Annals of the Royal College of Surgeons of England (1978) vol 6o
ASPECTS OF TREATMENT*
Immune response and non-specific immunotherapy in melanoma Ali A El-Domeiri MS FRCS FACS Associate Professor of Surgery, Abraham Lincoln School of Medicine, University of Illinois, Chicagot
Summary Tumour-associated antigens and specific immune response to these antigens have been clearly demonstrated in patients with melanoma. Impaired cellular immune reactions are evident in patients with progressive and disseminated disease. Immunotherapy is used to heighten the host resistance and hence prevent recurrence and spread of the tumour. BCG vaccine has been used to produce nonspecific stimulation of the immune system. Preliminary evaluation of the effect of adjuvant systemic BCG therapy suggests that the treatment mav have a beneficial effect on patients with early melanoma.
Introduction Accumulated evidence indicates that a relationship exists between host resistance and the development and progression of malignant tumours. A number of clinical observations and the results of several experimental studies in recent years strongly suggest that host resistance against cancer is a function of the immune system. Tumour antigens and specific immune responses to these antigens have been clearly demonstrated by serological methods in patients with various types of malignant neoplasm'-3. Furthermore, significant impairment of the immune responses has been encountered in patients with cancer, the degree of impairment being correlated with the extent of the disease and the response to treatment4'7. These findings provided the
rationale for using immunotherapy to heighten host resistance and hence inhibit the progression and spread of tumour. The purpose of this study was to evaluate the immune responses in patients with potentially curable melanoma as well as the effect of adjuvant non-specific immtunotherapy on the course of the disease.
Methods IN-VIVO SKIN TEST Cell-mediated reactivity was measured by the in-vivo procedure of skintesting, which produces a delayed localized allergic response (delayed hypersensitivity reaction). A battery of commonly encountered skin-test antigens was used to test for preexisting delayed hypersensitivity in 42 patients with various stages of melanoma. These antigens included dermatophytin 0 (I:I 00 candida antigen), mumps, purified protein derivative (PPD) of tuberculin (intermediate strength), and streptokinase (SK)/streptodornase (SD) (SK, 4U/SD, i U) and were injected intradermally in a volume of o.i ml. The tests were read at 24 and 48 h and the reaction considered positive if the diameter of induration was more than 5 mm. Also these patients were tested for their ability to develop
delayed hypersensitivity to dinitrochloroben-
zene (DNCB)5.
Cellular immunity was measured by an in-vitro assay of the reactivity of lymphocytes and macrophages in tisstue response of fAddress: Cook Coumty Hospital, 1825 West culture. Assay of the blastogenic as such to phytohaemmitogens lymphocytes Harrison, Chicago, Illinois, 60612. IN-VITRO TEST
*Fellows and Members interested in submitting papers for consideration with publication in this series should first write to the Editor.
a
view to
ii8
Ali A El-Domeiri
Results SKIN TEST Reaction to at least one of the common antigens was noted in 647o of patients with Stages I (local disease) and 2 (disease spread to regional lymph nodes) melanoma. In contrast a positive reaction was observed in only 25 7 of patients with Stage 3 disease (generalized metastases). A positive reaction to one or both test doses of DNCB was recorded in go97 of the patients with Stages I and 2 disease. In contrast, only 71 7 of those with Stage 3 disease showed a positive response. Assay of the blastogenic IN-VITRO TEST response of lymphocytes to mitoigens revealed that 3 out of 9 patients with regional metaFIG. I Vaccination with BCG using tine stases had an SR comparable to that of normal multipuncture technique to give 72 punctures. individuals, whereas all 5 patients with widespread metastases exhibited marked depression of the lymphocyte blastogenic response. agglutinin and concavalin A was used to evaluate immunocompetence in I 4 patients before and during immunotherapy. After Immunotherapy incubation of lymphocytes in tissue culture with mitogens for a few days the degree of A total of 42 patients received systemic BCG stimulation was measured by tritiated thy- therapy. Of these, 14 patients had Stage 2 midine incorporation and recorded as counts disease and were treated with systemic BCG per minute. The results of each assay were following surgical extirpation of the tumour. reported as stimulation ratio (SR) between the At the end of 2 years 7 of these I 4 patients were living free of disease. Neither tumour stimulated and control cultures. regression nor prolongation of survival could be observed in 28 melanoma patients who' had Immunotherapy distant metastases (Stage 3). Patients who The Tice strain of BCG vaccine* was admin- failed to convert a negative PPD reaction and istered in a dose of 0.2 mnIt using the tine those having a low SR of lymphocyte multipuncture technique, with double applica- blastogenic response had rapidly progressive tion of the disc to give 72 punctures (Fig. i). disease and were dead in less than 6 months. Vaccination was performed weekly for i Also aggravation of pre-existing symptoms was month, once every 2 weeks for 2 months, and noted in patients with visceral metastases. Five then once a month for a total period of i patients with lung metastases developed year. Also direct injection of BCG was used dyspnoea, persistent cough, and haemoptysis to treat cutaneous metastases. Depending on a few weeks after initiation of BCG therapy. the size of the lesion, 0.1-0.3 ml of the BCG Two more patients with suspected brain vaccine diluted to give 5XIO7 CFU/ml was metastases developed acute signs of increased used for intralesional injection. A maximum intracranial tension. of 4 lesions were treated at the same time. Intralesional injection of BCG was used in small group of patients who developed a *Developed by the Department of Microbiology, University of Illinois Medical Center, Tice Research cutaneous metastases after receiving a full Laboratories Chicago. course of adjuvant systemic BCG therapy. t ml contains 5X I 08 colony-forming organisms Although regression of the injected lesions was noted in 2 patients, more secondary deposits (CFU).
Immune response and non-specific immunotherapy in melanoma
metastases in patient FIG. 2 Cutaneous following wide excision of melanoma of the trunk and regional node dissection. Five cutaneous lesions were injected with BCG at 2 sittings. Two of the lesions, the uppermost and the one adjacent to the scar, were injected first. Although these lesions show evidence of regression more nodules continued to appear at other sites.
appeared at other sites (Fig. 2). Systemic reaction such as chills, fever, and malaise were encountered more frequently in patients treated with intralesional injections of BCG than in those receiving systemic therapy. Marked liver dysfunction and severe thrombocytopenia developed in one patient. Discussion The results of this study confirm that cutaneous delayed hypersensitivity reaction to common antigens as well as in-vitro assay of lymphocyte stimulation in tissue culture is useful in evaluating the immunocompetence of patients
I I9
with melanoma. The rationale of using nonspecific active immunization with BCG is based on the finding that the vaccine produces a generalized stimulation of the host's immune responses8' 9. Also there is experimental evidence to suggest that macrophages and histocytes activated by BCG may process tumour-associated antigens with the ultimate production of specific immunity"1' ". A beneficial effect of BCG therapy in patients with melanoma has been reported by Eilber et al."2. These authors noted that during a follow-up period of 30 months there was a lower incidence of recurrent and metastatic disease in patients receiving postoperative adjuvant BCG therapy than in a group of patients treated by operation alone. In the present study only 50% of patients with Stage 2 melanoma receiving adjuvant BCG therapy remain free of disease at 2 years. BCG immunotherapy alone did not seem to influence the course of the disease in patients with disseminated melanoma. Similar results. have been reported by other investigators in patients with advanced disease". Failure of immunotherapy in these patients was attributed to the lack of immunocompetence and the large tumour burden. Also it is conceivable that aggravation of symptoms in patients with visceral metastases is the result of stimulation of tumour growth by BCG; animal experiments have revealed that under certain conditions BCG may accelerate tumour growth'4. Intralesional injection of BCG, although effective in controlling cutaneous metastases, produced little or no effect on secondary deposits at other sites and may be accompanied by severe systemic reactions. Direct injection of BCG into, the tumour produced regression of cutaneous metastases of melanoma. Pinsky et al.7, using intralesional BCG, treated 29 melanoma patients with cutaneous metastases and observed regression of injected lesions in 2I. In 2 of these patients regression of uninjected lesions was also observed. The effect of intralesional injection of BCG is attributed to a local delayed hypersensitivity response induced by the vaccine within the tumour'6. It is possible that the adverse effect encountered with intralesioinal injection of the vaccine is due to the
I20
Ali A El Domeiri
release of a large volume of BCG into, the circulation through the rich vascular supply of the metastatic lesion. The use of smaller doses of BCG, as well as the prophylactic administration of antihistamines and antituberculosis agents, may reduce the incidence of these toxic manifestations. To avoid these severe reactions epilesional scarification with BCG has been advocated by Richman et al.17 as an alternative to intralesional injection.
References I
2
3 4 5 6
Hersh, E M, Freireich, E J, and McCredie, K B (1974) in Recent Results in Cancer Research, p. 25. New York, Springer. Lewis, M G, Ikonopisov, R L, Nairn, R C, Phillips, T M, Fairley, G H, Bodenham, D C, and Alexanider, P (I969) British Medical Journal, 3, 547. Fass, L, Herberman, R B, and Ziegler, J L (I970) New England Journal of Medicine, 282, 776. Al-Sarraf, M, Wong, P, Sardesai, S, and Vaitkelvicius, V K (1970) Cancer, 26, 262. El-Domeiri, A A (1972) International Surgery, 57, 617. Pinsky, C M, Oettgen, H F, and El-Domeiri, A A (5975) Proceedings of the American Association for Cancer Research, I2, 100.
7 Eilbe-r, F R, and Morton, D L (1970) Cancer, 25, 362. 8 Richards, V (I969) Ainerican Journal of Surgery,
ii8, 490. 9 Old, L J, ancl Clarke, D A
(1959) Nature, I84,
291.
Io
ii
12 13
I4 15 i6
17
Zbar, B, Bernstein, I D, Bartlett, G L, Hanna, M G, anid Rapp, H J (I972) Journal of the National Cancer Institute, 49, 119. Cleveland, R P, Meltzer, M' S, and Zbar, B (I974) Journal of the National Cancer Institute, 52, I887. Eilber, F R, Morton, D L, Holmes, C, Sparks, F C, and Ramming, K P (1976) New Enigland Journal of Medicine, 294, 237. Bast, R C, Zbar, B, Borsos, T, and Rapp, H J (I974) New England Journal of Medicine, 290, I458. Kaliss, N (I966) Annals of the New York Academy of Sciences, I29, I55. Pinsky, C, Hirshaut, Y, and Oettgen, H (I973) National Cancer Institute Monographs, 39, 225. Mastrangelo, M J, Sulit, H L, Prehn, L M, Bomstein, R S, Yarbo, J W, and Prehn, R T (1976) Cancer, 37, 684. Richman, S P, Mavligit, G M, Wolk, R, Gutterman, J U, and Hersh, E M (975) Journal of the American Medical Association, 234, I 233.
ASPECTS OF TREATMENT*
Immune response and non-specific immunotherapy in melanoma Ali A El-Domeiri MS FRCS FACS Associate Professor of Surgery, Abraham Lincoln School of Medicine, University of Illinois, Chicagot
Summary Tumour-associated antigens and specific immune response to these antigens have been clearly demonstrated in patients with melanoma. Impaired cellular immune reactions are evident in patients with progressive and disseminated disease. Immunotherapy is used to heighten the host resistance and hence prevent recurrence and spread of the tumour. BCG vaccine has been used to produce nonspecific stimulation of the immune system. Preliminary evaluation of the effect of adjuvant systemic BCG therapy suggests that the treatment mav have a beneficial effect on patients with early melanoma.
Introduction Accumulated evidence indicates that a relationship exists between host resistance and the development and progression of malignant tumours. A number of clinical observations and the results of several experimental studies in recent years strongly suggest that host resistance against cancer is a function of the immune system. Tumour antigens and specific immune responses to these antigens have been clearly demonstrated by serological methods in patients with various types of malignant neoplasm'-3. Furthermore, significant impairment of the immune responses has been encountered in patients with cancer, the degree of impairment being correlated with the extent of the disease and the response to treatment4'7. These findings provided the
rationale for using immunotherapy to heighten host resistance and hence inhibit the progression and spread of tumour. The purpose of this study was to evaluate the immune responses in patients with potentially curable melanoma as well as the effect of adjuvant non-specific immtunotherapy on the course of the disease.
Methods IN-VIVO SKIN TEST Cell-mediated reactivity was measured by the in-vivo procedure of skintesting, which produces a delayed localized allergic response (delayed hypersensitivity reaction). A battery of commonly encountered skin-test antigens was used to test for preexisting delayed hypersensitivity in 42 patients with various stages of melanoma. These antigens included dermatophytin 0 (I:I 00 candida antigen), mumps, purified protein derivative (PPD) of tuberculin (intermediate strength), and streptokinase (SK)/streptodornase (SD) (SK, 4U/SD, i U) and were injected intradermally in a volume of o.i ml. The tests were read at 24 and 48 h and the reaction considered positive if the diameter of induration was more than 5 mm. Also these patients were tested for their ability to develop
delayed hypersensitivity to dinitrochloroben-
zene (DNCB)5.
Cellular immunity was measured by an in-vitro assay of the reactivity of lymphocytes and macrophages in tisstue response of fAddress: Cook Coumty Hospital, 1825 West culture. Assay of the blastogenic as such to phytohaemmitogens lymphocytes Harrison, Chicago, Illinois, 60612. IN-VITRO TEST
*Fellows and Members interested in submitting papers for consideration with publication in this series should first write to the Editor.
a
view to
ii8
Ali A El-Domeiri
Results SKIN TEST Reaction to at least one of the common antigens was noted in 647o of patients with Stages I (local disease) and 2 (disease spread to regional lymph nodes) melanoma. In contrast a positive reaction was observed in only 25 7 of patients with Stage 3 disease (generalized metastases). A positive reaction to one or both test doses of DNCB was recorded in go97 of the patients with Stages I and 2 disease. In contrast, only 71 7 of those with Stage 3 disease showed a positive response. Assay of the blastogenic IN-VITRO TEST response of lymphocytes to mitoigens revealed that 3 out of 9 patients with regional metaFIG. I Vaccination with BCG using tine stases had an SR comparable to that of normal multipuncture technique to give 72 punctures. individuals, whereas all 5 patients with widespread metastases exhibited marked depression of the lymphocyte blastogenic response. agglutinin and concavalin A was used to evaluate immunocompetence in I 4 patients before and during immunotherapy. After Immunotherapy incubation of lymphocytes in tissue culture with mitogens for a few days the degree of A total of 42 patients received systemic BCG stimulation was measured by tritiated thy- therapy. Of these, 14 patients had Stage 2 midine incorporation and recorded as counts disease and were treated with systemic BCG per minute. The results of each assay were following surgical extirpation of the tumour. reported as stimulation ratio (SR) between the At the end of 2 years 7 of these I 4 patients were living free of disease. Neither tumour stimulated and control cultures. regression nor prolongation of survival could be observed in 28 melanoma patients who' had Immunotherapy distant metastases (Stage 3). Patients who The Tice strain of BCG vaccine* was admin- failed to convert a negative PPD reaction and istered in a dose of 0.2 mnIt using the tine those having a low SR of lymphocyte multipuncture technique, with double applica- blastogenic response had rapidly progressive tion of the disc to give 72 punctures (Fig. i). disease and were dead in less than 6 months. Vaccination was performed weekly for i Also aggravation of pre-existing symptoms was month, once every 2 weeks for 2 months, and noted in patients with visceral metastases. Five then once a month for a total period of i patients with lung metastases developed year. Also direct injection of BCG was used dyspnoea, persistent cough, and haemoptysis to treat cutaneous metastases. Depending on a few weeks after initiation of BCG therapy. the size of the lesion, 0.1-0.3 ml of the BCG Two more patients with suspected brain vaccine diluted to give 5XIO7 CFU/ml was metastases developed acute signs of increased used for intralesional injection. A maximum intracranial tension. of 4 lesions were treated at the same time. Intralesional injection of BCG was used in small group of patients who developed a *Developed by the Department of Microbiology, University of Illinois Medical Center, Tice Research cutaneous metastases after receiving a full Laboratories Chicago. course of adjuvant systemic BCG therapy. t ml contains 5X I 08 colony-forming organisms Although regression of the injected lesions was noted in 2 patients, more secondary deposits (CFU).
Immune response and non-specific immunotherapy in melanoma
metastases in patient FIG. 2 Cutaneous following wide excision of melanoma of the trunk and regional node dissection. Five cutaneous lesions were injected with BCG at 2 sittings. Two of the lesions, the uppermost and the one adjacent to the scar, were injected first. Although these lesions show evidence of regression more nodules continued to appear at other sites.
appeared at other sites (Fig. 2). Systemic reaction such as chills, fever, and malaise were encountered more frequently in patients treated with intralesional injections of BCG than in those receiving systemic therapy. Marked liver dysfunction and severe thrombocytopenia developed in one patient. Discussion The results of this study confirm that cutaneous delayed hypersensitivity reaction to common antigens as well as in-vitro assay of lymphocyte stimulation in tissue culture is useful in evaluating the immunocompetence of patients
I I9
with melanoma. The rationale of using nonspecific active immunization with BCG is based on the finding that the vaccine produces a generalized stimulation of the host's immune responses8' 9. Also there is experimental evidence to suggest that macrophages and histocytes activated by BCG may process tumour-associated antigens with the ultimate production of specific immunity"1' ". A beneficial effect of BCG therapy in patients with melanoma has been reported by Eilber et al."2. These authors noted that during a follow-up period of 30 months there was a lower incidence of recurrent and metastatic disease in patients receiving postoperative adjuvant BCG therapy than in a group of patients treated by operation alone. In the present study only 50% of patients with Stage 2 melanoma receiving adjuvant BCG therapy remain free of disease at 2 years. BCG immunotherapy alone did not seem to influence the course of the disease in patients with disseminated melanoma. Similar results. have been reported by other investigators in patients with advanced disease". Failure of immunotherapy in these patients was attributed to the lack of immunocompetence and the large tumour burden. Also it is conceivable that aggravation of symptoms in patients with visceral metastases is the result of stimulation of tumour growth by BCG; animal experiments have revealed that under certain conditions BCG may accelerate tumour growth'4. Intralesional injection of BCG, although effective in controlling cutaneous metastases, produced little or no effect on secondary deposits at other sites and may be accompanied by severe systemic reactions. Direct injection of BCG into, the tumour produced regression of cutaneous metastases of melanoma. Pinsky et al.7, using intralesional BCG, treated 29 melanoma patients with cutaneous metastases and observed regression of injected lesions in 2I. In 2 of these patients regression of uninjected lesions was also observed. The effect of intralesional injection of BCG is attributed to a local delayed hypersensitivity response induced by the vaccine within the tumour'6. It is possible that the adverse effect encountered with intralesioinal injection of the vaccine is due to the
I20
Ali A El Domeiri
release of a large volume of BCG into, the circulation through the rich vascular supply of the metastatic lesion. The use of smaller doses of BCG, as well as the prophylactic administration of antihistamines and antituberculosis agents, may reduce the incidence of these toxic manifestations. To avoid these severe reactions epilesional scarification with BCG has been advocated by Richman et al.17 as an alternative to intralesional injection.
References I
2
3 4 5 6
Hersh, E M, Freireich, E J, and McCredie, K B (1974) in Recent Results in Cancer Research, p. 25. New York, Springer. Lewis, M G, Ikonopisov, R L, Nairn, R C, Phillips, T M, Fairley, G H, Bodenham, D C, and Alexanider, P (I969) British Medical Journal, 3, 547. Fass, L, Herberman, R B, and Ziegler, J L (I970) New England Journal of Medicine, 282, 776. Al-Sarraf, M, Wong, P, Sardesai, S, and Vaitkelvicius, V K (1970) Cancer, 26, 262. El-Domeiri, A A (1972) International Surgery, 57, 617. Pinsky, C M, Oettgen, H F, and El-Domeiri, A A (5975) Proceedings of the American Association for Cancer Research, I2, 100.
7 Eilbe-r, F R, and Morton, D L (1970) Cancer, 25, 362. 8 Richards, V (I969) Ainerican Journal of Surgery,
ii8, 490. 9 Old, L J, ancl Clarke, D A
(1959) Nature, I84,
291.
Io
ii
12 13
I4 15 i6
17
Zbar, B, Bernstein, I D, Bartlett, G L, Hanna, M G, anid Rapp, H J (I972) Journal of the National Cancer Institute, 49, 119. Cleveland, R P, Meltzer, M' S, and Zbar, B (I974) Journal of the National Cancer Institute, 52, I887. Eilber, F R, Morton, D L, Holmes, C, Sparks, F C, and Ramming, K P (1976) New Enigland Journal of Medicine, 294, 237. Bast, R C, Zbar, B, Borsos, T, and Rapp, H J (I974) New England Journal of Medicine, 290, I458. Kaliss, N (I966) Annals of the New York Academy of Sciences, I29, I55. Pinsky, C, Hirshaut, Y, and Oettgen, H (I973) National Cancer Institute Monographs, 39, 225. Mastrangelo, M J, Sulit, H L, Prehn, L M, Bomstein, R S, Yarbo, J W, and Prehn, R T (1976) Cancer, 37, 684. Richman, S P, Mavligit, G M, Wolk, R, Gutterman, J U, and Hersh, E M (975) Journal of the American Medical Association, 234, I 233.
