Inhibition of Leukocyte Migration by Tumor-Associated Antigens in Soluble Extracts of Human Malignant Melanoma 1,2 J. L. MCCOy,3 L. F. Jerome," J. H. Dean,3 E. Perlin,4 R. K. Oldham," D. H. Char," M. H. Cohen," E. L. Felix,a and R. B. Herberman 5
!UMOR-A.SSOCIATED ANTI~ENS (TAA) and an Im!fiu~olo&lc host resp~mse agamst such antlget;Is may ex~st, m VI~W of ?CCaSlOna~ spontaneous regressions of pnmary lesions or metastatic nodules (1, 2). Recently, investig~tors ~ave us~d antibody techniques (3-8) and cell-medl.ate~ Immumty. (CMI) me.thods. (9-25). to deteet specific Immunologic reactions m patients With melanoma. A variety of CMI assays have been employed, i.e., in vivo delayed hypersensitivity skin reactions to membrane and solubilized extracts of autologous and allogeneic melanoma tumor cells (14-18), microcytotoxicity assays with tissue-culture target cells derived from melanomas (9-13), lymphocyte stimulation tests (19, 20), and migration inhibition (LMI) assays (21-25) with particulate saline extracts of melanomas. Cochran et al. (21, 23, 25) and Mackie et al. (22) analyzed their LMI data in . . I treatment 0 f tepa h tilen t an d re Ianon to t h e surglca found that positive preoperative LMI reactivity Iredi d h 1 f (1-4 d ) quent I y Isappeare sort yater surgery ays , but rapidly returned thereafter (5-22 days later). We applied the LMI assay recently developed in our • • laboratory (26) to study CMI reactivity agamst T AA in patients with malignant melanoma. In addition, the . . I d f . I' o b serve d LMI reactlVIty was ana yze or ItS re anon to the clinical stage of disease and to surgical therapy.
MATERIALS AND METHODS
Patients.-Whole blood and tissues from patients with malignant melanoma, other cancers, or benign nevi, and from normal adults were used. The patients were studied at the Surgery Branch of the National Cancer Institute at the Clinical Center of the National Institutes of Health or at the National Naval Medical Center. Normal donor leukocytes were obtained from the Blood Bank of the National Institutes of Health and from laboratory workers. Subjects were those in various stages of disease: only with local tumor (stage 1), with extension into the regional lymph nodes (stage 2) , and with metastatic disease (stage 3) . Some patients from the Naval Medical Center were on chemotherapy shortly before the tests. The total included 64 melanoma patients (9 were retested 2-5 times) and 43 normal donors. Virtually all individuals were immunocompetent and not anergic, as determined by delayed cutaneous skin reactivity against at least one standard antigen. Preparation of leukocytes.-Leukocyte migration was studied as previously described (26). Whole blood (50-60 ml) was collected in heparin and allowed to settle either by gravity sedimentation at 37 0 C for 1 hour or with the use of Plasmagel. The leukocytes (with some contamination with red blood cells) were resuspended in McCoy's 5A medium and washed twice with medium. The cell pellet was then resuspended in MeCoy's 5A medium supplemented with 10% heat-inactivated (56 0 C for 30 min) fetal calf serum, and the cell concentration was adjusted to 2-3 X 107 leukocytesyrnl. Antigen preparation.-Fresh melanoma or other type of tumor or benign nevus tissue (within 4-8 hr of surgery) was prepared as described in (26). Five to 20 ml (depending on the size of the original tumor, which was usually 1-5 g) of 3 M KCI was added to the cell suspension (prepared by mincing and homogenization in a Waring blender) and incubated at 4 0 C for 16-24 hours. This mixture was then centrifuged at 40,000 X g for 60 minutes in a Sorvall RC-2, desalted for several hours against phosphate-buffered saline, centrifuged an additional 15 minutes at 40,000 X g, and concentrated tenfold with an Amicon ultrafiltration unit (UM 10 membrane). Aliquots of each extract were stored at -70 0 C. The total was 11 melanom~ extracts, 7 other c~ncers (lung, breast, and colon carcinomas), and 1 bemgn nevus extract. . . . ~e were also concerned that ba~tenal.conta!fimatlon of tissues used for 3 M KCl extraction might yield soluReceived December 26, 1974; accepted March 26, 1975. Supported in part by Public Health Service contract NIH-NCI72-3227 from the National Cancer Institute. 3 Department of Immunology, Litton Bionetics, Inc., 5516 Nicholson Lane, Kensin~t.on, Md. 20795.. . 4 Internal Medicine Department, National Naval Medical Center, Bethesda, Md. 20014. 5 Laboratory of Immunodiagnosis, National Cancer Institute, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 6 Surgery Branch, National Cancer Institute. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO. I, JULY 1975
19
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SUMMARY-Direct leukocyte migration inhibition (LMI) assays were performed to investigate whether cell-mediated immune reactions could be detected In response to tumor-associated antigens of human melanoma. The antigens were 3 M KCI-soluble extracts of different fresh melanomas, other cancers, and benign nevus tissue. A total of 48 of the 79 (61 %) blood samples from melanoma patients (64 patients) reacted with extracts of melanoma tissue. Since the subjects were usually tested with two or three extracts, 57/134 (42%) tests with melanoma patients' leukocytes were inhibited by KCI extracts of melanoma tissue, whereas only 3/50 (6%) tests with leukocytes of normal donors and 4/27 (15%) with patients having other cancers gave positive results. No positive reactions were obtained when 13 melanoma patients were tested with a 3 M KCI extract of benign nevus tissue. Likewise, only 2/26 (8%) positive tests were obtained from melanoma patients tested with extracts of other cancers. Individuals in all stages of disease had similar incidences of positive reactions to the soluble melanoma extracts, except for patients with stage-l disease who exhibited a somewhat higher incidence of reactivity. The highest incidence of reactivity was observed in patients before surgical resection of the tumor, and somewhat decreased reactivity was seen 0-14 days post surgery. The results Indicate that the direct LMI assay may be used to measure cell immune reactivity against melanomaassociated antigens. Since many of the positive results were obtained with allogeneic extracts, the results also indicate that different melanomas possess common antigens.-J Natl Cancer Inst 55: 19-23, 1975.
20
MCCOY ET AL.
Mean of migration of 4 replicates in the presence of antigen M.I.= Mean of migration in the absence of antigen Replicate areas of migration were almost always within 10% of each other and were frequently within a
2-3% range. In statistical evaluation of the data, a twotailed ranking and Student's r-test were performed to establish P values (28). M.1. values of 0.80 or less were considered positive. We picked greater than or equal to 0.80 as a conservative value for positive LMI reactivity; this value has been used by other investigators (27, 29) . Because of the close values obtained with the four replicates, these values were always significantly different from an M.1. of 1.0 (P 1 year
_ _ _ _ _ _
14/19 1/4 7/10 6/12 4/7 16/27
TotaL
_
48/79 (61)
• See footnote to table 2.
(74) (25) (70) (50) (57) (59)
a/
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. l!l
IIELANGIIA IXTIIACTI WITH IIALIGIlANT IlILANOIlA L IUlCOCYTII
22
MCCOY ET AL.
The patients with melanoma were often tested with more than one melanoma extract. Degrees of reactivity were different with the various extracts. Table 4 presents the results obtained with four extracts. Three of the four gave good reactions (>40% of patients tested were positive), whereas one extract (5969) gave no positive reactions. DISCUSSION
TABLE 4.-LMI reactions of melanoma patients and normal donors with different melanoma extracts KCl extract
3194
Total positive/ total tested
4143
3539
5969
Normal donors
Melanoma patients
Normal donors
Melanoma patients
Normal donors
Melanoma patients
Normal donors
Melanoma patients
0.92 0.91 0.95 1.09 0.94 0.88 0.92 0.91 1.06 0.94
0.80 0.70 0.78 0.85 0.70 0.83 0.91 0.80 0.73 0.81 0.73 0.98 0.97 0.89 0.60 0.76 0.99 0.60 0.80
0.97 0.96 1.02 0.92 0.93 1.05 0.88
0.96 0.64 0.92 0.80 0.90 1.43 0.75 0.71 0.60 0.77 0.92 0.74 0.84 1.02 1.29 0.98 0.90 0.70 0.90
0.87 1.03 0.91 0.84 0.85 1.10 0.94
0.72 0.70 0.90 0.87 0.84 0.81 0.74 0.64 0.76 0.72 0.61 0.57 0.83 0.76 0.75
0.94 1.04 1.12 0.86 1.00 1.09 1.11 1.04
0.98 0.96 0.81 0.91 1.07
0/10
11/19 (53%)
0/7
8/19 (42%)
0/7
11/16 (62%)
0/8
0/5
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A series of reports (21-25) indicated the usefulness of the direct LMI assay for study of the cellular immune reactivity of melanoma patients to antigens in particulate saline extracts of melanoma cells. Our data extend the earlier reports and demonstrate that 48 of 79 (61%) tests with leukocytes of melanoma patients (64 patients) showed reactivity with 3 M KCl-soluble extracts of melanoma tissue. Although we tried to control the reactions with extracts of other types of cancer (25), the specificity of the reactions observed earlier was not extensively evaluated. We examined closely the specificity of the reactions elicited by soluble 3 M KCl extracts. Whereas 42% of tests of patients with malignant melanoma showed posi tive reactions wi th extracts of melanoma, only 6% of normal adults and 15% of patients with other tumors gave positive reactions with the same extracts. No melanoma patient reacted positively to an extract of a benign nevus, and only a few individuals reacted with extracts of other types of cancer. The breast cancer extracts used here gave considerably more reactions in patients with carcinoma of the breast (26). From these results, the reactions of melanoma patients with the KCl extracts of melanoma appear directed against melanoma-associated antigens. The observed inhibition did not seem due 'to toxicity of the tumor extracts, since at the concentrations of extracts used in the assays, migration of leukocytes from controls was usually not affected. The pattern of results is also not consistent with reactivity against histocompatibility antigens in the
extracts. In addition, 5 normal donors were multiparous females who did not react with the melanoma extracts. Also, our total experience with cancer patient reactivity against 3 M KCl extracts of normal tissues from liver, colon, lung, and breast has been that only 2/76 (3%) had positive LMI reactions. Further, none of 13 melanoma patients reacted against a 3 M KCl benign nevus extract. Since the tests were performed with allogeneic melanoma extracts, the results also indicate that different melanomas possess common antigens. This agrees with earlier reports of common T AA on human melanoma cells in migration inhibition assays (21-25), microcytotoxicity tests with cells cultured from melanomas (9-13, 30), lymphocyte stimulation assays (19, 20), and delayed hypersensitivity skin reactions (14-18). The Hellstroms (30) further suggested that melanoma cells may have more than one T AA, since lymphocytes from some patients were more reactive on some lines of cultivated melanoma cells than on others. Our results are consistent with this suggestion, because some melanoma patients reacted well with one KCl extract of melanoma tissue but failed to react well with a second or third extract. We found that leukocytes of normal black donors were not inhibited by melanoma extracts. The reports by the Hellstroms and their colleagues (10, 30) that lymphocytes from many normal black donors were cytotoxic to tissue culture cells derived from melanoma tissues suggested that this group has natural immunologic reactivity against melanoma cells. Although our data do not support this suggestion, the assays (LMI vs. microcytotoxicity) and the sources of antigen (extracts of fresh melanoma tissue vs, intact cultured cells) were sufficiently different to account for the divergent results. Variations in antigenicity of the 3 M KCl tissue extracts used in this study could have significantly affected the overall incidence of observed reactivity, since some extracts give much higher percentages of reactivity than
MIGRATION INHIBITION ASSAYS OF HUMAN MELANOMA
(8)
(9) (10)
(II) (12) (JJ)
(14) (15) (16) (17)
(18) (19) (20)
(21) (22) (23) (24) (25)
REFERENCES (1) EVERSON TC, COLE WF: Spontaneous Regression of Cancer. Philadelphia, WB Saunders, 1966 (2) BOYD W: The Spontaneous Regression of Cancer. Springfield, Illinois, CC Thomas, 1966 (3) MORTON DL, MALMGREN RA, HOLMES EC, et al: Demonstration of antibodies against malignant melanoma by immunofluorescence. Surgery 64:233-240, 1968 (4) MUNA NM, MARCUS S, SMART C: Detection by immunofluorescence of antibodies specific for human malignant melanoma cells. Cancer 23:88-93, 1969 (5) LEWIS MG, IKONOPISOV RL, NAIRN RC, et al: Tumour specific antibodies in human malignant melanoma and their relationships to the extent of the disease. Br Med J 3:547-552, 1969 (6) LEWIS MG: Malignant melanoma of the sole of the foot in Uganda: The role of pigmentation. Br J Cancer 21:483, 1967 (7) NAIRN RC, NIND APP, GULl EPG, et al: Specific immune
(26)
(27)
(28) (29) (30) (31)
response to human skin carcinoma. Br Med J 4:701-705. 1971 FEDERMAN JL, LEWIS MG, CLARK WH: Tumor-associated antibodies to ocular and cutaneous malignant melanomas: Negative interactions with normal choroidal melanocytes. J Nat! Cancer Inst 52:587-589, 1974 HELLSTROM I, HELLSTROM KE, SJOGREN HO, et al: Demonstration of cell-mediated immunity to human neoplasms of various histological types. Int J Cancer 7: 1-16, 1971 HELLSTROM I, HELLSTROM KE, SJOGREN HO, et al: Destruction of cultivated melanoma cells by lymphocytes from healthy Black (North American Negro) donors. Int J Cancer 11:116-123, 1973 FOSSATI G, COLNAGHI MI, DELLA PORTA G, et al: Cellular and humoral immunity against human malignant melanoma. Int J Cancer 8:344-350, 1971 DE VRIES JE, RUMKE P, BERNHEIM JL: Cytotoxic lymphocytes in melanoma patients. Int J Cancer 9:567-576, 1972 BYRNE M, HEPPNER G, STOLBACH L, et al: Tumor immunity in melanoma patients as assessed by colony inhibition and microcytotoxicity methods: A preliminary report. Natl Cancer Inst Monogr 37:3-8, 1973 STEWART TH: The presence of delayed hypersensitivity reactions in patients toward cellular extracts of their malignant tumors. Cancer 23: 1368-1379, 1969 FASS L, HERBERMAN RB, ZIEGLER JL, et al: Cutaneous hypersensitivity reactions to autologous extracts of malignant melanoma cells. Lancet 1:116--118, 1970 BLUMING AZ. VOGEL CL, ZIEGLF.R ]L. et al: Delayed hypersensitivity reactions to extracts of autologous malignant melanoma: A second look. J Natl Cancer Inst 48:17-24,1972 CHAR DH, HOLLINSHEAD A, COGAN EG, et al: Cutaneous delayed hypersensitivity reactions to soluble melanoma antigen in patients with acute malignant melanoma. N Engl J Med 291:274-277, 1974 HOLLINSHEAD AC, HERBERMAN RB, JAFFURS WJ, et al: Soluble membrane antigens of human malignant melanoma cells. Cancer 34: 1235-1243, 1974 JEHN UW, NATHANSON L, SCHWARTZ RS, et al: In vitro lymphocyte stimulation by a soluble antigen from malignant melanoma. N Engl J Med 283:329-333, 1970 NAGEL GA, PIESSENS WF, STiLMENT MM, et al: Evidence for tumour-specific immunity in human malignant melanoma. Eur J Cancer 7:41--47, 1971 COCHRAN AJ, JEHN UW, GOTHOSKAR BP: Cell-mediated immunity to malignant melanoma. Lancet 1:134(}-1341, 1972 MACKIE RM. SPILG WG, THOMAS CE, et al: Cell-mediated immunity in patients with malignant melanoma. Br J Derrnatol 37:523-528, 1972 COCHRAN AJ, SPILG WG, MACKIE RM, et al: Post-operative depression of tumour-directed cell-mediated immunity in patients with malignant disease. Br Med J 4:67-70, 1972 SEGALL A, WEILER 0, GENIN J, et al: In vitro study of cellular immunity against autochthonous human cancer. Int J Cancer 9:417--425, 1972 COCHRAN AJ, RHOMAS CE, SPILG WG, et al: Tumor directed cellular immunity in malignant melanoma and its modification by surgical treatment. Yale J BioI Med 46:65(}-654, 1973 McCoy JL, JEROME LF, DEAN JH, et al: Inhibition of leukocyte migration by tumor-associated antigens in soluble extracts of human breast carcinoma. J Nat! Cancer Inst 53:1117, 1974 ROSENBERG SA, DAVID JR: Inhibition of leukocyte migration: An evaluation of this in vitro assay of delayed hypersensitivity in man to soluble antigen. J Immunol 105:14471452, 1970 SNEDECOR GW, COCHRAN WG: Statistical Methods. Ames, Iowa, Iowa State Univ Press, 1971, pp 13(}-131 ANDERSEN U, BJERRUM 0, BENDIXEN G, et al: Effect of autologous mammary tumour extracts on human -Ieukocvte migration in vitro. Int J Cancer 5:357-363, 1970 HELLSTROM I, HELLSTROM KE: Some recent studies on cellular immunity to human melanomas. Fed Proc 32:156--159, 1973 ROCKLIN R: Products of activated lymphocytes: Leukocyte inhibitory factor (LIF) distinct from migration inhibitory factor (MIF). J Immunol 112:1461-1466, 1974
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others. This may have been due to variations in the proportion of melanoma cells versus normal cells in the tumor mass used for antigen extraction, the stage of the tumor (e.g., primary vs. metastatic), or various other factors. It will be particularly important to identify large extracts that have good demonstrable LMI reactivity with melanoma patients and to use these as the standard preparations. We found no definitive correlation between LMI reactivity and stage of disease. Some previous studies of CMI and humoral immunity in melanoma patients reported correlations with the clinical stage, but others did not (3, 5, 15, 20-23, 25). Such differences could be ascribed to variations in the patient populations or the therapy, or to important factors related to the assay systems or the antigens used. In regard to reactivity in relation to surgery, Cochran et al. (23) found depressed reactivity in patients at 1-4 days after surgery and then a rapid increase in reactivity after 5-22 days. Our data, with depressed reactivity possibly seen in the 0- to 10-day postoperative period with 4 patients, and showing recovery during the 11- to 30-day postsurgery period, are in agreement with those of Cochran et al. We, however, noted good reactivity in many patients with metastatic disease. The effects of disease status and therapy on the reactivity in LMI will be satisfactorily determined only when serial studies on the same patients with the same antigen extracts for each test are performed. Also, it will be important to determine whether correlations in delayed cutaneous skin reactivity of the patients correlate with the LMI results. Some of this has been done, but most skin tests have been performed with extracts other than 3 M KCl material. We are currently conducting a study directly testing the same patients in both skin and LMI assays with the same extracts. Finally, our study used the direct migration inhibition assay rather than the indirect method which uses guinea pig macrophages as the indicator cells. Possibly, the direct method with leukocytes measures different factors, as described by Rocklin (31). Studies are now in progress to look at supernatants generated by the interaction of patient leukocytes with 3 M KCl extracts to determine whether the observed results were due to production of leukocyte inhibitory factor and/or macrophage inhibitory factor.
23
!UMOR-A.SSOCIATED ANTI~ENS (TAA) and an Im!fiu~olo&lc host resp~mse agamst such antlget;Is may ex~st, m VI~W of ?CCaSlOna~ spontaneous regressions of pnmary lesions or metastatic nodules (1, 2). Recently, investig~tors ~ave us~d antibody techniques (3-8) and cell-medl.ate~ Immumty. (CMI) me.thods. (9-25). to deteet specific Immunologic reactions m patients With melanoma. A variety of CMI assays have been employed, i.e., in vivo delayed hypersensitivity skin reactions to membrane and solubilized extracts of autologous and allogeneic melanoma tumor cells (14-18), microcytotoxicity assays with tissue-culture target cells derived from melanomas (9-13), lymphocyte stimulation tests (19, 20), and migration inhibition (LMI) assays (21-25) with particulate saline extracts of melanomas. Cochran et al. (21, 23, 25) and Mackie et al. (22) analyzed their LMI data in . . I treatment 0 f tepa h tilen t an d re Ianon to t h e surglca found that positive preoperative LMI reactivity Iredi d h 1 f (1-4 d ) quent I y Isappeare sort yater surgery ays , but rapidly returned thereafter (5-22 days later). We applied the LMI assay recently developed in our • • laboratory (26) to study CMI reactivity agamst T AA in patients with malignant melanoma. In addition, the . . I d f . I' o b serve d LMI reactlVIty was ana yze or ItS re anon to the clinical stage of disease and to surgical therapy.
MATERIALS AND METHODS
Patients.-Whole blood and tissues from patients with malignant melanoma, other cancers, or benign nevi, and from normal adults were used. The patients were studied at the Surgery Branch of the National Cancer Institute at the Clinical Center of the National Institutes of Health or at the National Naval Medical Center. Normal donor leukocytes were obtained from the Blood Bank of the National Institutes of Health and from laboratory workers. Subjects were those in various stages of disease: only with local tumor (stage 1), with extension into the regional lymph nodes (stage 2) , and with metastatic disease (stage 3) . Some patients from the Naval Medical Center were on chemotherapy shortly before the tests. The total included 64 melanoma patients (9 were retested 2-5 times) and 43 normal donors. Virtually all individuals were immunocompetent and not anergic, as determined by delayed cutaneous skin reactivity against at least one standard antigen. Preparation of leukocytes.-Leukocyte migration was studied as previously described (26). Whole blood (50-60 ml) was collected in heparin and allowed to settle either by gravity sedimentation at 37 0 C for 1 hour or with the use of Plasmagel. The leukocytes (with some contamination with red blood cells) were resuspended in McCoy's 5A medium and washed twice with medium. The cell pellet was then resuspended in MeCoy's 5A medium supplemented with 10% heat-inactivated (56 0 C for 30 min) fetal calf serum, and the cell concentration was adjusted to 2-3 X 107 leukocytesyrnl. Antigen preparation.-Fresh melanoma or other type of tumor or benign nevus tissue (within 4-8 hr of surgery) was prepared as described in (26). Five to 20 ml (depending on the size of the original tumor, which was usually 1-5 g) of 3 M KCI was added to the cell suspension (prepared by mincing and homogenization in a Waring blender) and incubated at 4 0 C for 16-24 hours. This mixture was then centrifuged at 40,000 X g for 60 minutes in a Sorvall RC-2, desalted for several hours against phosphate-buffered saline, centrifuged an additional 15 minutes at 40,000 X g, and concentrated tenfold with an Amicon ultrafiltration unit (UM 10 membrane). Aliquots of each extract were stored at -70 0 C. The total was 11 melanom~ extracts, 7 other c~ncers (lung, breast, and colon carcinomas), and 1 bemgn nevus extract. . . . ~e were also concerned that ba~tenal.conta!fimatlon of tissues used for 3 M KCl extraction might yield soluReceived December 26, 1974; accepted March 26, 1975. Supported in part by Public Health Service contract NIH-NCI72-3227 from the National Cancer Institute. 3 Department of Immunology, Litton Bionetics, Inc., 5516 Nicholson Lane, Kensin~t.on, Md. 20795.. . 4 Internal Medicine Department, National Naval Medical Center, Bethesda, Md. 20014. 5 Laboratory of Immunodiagnosis, National Cancer Institute, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 6 Surgery Branch, National Cancer Institute. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO. I, JULY 1975
19
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SUMMARY-Direct leukocyte migration inhibition (LMI) assays were performed to investigate whether cell-mediated immune reactions could be detected In response to tumor-associated antigens of human melanoma. The antigens were 3 M KCI-soluble extracts of different fresh melanomas, other cancers, and benign nevus tissue. A total of 48 of the 79 (61 %) blood samples from melanoma patients (64 patients) reacted with extracts of melanoma tissue. Since the subjects were usually tested with two or three extracts, 57/134 (42%) tests with melanoma patients' leukocytes were inhibited by KCI extracts of melanoma tissue, whereas only 3/50 (6%) tests with leukocytes of normal donors and 4/27 (15%) with patients having other cancers gave positive results. No positive reactions were obtained when 13 melanoma patients were tested with a 3 M KCI extract of benign nevus tissue. Likewise, only 2/26 (8%) positive tests were obtained from melanoma patients tested with extracts of other cancers. Individuals in all stages of disease had similar incidences of positive reactions to the soluble melanoma extracts, except for patients with stage-l disease who exhibited a somewhat higher incidence of reactivity. The highest incidence of reactivity was observed in patients before surgical resection of the tumor, and somewhat decreased reactivity was seen 0-14 days post surgery. The results Indicate that the direct LMI assay may be used to measure cell immune reactivity against melanomaassociated antigens. Since many of the positive results were obtained with allogeneic extracts, the results also indicate that different melanomas possess common antigens.-J Natl Cancer Inst 55: 19-23, 1975.
20
MCCOY ET AL.
Mean of migration of 4 replicates in the presence of antigen M.I.= Mean of migration in the absence of antigen Replicate areas of migration were almost always within 10% of each other and were frequently within a
2-3% range. In statistical evaluation of the data, a twotailed ranking and Student's r-test were performed to establish P values (28). M.1. values of 0.80 or less were considered positive. We picked greater than or equal to 0.80 as a conservative value for positive LMI reactivity; this value has been used by other investigators (27, 29) . Because of the close values obtained with the four replicates, these values were always significantly different from an M.1. of 1.0 (P 1 year
_ _ _ _ _ _
14/19 1/4 7/10 6/12 4/7 16/27
TotaL
_
48/79 (61)
• See footnote to table 2.
(74) (25) (70) (50) (57) (59)
a/
Downloaded from https://academic.oup.com/jnci/article-abstract/55/1/19/918421 by INSEAD user on 01 December 2018
. l!l
IIELANGIIA IXTIIACTI WITH IIALIGIlANT IlILANOIlA L IUlCOCYTII
22
MCCOY ET AL.
The patients with melanoma were often tested with more than one melanoma extract. Degrees of reactivity were different with the various extracts. Table 4 presents the results obtained with four extracts. Three of the four gave good reactions (>40% of patients tested were positive), whereas one extract (5969) gave no positive reactions. DISCUSSION
TABLE 4.-LMI reactions of melanoma patients and normal donors with different melanoma extracts KCl extract
3194
Total positive/ total tested
4143
3539
5969
Normal donors
Melanoma patients
Normal donors
Melanoma patients
Normal donors
Melanoma patients
Normal donors
Melanoma patients
0.92 0.91 0.95 1.09 0.94 0.88 0.92 0.91 1.06 0.94
0.80 0.70 0.78 0.85 0.70 0.83 0.91 0.80 0.73 0.81 0.73 0.98 0.97 0.89 0.60 0.76 0.99 0.60 0.80
0.97 0.96 1.02 0.92 0.93 1.05 0.88
0.96 0.64 0.92 0.80 0.90 1.43 0.75 0.71 0.60 0.77 0.92 0.74 0.84 1.02 1.29 0.98 0.90 0.70 0.90
0.87 1.03 0.91 0.84 0.85 1.10 0.94
0.72 0.70 0.90 0.87 0.84 0.81 0.74 0.64 0.76 0.72 0.61 0.57 0.83 0.76 0.75
0.94 1.04 1.12 0.86 1.00 1.09 1.11 1.04
0.98 0.96 0.81 0.91 1.07
0/10
11/19 (53%)
0/7
8/19 (42%)
0/7
11/16 (62%)
0/8
0/5
Downloaded from https://academic.oup.com/jnci/article-abstract/55/1/19/918421 by INSEAD user on 01 December 2018
A series of reports (21-25) indicated the usefulness of the direct LMI assay for study of the cellular immune reactivity of melanoma patients to antigens in particulate saline extracts of melanoma cells. Our data extend the earlier reports and demonstrate that 48 of 79 (61%) tests with leukocytes of melanoma patients (64 patients) showed reactivity with 3 M KCl-soluble extracts of melanoma tissue. Although we tried to control the reactions with extracts of other types of cancer (25), the specificity of the reactions observed earlier was not extensively evaluated. We examined closely the specificity of the reactions elicited by soluble 3 M KCl extracts. Whereas 42% of tests of patients with malignant melanoma showed posi tive reactions wi th extracts of melanoma, only 6% of normal adults and 15% of patients with other tumors gave positive reactions with the same extracts. No melanoma patient reacted positively to an extract of a benign nevus, and only a few individuals reacted with extracts of other types of cancer. The breast cancer extracts used here gave considerably more reactions in patients with carcinoma of the breast (26). From these results, the reactions of melanoma patients with the KCl extracts of melanoma appear directed against melanoma-associated antigens. The observed inhibition did not seem due 'to toxicity of the tumor extracts, since at the concentrations of extracts used in the assays, migration of leukocytes from controls was usually not affected. The pattern of results is also not consistent with reactivity against histocompatibility antigens in the
extracts. In addition, 5 normal donors were multiparous females who did not react with the melanoma extracts. Also, our total experience with cancer patient reactivity against 3 M KCl extracts of normal tissues from liver, colon, lung, and breast has been that only 2/76 (3%) had positive LMI reactions. Further, none of 13 melanoma patients reacted against a 3 M KCl benign nevus extract. Since the tests were performed with allogeneic melanoma extracts, the results also indicate that different melanomas possess common antigens. This agrees with earlier reports of common T AA on human melanoma cells in migration inhibition assays (21-25), microcytotoxicity tests with cells cultured from melanomas (9-13, 30), lymphocyte stimulation assays (19, 20), and delayed hypersensitivity skin reactions (14-18). The Hellstroms (30) further suggested that melanoma cells may have more than one T AA, since lymphocytes from some patients were more reactive on some lines of cultivated melanoma cells than on others. Our results are consistent with this suggestion, because some melanoma patients reacted well with one KCl extract of melanoma tissue but failed to react well with a second or third extract. We found that leukocytes of normal black donors were not inhibited by melanoma extracts. The reports by the Hellstroms and their colleagues (10, 30) that lymphocytes from many normal black donors were cytotoxic to tissue culture cells derived from melanoma tissues suggested that this group has natural immunologic reactivity against melanoma cells. Although our data do not support this suggestion, the assays (LMI vs. microcytotoxicity) and the sources of antigen (extracts of fresh melanoma tissue vs, intact cultured cells) were sufficiently different to account for the divergent results. Variations in antigenicity of the 3 M KCl tissue extracts used in this study could have significantly affected the overall incidence of observed reactivity, since some extracts give much higher percentages of reactivity than
MIGRATION INHIBITION ASSAYS OF HUMAN MELANOMA
(8)
(9) (10)
(II) (12) (JJ)
(14) (15) (16) (17)
(18) (19) (20)
(21) (22) (23) (24) (25)
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others. This may have been due to variations in the proportion of melanoma cells versus normal cells in the tumor mass used for antigen extraction, the stage of the tumor (e.g., primary vs. metastatic), or various other factors. It will be particularly important to identify large extracts that have good demonstrable LMI reactivity with melanoma patients and to use these as the standard preparations. We found no definitive correlation between LMI reactivity and stage of disease. Some previous studies of CMI and humoral immunity in melanoma patients reported correlations with the clinical stage, but others did not (3, 5, 15, 20-23, 25). Such differences could be ascribed to variations in the patient populations or the therapy, or to important factors related to the assay systems or the antigens used. In regard to reactivity in relation to surgery, Cochran et al. (23) found depressed reactivity in patients at 1-4 days after surgery and then a rapid increase in reactivity after 5-22 days. Our data, with depressed reactivity possibly seen in the 0- to 10-day postoperative period with 4 patients, and showing recovery during the 11- to 30-day postsurgery period, are in agreement with those of Cochran et al. We, however, noted good reactivity in many patients with metastatic disease. The effects of disease status and therapy on the reactivity in LMI will be satisfactorily determined only when serial studies on the same patients with the same antigen extracts for each test are performed. Also, it will be important to determine whether correlations in delayed cutaneous skin reactivity of the patients correlate with the LMI results. Some of this has been done, but most skin tests have been performed with extracts other than 3 M KCl material. We are currently conducting a study directly testing the same patients in both skin and LMI assays with the same extracts. Finally, our study used the direct migration inhibition assay rather than the indirect method which uses guinea pig macrophages as the indicator cells. Possibly, the direct method with leukocytes measures different factors, as described by Rocklin (31). Studies are now in progress to look at supernatants generated by the interaction of patient leukocytes with 3 M KCl extracts to determine whether the observed results were due to production of leukocyte inhibitory factor and/or macrophage inhibitory factor.
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