to alter growth rates of recurrent tumors, however. Although immunity is not markedly potentiated, it is sufficiently augmented to be effective in protecting against small doses of tumor cells of up to 106 cells, and presumably in the presence of minimal residual disease. It is possible that immunity could be potentiated further by the injection of accessible tumors with a nonspecific immunoadjuvant one to five days before cryosurgery. REFERENCES l. Attia, M.A.M., and Weiss, D.W.: Immunology of spontaneous mammary carcinomas
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
in mice. V: Acquired tumor resistance and enhancement in strain A mice infected with mammary tumor virus. Cancer Res., 26:1787, 1966. Faraci, R.P., Bagley, D. H., Marrone, J., eta!.: In vitro demonstration of cryosurgical augmentation of tumor immunity. Surgery, 77:433, 1975. Moore, F.T., Blackwood, J., Sanzenbacher, L., et a!.: Cryotherapy for malignant tumors: Immunologic response. Arch. Surg., 96:527, 1968. Nee!, H.B., III, and DeSanto, L.W.: Cryosurgical control of cancer: Effects of freeze rates, tumor temperatures, and ischemia. Ann. Otol. Rhino!. Laryngol., 82:716, 1973. Nee!, H.B., III, Ketcham, A.S., and Hammond, W.G.: Requisites for successful cryogenic surgery of cancer. Arch. Surg., I 02:45, 1971. Nee!, H.B., III, Ketcham, A.S., and Hammond, W.G.: Experimental evaluation of in situ oncocide for primary tumor therapy: Comparison of tumor-specific immunity after complete excision, cryonecrosis and ligation. Laryngoscope, 83:376, 1973. Nee!, H.B., III, and Ritts, R.E., Jr.: Immunotherapeutic effect of tumor necrosis in situ. Trans. Am. Acad. Ophthalmol. Otolaryngol., 84:285, 1977. Neel, H.B., III, and Ritts, R.E., Jr.: Transfer of tumor-specific immunity with syngeneic spleen cells and serum from mice that have large tumors and metastases. Cancer, 40:1643, 1977. Neel, H.B., Ill, and Ritts, R.E.,Jr.: Immunotherapeutic effect of tumor necrosis after cryosurgery, electrocoagulation, and ligation. J. Surg. Oncol., II :45, 1979. Ritts, R.E., Jr., and Neel, H. B., Ill: An overview of cancer immunology. Mayo Clin. Proc., 49:118, 1974. Soanes, W.A., Ahlin, R.J., and Gonder, M.J.: Remission of metastatic lesions following cryosurgery in prostatic cancer: Immunologic considerations. J. Urol., 104:154, 1970. Sobel, S.H., and Patterson, W.B.: Electrocoagulation vs. excision: Comparison of degree of immunity engendered by treatment of induced tumors in syngeneic mice. Surg. Forum, 23:85, 1972. Strauss, A.A., Appel, M., Saphir, 0., et al.: Immunologic resistance to carcinoma produced by electrocoagulation. Surg. Gynecol. Obstet., 121 :989, 1965.
Department of Otorhinolaryngology Mayo Clinic and Mayo Foundation Rochester, Minnesota 55901
Immunotherapeutic Effect of Cryosurgical Tumor Necrosis H. Bryan Neel III, M.D., Ph.D.*
Cryosurgery has been applied clinically for both cure and palliation of benign and malignant neoplasms. Detailed studies of temporal and physical factors have provided the information necessary to ensure a reasonable chance of therapeutic success. 4 •5 Cryosurgery not only has a local tumoricidal effect but also has systemic immunologic antitumor effects. Of particular interest has been the observation that destruction of tumor by cryosurgery leads to increased tumor-specific transplantation immunity. Tumor-specific transplantation immunity to tumors can be induced in animals by a variety of means, and early studies along these lines constitute one of the most important advances in cancer biology. Basic to this achievement was the development and ready availability of highly inbred, syngeneic strains of rodents. Now there is evidence that many human tumors contain tumorspecific antigens that are accessible for immunologic recognition and capable of inducing responses that are cytotoxic to tumor cells. 10 This has led to a resurgence of interest and investigation of immunotherapy for cancer and has provided the impetus to study, in the laboratory, the effects of tumor destruction by various means, including cryosurgery, on tumor-specific transplantation immunity and related immunologic parameters. Immunotherapy has the theoretical capacity of destroying all tumor cells, provided that the tumor burden is minimal-such as after a "curative" surgical procedure in which the primary tumor is completely excised yet tumor cells remain in circulation or in the form of clinically undetectable microscopic metastases. Modest or minimal toxicity is associated with immunotherapy in contrast to chemotherapy, which, in addition, may impair cell-mediated immunity directed against tumor. Unfortunately, immunotherapy may facilitate or enhance tumor *Consultant, Department of Otorhinolaryngology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota; Associate Professor, Mayo Medical School, Rochester, Minnesota Veterinary Clinics of North America: Small Animal Practice-Val. 10, No.4, November 1980
763
764
H.
BRYAN NEEL
III
growth, a hazard that must be considered, particularly if specific immunization is to be used. Therefore, cryosurgery is an attractive form of cancer treatment because it is highly successful in selected patients and it has a nontoxic immunotherapeutic effect. Necrosis of tumor 'in the host by cryosurgery or other methods, alone or in combination with excision, is unlikely to result in facilitation of tumor growth and will have a protective effect.
HISTORICAL PERSPECTIVE Little is known about the relationship between the nature and duration of the host's exposure to tumor antigen and the subsequent effectiveness of the host's immune response. Strauss et al. were the first to propose that destruction of tumor in the host augmented that host's resistance to tumor. 13 They found that partial electrocoagulation of human rectal tumors was followed by regression and disappearance of residual tumor in some cases. From this observation, they inferred that both local and systemic effects were induced by prolonged absorption of dead tumor tissue, including tumor-specific antigens presumably. Immunologic mechanisms in explanation of these observations in man were adduced from tumor challenge data in rabbits after electrocoagulation of Brown-Pearce tumors. Unfortunately, this host is not syngeneic and the tumor was not of recent origin, and furthermore immunogenicity of the tumor was not demonstrated; these are recurrent deficiencies in many studies of tumor immunobiology even today. However, these observations were confirmed by Sobel and Patterson, who found that electrocoagulation of tumor in a syngeneic murine tumor-bearing host produced greater tumor-specific transplantation immunity than that observed after amputation (excision) of tumor. 12 Others have since provided further confirmation. Moore et al. reported that cryosurgical treatment of several different human tumors induced an immune response that was apparently directed against the tumors. 3 Soanes et al. observed roentgenographic regression of pulmonary metastases in patients with prostatic carcinoma after cryosurgery of the primary tumor.U Addressing the issue directly in the laboratory, Neel and associates showed that tumor-specific transplantation immunity after challenge with the same tumor was greater after cryosurgery than it was after simple excision of the primary tumor. 5
RECENT STUDIES Several studies have been done to determine the relative effectiveness of cryosurgery and other methods of tumor necrosis in situ. The
765
IMMUNOTHERAPEUTIC EFFECT OF CRYOSURGICAL TUMOR NECROSIS
materials, methods, and experimental design have been described. 5 - 7 •9 Briefly, inbred strains of mice and tumors with demonstrable immunogenicity were used. Viral and chemically induced tumors were maintained by serial transplantation in their strains of origin. Appropriate dilutions of single-cell suspensions of tumor were prepared and used for transplantation or challenge of treated mice. In the most recent studies, tumors were treated in one of the following ways: (1) completely excised, (2) frozen through the intact overlying skin, (3) frozen through the intact overlying skin and excised four hours after cryosurgery, (4) frozen through the intact overlying skin and excised 24 hours after cryosurgery, and (5) infarcted by ligation of the tumor. All groups were challenged 7 to 21 days after treatment of the primary tumor. Groups to be compared were all inoculated on the same day with appropriate dilutions of single-cell suspensions of tumor. The cumulative incidence of palpable tumor at the challenge site was recorded every three or four days. From the long and short diameters, individual tumor volumes were calculated from the formula V = 0.4 ab2 • 1 The significance of the differences when mean tumor volumes in the various groups were compared was tested with Student's t test.
Figure 1. Cumulative tumor incidences and mean tumor volumes after challenge with 104 viable tumor cells 21 days after treatment in C57BL/ 6J-sarcoma system. (From Nee!, H. B., Ill: Tumor immunology: An overview. In Ballenger, J. (ed.): Diseases of the Nose, Throat, and Ears. Edition 12. Philadelphia, Lea & Febiger, 1977, p. 529; reprinted with permission.)
BOO
o--o Control
.....__
......__..... Excised
E: ~
..--.Ligated
.., 700
~
~ ~
.....
C)
§ .....
600
- . . . Frozen
o--o
Coagulated
500 400
§ 300
~
200 100 8 1
30
766
H.
BRYAN NEEL
III
The relative strength of immunity can be interpreted from the percentage of mice in which tumor develops from the challenge inoculum, the latent period from the time of challenge and the appearance of tumor, and the mean sequential measurements of volume at the challenge site in those animals in which tumors develop at the challenge site. That information is shown in Figure 1, which represents a typical experiment. Tumors appeared early and enlarged rapidly in control groups (mice not previously exposed to tumor) and after excision. Either no tumor developed or the appearance of the tumor at the challenge site was less frequent after cryosurgery, ligation, and electrocoagulation of the primary tumor. There was a longer latent period before the tumor appeared in mice in which tumors had been frozen, ligated, or coagulated than in the mice in which tumors had been excised and in the control mice. Mean tumor volumes after ligation, electrocoagulation, or cryosurgery were significantly less than those observed in mice that had had tumor excised and in controls (P<0.05). Of the three methods of producing necrosis, electrocoagulation seemed to induce the least immunity, particularly as reflected in mean tumor volumes. This pattern of differential immunity was generally consistent after challenge with the various doses of tumor inocula in all the tumor-host systems that were studied.
1400
Figure 2. Cumulative tumor incidences and mean tumor volumes after challenge with 5 x 105 viable tumor cells 21 days after treatment in C57BLI 6J-sarcoma system. Frz-exc 4 h = frozen-excision 4 hours; Frz-exc 24 h = frozen-excision 24 hours. (From Nee!, H. B., III, and Ritts, R. E., Jr.: Immunotherapeutic effect of tumor necrosis after cryosurgery, electrocoagulation, and ligation. ]. Surg. Oncol., 11:45, 1979; reprinted with permission of Alan R. Liss, Inc.)
o--o Control .............., Excised
x-
Frz-exc 4 h
o----¢ Frz-exc 24 h
1000
....
0
--Frozen
600
§
.....
200 5
10
15
20
Days after challenge
25
IMMUNOTHERAPEUTIC EFFECT OF CRYOSURGICAL TUMOR NECROSIS
767
The differential immunity was somewhat less when the primary tumors were excised 4 or 24 hours after cryosurgical necrosis. Immunity was present but it was less than that seen when the necrotic tumor remained in the host and similar in degree to that which followed simple excision without cryosurgery (Fig. 2). 9 Both the cellular and the humoral arms of the immune mechanism are augmented by cryosurgery, as was shown by Faraci and associates in an elegant series of experiments that were performed in vitro. 2 They assessed differential cellular and humoral immunity after cryosurgery and excision, using an immunogenic methylcholanthrene-induced fibrosarcoma in C57BL mice. Lymphocyte-mediated cytotoxicity against tumor cells was markedly greater when cells from the cryosurgically treated mice were used as compared with cells from the excision (amputation) and control groups (Fig. 3). This immunity, which was markedly augmented for more than 14 days after cryosurgery, was shown to be specifically directed against the tumor because lymphocytes from cryosurgically treated tumor-bearing and normal mice were not cytotoxic to another type of tumor. Serum-mediated (humoral) cytotoxicity tests were also done. Serum containing complement-dependent antibodies from cryosurgically treated mice was extremely cytotoxic against tumor cells in vitro for the -
Fl Lymphocytes
----- A Lymphocytes TB Lymphocytes
03
- - FN Lymphocytes
u.J <..)
Ci ~
02
~ X
0
>0 >-
><..)
01
''
OL-------------------~~~------------~--~~~-
7
14
TIME FOLLOWING TREATMENT (days)
Figure 3. Results of lymphocyte-mediated cytotoxicity in frozen-immune (FI), amputation/excision (A), untreated tumor-bearing (TB), and frozen normal (FN) mice. (From Faraci, R. P., Bagley, D. H., Marrone, ]., et al.: In vitro demonstration of cryosurgical augmentation of tumor immunity. Surgery, 77:433, 1975; reprinted with permission of C. V. Mosbv Companv.)
768
H.
BRYAN NEEL
III
0.7
06
05
--
04
03
·~
····-I------FI A TB FN
02
I
.. -~
-----------1
Sere Sera Sera Sera
01
Figure 4. Results of serum-mediated cytotoxicity in frozen-immune (Fl), amputation/excision (A), untreated tumor-bearing (TB), and frozen normal (FN) mice. (From Faraci, R. P., Bagley, D. H., Marrone,]., et al.: In vitro demonstration of cryosurgical augmentation of tumor immunity. Surgery, 77:433, 197 5; reprinted with permission of C. V. Mosby Company.)
---~
--1. 0~~--~~--=----------------~---------------~ 14 21 7
TIME FOLLOWING TREATMENT (doy•l
21-day duration of the experiments (Fig. 4). 2 Serum from cryosurgically treated normal mice (no exposure to tumor) was not cytotoxic and therefore did not contain any antibodies directed against the tumor. These experiments in mice show that cell-mediated cytotoxicity and humoral cytotoxicity are augmented by cryosurgery. Neel and Ritts designed experiments to assess the relative immunotherapeutic effects of adoptive transfer of either viable lymphoid spleen cells or serum from normal and tumor-bearing mice. 8 In one of these experiments it was shown that the protective effect against challenge with the same tumor after "immune" serum or cells had been given was similar to that seen in mice after tumors had been destroyed cryosurgically. Cells and serum from normal animals did not convey a protective effect. In other words, a tumor-specific protective effect was evoked by augmentation of immunity, and the protection seen after cryosurgery of tumor was equivalent to that which could be transferred passively to normal animals by either immune lymphoid cells or serum.
SUMMARY It is clear, then, that cryosurgery augments immunity that is specifically directed against the tumor in several murine tumor systems. Both cell-mediated and humoral immunity are involved. The degree of augmentation of immunity is greater than that following a period of
IMMUNOTHERAPEUTIC EFFECT OF CRYOSURGICAL TUMOR NECROSIS
769
tumor growth and cold-knife excision and that following electrocoagulation of tumor. The degree of tumor-specific immunity after cryosurgery is not sufficient to alter growth rates of recurrent tumors, however. Although immunity is not markedly potentiated, it is sufficiently augmented to be effective in protecting against small doses of tumor cells of up to 106 cells, and presumably in the presence of minimal residual disease. It is possible that immunity could be potentiated further by the injection of accessible tumors with a nonspecific immunoadjuvant one to five days before cryosurgery. REFERENCES l. Attia, M.A.M., and Weiss, D.W.: Immunology of spontaneous mammary carcinomas
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
in mice. V: Acquired tumor resistance and enhancement in strain A mice infected with mammary tumor virus. Cancer Res., 26:1787, 1966. Faraci, R.P., Bagley, D. H., Marrone, J., eta!.: In vitro demonstration of cryosurgical augmentation of tumor immunity. Surgery, 77:433, 1975. Moore, F.T., Blackwood, J., Sanzenbacher, L., et a!.: Cryotherapy for malignant tumors: Immunologic response. Arch. Surg., 96:527, 1968. Nee!, H.B., III, and DeSanto, L.W.: Cryosurgical control of cancer: Effects of freeze rates, tumor temperatures, and ischemia. Ann. Otol. Rhino!. Laryngol., 82:716, 1973. Nee!, H.B., III, Ketcham, A.S., and Hammond, W.G.: Requisites for successful cryogenic surgery of cancer. Arch. Surg., I 02:45, 1971. Nee!, H.B., III, Ketcham, A.S., and Hammond, W.G.: Experimental evaluation of in situ oncocide for primary tumor therapy: Comparison of tumor-specific immunity after complete excision, cryonecrosis and ligation. Laryngoscope, 83:376, 1973. Nee!, H.B., III, and Ritts, R.E., Jr.: Immunotherapeutic effect of tumor necrosis in situ. Trans. Am. Acad. Ophthalmol. Otolaryngol., 84:285, 1977. Neel, H.B., III, and Ritts, R.E., Jr.: Transfer of tumor-specific immunity with syngeneic spleen cells and serum from mice that have large tumors and metastases. Cancer, 40:1643, 1977. Neel, H.B., Ill, and Ritts, R.E.,Jr.: Immunotherapeutic effect of tumor necrosis after cryosurgery, electrocoagulation, and ligation. J. Surg. Oncol., II :45, 1979. Ritts, R.E., Jr., and Neel, H. B., Ill: An overview of cancer immunology. Mayo Clin. Proc., 49:118, 1974. Soanes, W.A., Ahlin, R.J., and Gonder, M.J.: Remission of metastatic lesions following cryosurgery in prostatic cancer: Immunologic considerations. J. Urol., 104:154, 1970. Sobel, S.H., and Patterson, W.B.: Electrocoagulation vs. excision: Comparison of degree of immunity engendered by treatment of induced tumors in syngeneic mice. Surg. Forum, 23:85, 1972. Strauss, A.A., Appel, M., Saphir, 0., et al.: Immunologic resistance to carcinoma produced by electrocoagulation. Surg. Gynecol. Obstet., 121 :989, 1965.
Department of Otorhinolaryngology Mayo Clinic and Mayo Foundation Rochester, Minnesota 55901