DISCUSSION PAPER: EFFECT OF HEMOLYTIC STREPTOCOCCI ON LYMPHOCYTIC LEUKEMIA IN AKR MICE H. F. Havas Department oj Microbiology und Itnmirnology Temple University School of Medicine Philadelphia, Pcnnsylvanirr I9140
In this afternoon’s session, the effectiveness of nonspecific immunostimulation by Bacillus Calmette-GuCrin (BCG) as well as its limitations were discussed.’. The speakers stressed the necessity of a small tumor burden and high dose of BCG for successful therapy. The therapeutic effects, particularly when used in conjunction with chemotherapy, were prolongation of periods of remission and survival for patients with postoperative breast cancer and with melanoma and myelogenous leukemia. BCG injection resulted in a granulocytomatous reaction at both the tumor site and at regional lymph nodes. One speaker who speculated on the mechanism of BCG action postulated that the “nonspecific” immunostimulants may increase “specific” immunity to tumors that share antigenic determinants with the bacterial adjuvant.:’ Previous reports of remission of tumors in patients after streptococcal infection, notably erysipelas,’ and the successful in vitro treatment of Ehrlich ascites tumor prompted us to reinvestigate the effect of living streptococci on several transplantable murine tumors and on a spontaneously occurring lymphocytic leukemia that arises in AKR mice.’; The experiments I will report here include both in vivo and in vitro streptococcal treatment of spontaneous and transplanted lymphocytic leukemias of AKR mice. In Vivo TREATMENT
Mice with established spontaneous lymphocytic leukemia (ascertained by their white blood cell and differential counts) were injected with 0.20 ml of a 20-hr streptococcal suspension (stationary phase), followed by three daily injections of 10,000 U of penicillin to control the streptococcal infection. This procedure produced no cures or prolongation of survival compared to untreated mice. However, when mice were infected with streptococci 1 day prior to or up to 2 days past a 1-3 x 106 leukemic spleen cell injection, they showed some increase in survival compared to recipient control mice that received no treatment. However, eventually all mice succumbed to leukemia.6 In Vitro PRETREATMENT
Following the protocol of Koshirnura et a!.,: spleen cell suspensions were incubated in vitro for 1.5-2.5 hr at 37” C with a 20-hr culture of Streptococcus pyogenes group A (of a strain designated “SA” by Koshimura) . Subsequently, 1-3 X 10” of these streptococcally pretreated spleen cells were in-
299
300
Annals New York Academy of Sciences
jected into recipient mice in groups of eight. The two control groups received either untreated spleen cells o r streptococcal suspensions without spleen cells. The parameters followed in the recipient mice were leukemia incidence, survival time, peripheral lymphocyte and differential counts, and histopathologic findings of lymphoid tissue on autopsy. I n five similar experiments, we found that 24 of 40 mice that received pretreated spleen cells showed no signs of leukemia up to 4% months later, whereas all 40 control mice succumbed to leukemia. Some of the former group died of infection several days after receiving the streptococcal spleen cell mixture, whereas others proceeded to develop leukemia with some delay. There was an average increase in survival time from 14 days in the controls to 30 days in recipients of pretreated cells and a delay in the increase of peripheral white blood cells and lymphoblasts. At autopsy, control mice typically showed greatly enlarged spleens (up to 1 g), livers, and lymph nodes. Histopathology of these enlarged organs showed extensive leukemic cell infiltration by lymphoblasts of lymph nodes, liver, spleen, thymus, and, sometimes, lungs. Peripheral WBCs numbered up to 299,000, of which as many as 10% were immature blast cells. Leukemic infiltration of bone marrow could not be ascertained. I n contrast, animals that received streptococcally treated tumor cells had normal sized organs but showed an intensive inflammatory response in the lymphoid organs. This inflammatory response may well have been a factor that contributed to the absence of leukemia in 24 of 40 mice and to prolongation of survival in others. (Autopsy findings of these 24 mice up to 4 months after injection of leukemic cells showed no leukemic infiltration.) Another group of mice injected only with streptococci and penicillin also showed inflammatory responses in spleen and lymphoid tissue. Pretreatment of spleen cells of 49 mice (in groups of eight) with either heat-killed streptococci or mixed bacterial toxins from Serratia and Streptococcus were totally ineffective. The effectiveness of hemolytic streptococci against leukemia spleen cells paralleled that found against other transplantable tumors.?. Pretreatment of sarcoma 37 or Krebs-2 carcinoma ascites tumor cells with several strains of hemolytic streptococci resulted in oncolysis of the tumor cells within minutes, whereas treatment with Streptococcirs faecalis or Serrutia marcescens had no effect.'. Purified streptococcal extracts and streptolysin S were as effective as the living streptococci.' If streptococci are effective in vitro in lysing tumor o r leukemic spleen cells, what are the possible reasons for the failure in our experiments to affect the final outcome in the in vivo-treated leukemic host? The tumor load may have been too great for the number of streptococci injected. Rapp mentioned that BCG is only effective against a small number of tumor cells (108).9 The injection of 1.5-3.5 x 10'; leukemic spleen cells per mouse may have provided a leukemic cell overload. A critical ratio of streptococci to tumor cells may be a prerequisite for successful immunotherapy. This critical ratio may not have been reached in the unsuccessfully in vitro-pretreated cells and was not achieved in the in vivo experiments. The ratio of streptococci to tumor cells measured in some experiments was approximately 190:1 and may have been close to borderline. The treatment of the host with penicillin decreased the number of viable
Havas: Hemolytic Streptococci
30 1
streptococci, and this decrease further altered the ratio of bacterial to tumor cells, which reduced their effectiveness.
It is also known that penicillin suppresses the immune response.1o Administration of repeated injection of smaller dosages of streptococci might obviate the subsequent penicillin treatment. What is the lesson from these studies? Obviously, preincubation of a patient’s leukemic cells with streptococci is not a practical measure, unless this treatment would enhance their antigenicity for specific immunization. However, the demonstrated oncolytic ability, albeit in vitro, of certain strains of hemolytic streptococci on several transplantable mouse tumors and on leukemic spleen cells, probably due to the vast array of proteolytic enzymes, warrants further investigation of streptococci as possible oncolytic agents in the living host, despite the initial negative results in in vivo-treated mice. The intratumor injection of streptococci, their use in combination with chemotherapy, shown to be effective with BCG, also warrants further investigation. In this Conference, it was quite apparent that although we have achieved some successes, we still do not understand the mode of action of these “nonspecific” immunostimulants nor whether they are truly nonspecific. A greater emphasis should be placed on studying the mechanism of action of the “nonspecific’’ bacterial agents if we are to develop a rational approach to the immunotherapy of cancer and leukemia. REFERENCES 1. GUTTERMAN, J. U., G. M. MAVLIGIT, M. A. BURGESS, C. M. MCBRIDE& E. M.
HERSH.This monograph. 2. PINSKY, C. This monograph. 3. BAST,R. C., JR., B. S. BAST& H. J . RAPP.This monograph. 4. COLEY,W. B. 1893. The treatment of malignant tumors by repeated inoculations of erysipelas, with a report of original cases. Amer. J. Med. Sci. 105: 487-5 11. 5. KOSHIMURA, S., K. MURASAWA, E. NAKAGAWA, M. VEDA, M. BANDOY & R. HIRATA.1955. Experimental anticancer studies. Part 111. On the influence of 6. 7. 8. 9. 10.
living hemolytic streptococci upon the invasion power of Ehrlich ascites carcinoma in mice. Jap. J. Exp. Med. 25: 93-102. HAVAS, H. F. & A. J. DONNELLY. 1963. Unpublished data. HAVAS,H. F. 1964. The cytotoxic effects of hemolytic streptococci and streptococcal products on ascites tumor cells. Third International Congress of Chemotherapy. : 1096-1 103. George Thieme Verlag. HAVAS, H. F., A. J. DONNELLY & A. V. PORRECA. 1963. The cytotoxic effects of hemolytic streptococci on ascites tumor cells. Cancer Res. 23: 700-706. RAPP, H., S. J. KLEINSCHUSTER, D. C. LUEKER& R. A. KAINER.1976. Immunotherapy of experimental cancer as a guide to the treatment of human cancer. Ann. N.Y. Acad. Sci. 276: 550-556. RAEBURN, J. A. 1972. Antibiotics and immunodeficiency 19. Lancet.
In this afternoon’s session, the effectiveness of nonspecific immunostimulation by Bacillus Calmette-GuCrin (BCG) as well as its limitations were discussed.’. The speakers stressed the necessity of a small tumor burden and high dose of BCG for successful therapy. The therapeutic effects, particularly when used in conjunction with chemotherapy, were prolongation of periods of remission and survival for patients with postoperative breast cancer and with melanoma and myelogenous leukemia. BCG injection resulted in a granulocytomatous reaction at both the tumor site and at regional lymph nodes. One speaker who speculated on the mechanism of BCG action postulated that the “nonspecific” immunostimulants may increase “specific” immunity to tumors that share antigenic determinants with the bacterial adjuvant.:’ Previous reports of remission of tumors in patients after streptococcal infection, notably erysipelas,’ and the successful in vitro treatment of Ehrlich ascites tumor prompted us to reinvestigate the effect of living streptococci on several transplantable murine tumors and on a spontaneously occurring lymphocytic leukemia that arises in AKR mice.’; The experiments I will report here include both in vivo and in vitro streptococcal treatment of spontaneous and transplanted lymphocytic leukemias of AKR mice. In Vivo TREATMENT
Mice with established spontaneous lymphocytic leukemia (ascertained by their white blood cell and differential counts) were injected with 0.20 ml of a 20-hr streptococcal suspension (stationary phase), followed by three daily injections of 10,000 U of penicillin to control the streptococcal infection. This procedure produced no cures or prolongation of survival compared to untreated mice. However, when mice were infected with streptococci 1 day prior to or up to 2 days past a 1-3 x 106 leukemic spleen cell injection, they showed some increase in survival compared to recipient control mice that received no treatment. However, eventually all mice succumbed to leukemia.6 In Vitro PRETREATMENT
Following the protocol of Koshirnura et a!.,: spleen cell suspensions were incubated in vitro for 1.5-2.5 hr at 37” C with a 20-hr culture of Streptococcus pyogenes group A (of a strain designated “SA” by Koshimura) . Subsequently, 1-3 X 10” of these streptococcally pretreated spleen cells were in-
299
300
Annals New York Academy of Sciences
jected into recipient mice in groups of eight. The two control groups received either untreated spleen cells o r streptococcal suspensions without spleen cells. The parameters followed in the recipient mice were leukemia incidence, survival time, peripheral lymphocyte and differential counts, and histopathologic findings of lymphoid tissue on autopsy. I n five similar experiments, we found that 24 of 40 mice that received pretreated spleen cells showed no signs of leukemia up to 4% months later, whereas all 40 control mice succumbed to leukemia. Some of the former group died of infection several days after receiving the streptococcal spleen cell mixture, whereas others proceeded to develop leukemia with some delay. There was an average increase in survival time from 14 days in the controls to 30 days in recipients of pretreated cells and a delay in the increase of peripheral white blood cells and lymphoblasts. At autopsy, control mice typically showed greatly enlarged spleens (up to 1 g), livers, and lymph nodes. Histopathology of these enlarged organs showed extensive leukemic cell infiltration by lymphoblasts of lymph nodes, liver, spleen, thymus, and, sometimes, lungs. Peripheral WBCs numbered up to 299,000, of which as many as 10% were immature blast cells. Leukemic infiltration of bone marrow could not be ascertained. I n contrast, animals that received streptococcally treated tumor cells had normal sized organs but showed an intensive inflammatory response in the lymphoid organs. This inflammatory response may well have been a factor that contributed to the absence of leukemia in 24 of 40 mice and to prolongation of survival in others. (Autopsy findings of these 24 mice up to 4 months after injection of leukemic cells showed no leukemic infiltration.) Another group of mice injected only with streptococci and penicillin also showed inflammatory responses in spleen and lymphoid tissue. Pretreatment of spleen cells of 49 mice (in groups of eight) with either heat-killed streptococci or mixed bacterial toxins from Serratia and Streptococcus were totally ineffective. The effectiveness of hemolytic streptococci against leukemia spleen cells paralleled that found against other transplantable tumors.?. Pretreatment of sarcoma 37 or Krebs-2 carcinoma ascites tumor cells with several strains of hemolytic streptococci resulted in oncolysis of the tumor cells within minutes, whereas treatment with Streptococcirs faecalis or Serrutia marcescens had no effect.'. Purified streptococcal extracts and streptolysin S were as effective as the living streptococci.' If streptococci are effective in vitro in lysing tumor o r leukemic spleen cells, what are the possible reasons for the failure in our experiments to affect the final outcome in the in vivo-treated leukemic host? The tumor load may have been too great for the number of streptococci injected. Rapp mentioned that BCG is only effective against a small number of tumor cells (108).9 The injection of 1.5-3.5 x 10'; leukemic spleen cells per mouse may have provided a leukemic cell overload. A critical ratio of streptococci to tumor cells may be a prerequisite for successful immunotherapy. This critical ratio may not have been reached in the unsuccessfully in vitro-pretreated cells and was not achieved in the in vivo experiments. The ratio of streptococci to tumor cells measured in some experiments was approximately 190:1 and may have been close to borderline. The treatment of the host with penicillin decreased the number of viable
Havas: Hemolytic Streptococci
30 1
streptococci, and this decrease further altered the ratio of bacterial to tumor cells, which reduced their effectiveness.
It is also known that penicillin suppresses the immune response.1o Administration of repeated injection of smaller dosages of streptococci might obviate the subsequent penicillin treatment. What is the lesson from these studies? Obviously, preincubation of a patient’s leukemic cells with streptococci is not a practical measure, unless this treatment would enhance their antigenicity for specific immunization. However, the demonstrated oncolytic ability, albeit in vitro, of certain strains of hemolytic streptococci on several transplantable mouse tumors and on leukemic spleen cells, probably due to the vast array of proteolytic enzymes, warrants further investigation of streptococci as possible oncolytic agents in the living host, despite the initial negative results in in vivo-treated mice. The intratumor injection of streptococci, their use in combination with chemotherapy, shown to be effective with BCG, also warrants further investigation. In this Conference, it was quite apparent that although we have achieved some successes, we still do not understand the mode of action of these “nonspecific” immunostimulants nor whether they are truly nonspecific. A greater emphasis should be placed on studying the mechanism of action of the “nonspecific’’ bacterial agents if we are to develop a rational approach to the immunotherapy of cancer and leukemia. REFERENCES 1. GUTTERMAN, J. U., G. M. MAVLIGIT, M. A. BURGESS, C. M. MCBRIDE& E. M.
HERSH.This monograph. 2. PINSKY, C. This monograph. 3. BAST,R. C., JR., B. S. BAST& H. J . RAPP.This monograph. 4. COLEY,W. B. 1893. The treatment of malignant tumors by repeated inoculations of erysipelas, with a report of original cases. Amer. J. Med. Sci. 105: 487-5 11. 5. KOSHIMURA, S., K. MURASAWA, E. NAKAGAWA, M. VEDA, M. BANDOY & R. HIRATA.1955. Experimental anticancer studies. Part 111. On the influence of 6. 7. 8. 9. 10.
living hemolytic streptococci upon the invasion power of Ehrlich ascites carcinoma in mice. Jap. J. Exp. Med. 25: 93-102. HAVAS, H. F. & A. J. DONNELLY. 1963. Unpublished data. HAVAS,H. F. 1964. The cytotoxic effects of hemolytic streptococci and streptococcal products on ascites tumor cells. Third International Congress of Chemotherapy. : 1096-1 103. George Thieme Verlag. HAVAS, H. F., A. J. DONNELLY & A. V. PORRECA. 1963. The cytotoxic effects of hemolytic streptococci on ascites tumor cells. Cancer Res. 23: 700-706. RAPP, H., S. J. KLEINSCHUSTER, D. C. LUEKER& R. A. KAINER.1976. Immunotherapy of experimental cancer as a guide to the treatment of human cancer. Ann. N.Y. Acad. Sci. 276: 550-556. RAEBURN, J. A. 1972. Antibiotics and immunodeficiency 19. Lancet.
