Specific Active Immunotherapy for Melanoma H. F. SEIGLER, M..D., E. COX, F. MUTZNER, L. SHEPHERD, E. NICHOLSON, W. W. SHINGLETON
The most effective immunization historically has been active immunization against a specific disease process. We report here the results of specific active immunization of 719 patients with invasive melanoma. The patients were sequentially immunized with approximately 2.5 x 107 x-irradiated, neuraminidase treated melanoma cells and BCG was used for its adjuvant effect. The sites of primary tumor location included trunk (42%), extremity (35%), head and neck (15%), unknown (3%), eye (2%) and mucocutaneous (1%). Two hundred thirty-six Clark's Level III patients with Stage I disease experienced an 88% survival ar four years while 47 Stage II patients had an observed survival of 82% during this same time period. Very similar statistics were observed for 124 State I Clark's Level IV patients and 70 Stage II patients. Nineteen and 26 Clark's Level V patients with Stage I and Stage II disease realized a 100% and 55% observed survival at four years respectively. Fifty-two patients with either an unknown primary and metastatic disease prior to immunotherapy or mucous membrane primaries with metastatic disease prior to immunization were observed to have a 65% survival at four years while invasive ocular primary patients without metastatic disease prior to immunotherapy had a 70% four year survival in the 19 patients evaluable. The largest number of patients fall in the first two years of follow-up, therefore, the degree of confidence concerning the survival statistics for years 3 and 4 is broad at this point in time. Further follow-up with careful evaluation will be required before definition statements can be made and comparisons can be elicited. The overall survival statistics do, however, encourage continued evaluation of specific active immunotherapy for patients with invasive melanoma with both Stage I and Stage II disease.
g UCH OF THE HOST-TUMOR interaction in man must IVi be studied in terms of membrane associated antigenic expression and the complexity of the various immune responses to these antigens. Normal human cells express both blood group antigens, and HLA-A, -B, -C and -D antigens. Tumor cells express these same blood group and HLA alloantigens but also express, in different concentrations, fetal antigens, Presented at the Annual Meeting of the American Surgical Association. Hot Springs, Virginia, April 26-28, 1979. Reprint requests: H. F. Seigler, M.D., Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710. Supported in part by CA 11265, General Clinical Cancer Research Center Grant, National Institutes of Health, and CA 20364, National Cancer Institute. Also supported by Program #821, Veterans Administration Hospital, Durham, North Carolina.
From the Departments of Surgery and Medicine, Duke University Medical Center and Veterans Administration Hospital, Durham, North Carolina
differentiation antigens, and tumor associated antigens that are both cross-reactive and individually specific. The HLA antigens segregate independently from tumor associated antigens. In recent hybridoma studies5 fused cells with deletion of the C6 chromosome do not express the serologically detectable HLA antigens but do express tumor associated antigens. The human histocompatibility Dr antigens have been reported to be important in both cell-cell interactions and for control of cell proliferation.7 Their recent detection on melanoma cells7 may indeed relate to the growth characteristics of these tumor cells and indicate their escape from immune destruction. The host responses to the membrane antigens of tumor cells involve lymphocytes, monocytes, and macrophages as well as other cell types. The immunity is not purely a cell mediated one for a variety of humoral immune responses have been described. Antimelanoma antibodies have been described in man"6 and their presence has been correlated with a favorable clinical course. The role of serum blocking factors, anti-idiotypic antibodies and immune complexes have been implicated in the abnormal immune regulation in the tumorbearing host. Transfer factor, immune RNA, and thymosin have had only ancedotal experience in clinical immunotherapy trials. Passive humoral and passive cellular attempts have had rather dramatic results in experimental animal models, but again clinical trials have been inconclusive. A number of nonspecific agents have been used as active immunopotentiators in clinical trials. The most popular ones have included levamisole, vaccinia, Corynebacterium Parvum and Bacillus Calmette-Guerin (BCG). Only limited success has been realized using these nonspecific agents. Specific active immunization has historically been our most efficient way of producing host immunity. For specific active immunization to be effective, the immunizing antigen should be accessible to host recog-
0003-4932/79/0900/0366 $00.85 ( J. B. Lippincott Company
366
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nition and possess a degree of immunogenicity. Host immune responsiveness must also be unimpaired. We have previously shown that human melanoma cells do indeed express tumor associated antigen(s) and this antigen(s) is immunogenic to most patients diagnosed with invasive melanomas. We report here our experience with specific active immunization in patients with Stage I and Stage II melanoma.
Materials and Methods Melanoma Cells The patients were actively immunized with approximately 2.5 x 107 x-irradiated (10,OOOr) neuraminidase treated melanoma cells, with BCG used as an adjuvant. Each immunization was spaced approximately four to six weeks apart, and appropriate clinical assessment was made to permit accurate staging of the disease. The tumor cells used in this study were established as primary tissue cultures. Both pigmented and nonpigmented melanoma cell lines were used for all experiments. The cultures were maintained in Eagle's Minimum Essential Medium plus 20% fetal calf serum. Each patient was immunized with only one tissue culture cell line. Both allogeneic and autochthonous immunizations were included.
Immunization Schedule All patients were immunized subcutaneously with approximately 2.5 x 107 x-irradiated, neuraminidase treated melanoma cells, either allogeneic or autochthonous. The tumor vaccine was admixed with 0.1 cc Glaxo BCG for its adjuvant effect. No patient experienced any serious side effect to the vaccine and no local tumors at the injection site have been produced. Each immunization was scheduled at intervals of approximately six weeks. Statistical Methods Survival curves are presented as determined by both the direct method and the product-limit acturial method.4 The latter method gives narrower 95% TABLE 1. Distribution of Primary Melanoma
Primary Site Trunk
Extremity Head and Neck Unk. Primary Eye
Mucocutaneous
No. Patients 411 (42%) 346 (35%) 151 (15%) 46 (3%) 19 (2%) 6 (1%) 979
367
TABLE 2. Melanoma Patients Receiving Spec ific Ac tive Immunotherapy
Clark III Clark IV Clark V Unk. Primary Mucocutaneous Ocular Evaluable Clark I-II Clark's Unk. Incomplete Data Total
Stage II
Stage I
Total
54 75 30
297 170 22
351 245 52 46 6 19 719
41
98
109 139 12 979
confidence intervals compared to the direct method. This effect is especially seen at the third and fourth year points for which relatively few patients are at risk because the majority of patients have entered the study within the past three years. Survival comparisons between subgroups were tested for significance by the Cox-Mantel test.2 Continuity corrected Chisquare was used for comparison of discrete variables. Results
Nine hundred seventy-nine patients entered the
study between 1972 and March, 1976. Incidence by
site of the primary tumor given in Table 1 reflects the distribution observed in most studies of primary melanoma. Four hundred and eleven patients had truncal primary lesions while 346 originated on the extremities, 151 had a head and neck primary, and in 46 the primary site was unknown. Eye primaries and mucocutaneous lesions were observed in 19 and six patients respectively. Of the 979 patients, the 109 patients with Clarks Level I or II lesions did not receive immunotherapy. An additional 139 patients (41 stage I, 98 stage II) with known primary site were excluded from analysis because of undetermined Clark's Level. Twelve additional patients were not evaluable for survival because of incomplete information. The remaining 719 patients were evaluable for survival. Of these, 351 had Clark's Level III lesions, 245 penetrated to Level IV and 52 were Level V lesions. Seventy-one patients could not be classified by Clark's Level because of an ocular (19), mucous membrane (6) or unknown primary (46) lesion. Direct survival rates calculated through the fourth year from initial surgical treatment are presented in Figures 1-5. The vertical bars which enclose 95% confidence intervals broaden noticeably beyond the second year of follow-up, emphasizing that the great majority of patients have been at risk for less than four years. Actuarial survival estimates are given in Figures 6-9.
368
SEIGLER AND OTHERS
MELANOMA PATIENTS (719) COMPLETED FOUR STAGES IMMUNOTHERAPY
MELANOMA PATIENTS (119) COMPLETED FOUR STAGES IMMUNOTHERAPY
lOOr
14 2
236 2
58
Ann. Surg. * September 1979
3
loor
26
19 1026
80j
1 41
.
80 132
t- 41
118
11i
t{28
4
p14
--J
I
60F
601 CLARK'S LEVEL m (351) ono met disease pre-immuno. e met disease pre- immuno
>
0)
0-
401
201
,
11
>e D
Lr)
401 CLARK'S LEVEL 2 (52) o no met. disease pre-immuno.
201
* met. disease pre-immuno.
OL
nL
v1/2
2 3 YEARS
1
4
FiG. 1. Direct survival rates-Clark's Level 111 (351). Melanoma patients completing four stages of immunotherapy.
MELANOMA PATIENTS (119) COMPLETED FOUR STAGES IMMUNOTHERAPY
100
124
452 T3
80~ {43
31
60F
21
The most effective immunization historically has been active immunization against a specific disease process. We report here the results of specific active immunization of 719 patients with invasive melanoma. The patients were sequentially immunized with approximately 2.5 x 107 x-irradiated, neuraminidase treated melanoma cells and BCG was used for its adjuvant effect. The sites of primary tumor location included trunk (42%), extremity (35%), head and neck (15%), unknown (3%), eye (2%) and mucocutaneous (1%). Two hundred thirty-six Clark's Level III patients with Stage I disease experienced an 88% survival ar four years while 47 Stage II patients had an observed survival of 82% during this same time period. Very similar statistics were observed for 124 State I Clark's Level IV patients and 70 Stage II patients. Nineteen and 26 Clark's Level V patients with Stage I and Stage II disease realized a 100% and 55% observed survival at four years respectively. Fifty-two patients with either an unknown primary and metastatic disease prior to immunotherapy or mucous membrane primaries with metastatic disease prior to immunization were observed to have a 65% survival at four years while invasive ocular primary patients without metastatic disease prior to immunotherapy had a 70% four year survival in the 19 patients evaluable. The largest number of patients fall in the first two years of follow-up, therefore, the degree of confidence concerning the survival statistics for years 3 and 4 is broad at this point in time. Further follow-up with careful evaluation will be required before definition statements can be made and comparisons can be elicited. The overall survival statistics do, however, encourage continued evaluation of specific active immunotherapy for patients with invasive melanoma with both Stage I and Stage II disease.
g UCH OF THE HOST-TUMOR interaction in man must IVi be studied in terms of membrane associated antigenic expression and the complexity of the various immune responses to these antigens. Normal human cells express both blood group antigens, and HLA-A, -B, -C and -D antigens. Tumor cells express these same blood group and HLA alloantigens but also express, in different concentrations, fetal antigens, Presented at the Annual Meeting of the American Surgical Association. Hot Springs, Virginia, April 26-28, 1979. Reprint requests: H. F. Seigler, M.D., Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710. Supported in part by CA 11265, General Clinical Cancer Research Center Grant, National Institutes of Health, and CA 20364, National Cancer Institute. Also supported by Program #821, Veterans Administration Hospital, Durham, North Carolina.
From the Departments of Surgery and Medicine, Duke University Medical Center and Veterans Administration Hospital, Durham, North Carolina
differentiation antigens, and tumor associated antigens that are both cross-reactive and individually specific. The HLA antigens segregate independently from tumor associated antigens. In recent hybridoma studies5 fused cells with deletion of the C6 chromosome do not express the serologically detectable HLA antigens but do express tumor associated antigens. The human histocompatibility Dr antigens have been reported to be important in both cell-cell interactions and for control of cell proliferation.7 Their recent detection on melanoma cells7 may indeed relate to the growth characteristics of these tumor cells and indicate their escape from immune destruction. The host responses to the membrane antigens of tumor cells involve lymphocytes, monocytes, and macrophages as well as other cell types. The immunity is not purely a cell mediated one for a variety of humoral immune responses have been described. Antimelanoma antibodies have been described in man"6 and their presence has been correlated with a favorable clinical course. The role of serum blocking factors, anti-idiotypic antibodies and immune complexes have been implicated in the abnormal immune regulation in the tumorbearing host. Transfer factor, immune RNA, and thymosin have had only ancedotal experience in clinical immunotherapy trials. Passive humoral and passive cellular attempts have had rather dramatic results in experimental animal models, but again clinical trials have been inconclusive. A number of nonspecific agents have been used as active immunopotentiators in clinical trials. The most popular ones have included levamisole, vaccinia, Corynebacterium Parvum and Bacillus Calmette-Guerin (BCG). Only limited success has been realized using these nonspecific agents. Specific active immunization has historically been our most efficient way of producing host immunity. For specific active immunization to be effective, the immunizing antigen should be accessible to host recog-
0003-4932/79/0900/0366 $00.85 ( J. B. Lippincott Company
366
VOl. 190.o NO. 3
IMMUNOTHERAPY FOR MELANOMA
nition and possess a degree of immunogenicity. Host immune responsiveness must also be unimpaired. We have previously shown that human melanoma cells do indeed express tumor associated antigen(s) and this antigen(s) is immunogenic to most patients diagnosed with invasive melanomas. We report here our experience with specific active immunization in patients with Stage I and Stage II melanoma.
Materials and Methods Melanoma Cells The patients were actively immunized with approximately 2.5 x 107 x-irradiated (10,OOOr) neuraminidase treated melanoma cells, with BCG used as an adjuvant. Each immunization was spaced approximately four to six weeks apart, and appropriate clinical assessment was made to permit accurate staging of the disease. The tumor cells used in this study were established as primary tissue cultures. Both pigmented and nonpigmented melanoma cell lines were used for all experiments. The cultures were maintained in Eagle's Minimum Essential Medium plus 20% fetal calf serum. Each patient was immunized with only one tissue culture cell line. Both allogeneic and autochthonous immunizations were included.
Immunization Schedule All patients were immunized subcutaneously with approximately 2.5 x 107 x-irradiated, neuraminidase treated melanoma cells, either allogeneic or autochthonous. The tumor vaccine was admixed with 0.1 cc Glaxo BCG for its adjuvant effect. No patient experienced any serious side effect to the vaccine and no local tumors at the injection site have been produced. Each immunization was scheduled at intervals of approximately six weeks. Statistical Methods Survival curves are presented as determined by both the direct method and the product-limit acturial method.4 The latter method gives narrower 95% TABLE 1. Distribution of Primary Melanoma
Primary Site Trunk
Extremity Head and Neck Unk. Primary Eye
Mucocutaneous
No. Patients 411 (42%) 346 (35%) 151 (15%) 46 (3%) 19 (2%) 6 (1%) 979
367
TABLE 2. Melanoma Patients Receiving Spec ific Ac tive Immunotherapy
Clark III Clark IV Clark V Unk. Primary Mucocutaneous Ocular Evaluable Clark I-II Clark's Unk. Incomplete Data Total
Stage II
Stage I
Total
54 75 30
297 170 22
351 245 52 46 6 19 719
41
98
109 139 12 979
confidence intervals compared to the direct method. This effect is especially seen at the third and fourth year points for which relatively few patients are at risk because the majority of patients have entered the study within the past three years. Survival comparisons between subgroups were tested for significance by the Cox-Mantel test.2 Continuity corrected Chisquare was used for comparison of discrete variables. Results
Nine hundred seventy-nine patients entered the
study between 1972 and March, 1976. Incidence by
site of the primary tumor given in Table 1 reflects the distribution observed in most studies of primary melanoma. Four hundred and eleven patients had truncal primary lesions while 346 originated on the extremities, 151 had a head and neck primary, and in 46 the primary site was unknown. Eye primaries and mucocutaneous lesions were observed in 19 and six patients respectively. Of the 979 patients, the 109 patients with Clarks Level I or II lesions did not receive immunotherapy. An additional 139 patients (41 stage I, 98 stage II) with known primary site were excluded from analysis because of undetermined Clark's Level. Twelve additional patients were not evaluable for survival because of incomplete information. The remaining 719 patients were evaluable for survival. Of these, 351 had Clark's Level III lesions, 245 penetrated to Level IV and 52 were Level V lesions. Seventy-one patients could not be classified by Clark's Level because of an ocular (19), mucous membrane (6) or unknown primary (46) lesion. Direct survival rates calculated through the fourth year from initial surgical treatment are presented in Figures 1-5. The vertical bars which enclose 95% confidence intervals broaden noticeably beyond the second year of follow-up, emphasizing that the great majority of patients have been at risk for less than four years. Actuarial survival estimates are given in Figures 6-9.
368
SEIGLER AND OTHERS
MELANOMA PATIENTS (719) COMPLETED FOUR STAGES IMMUNOTHERAPY
MELANOMA PATIENTS (119) COMPLETED FOUR STAGES IMMUNOTHERAPY
lOOr
14 2
236 2
58
Ann. Surg. * September 1979
3
loor
26
19 1026
80j
1 41
.
80 132
t- 41
118
11i
t{28
4
p14
--J
I
60F
601 CLARK'S LEVEL m (351) ono met disease pre-immuno. e met disease pre- immuno
>
0)
0-
401
201
,
11
>e D
Lr)
401 CLARK'S LEVEL 2 (52) o no met. disease pre-immuno.
201
* met. disease pre-immuno.
OL
nL
v1/2
2 3 YEARS
1
4
FiG. 1. Direct survival rates-Clark's Level 111 (351). Melanoma patients completing four stages of immunotherapy.
MELANOMA PATIENTS (119) COMPLETED FOUR STAGES IMMUNOTHERAPY
100
124
452 T3
80~ {43
31
60F
21
