- Email: [email protected]
CUTANEOUS HYPERSENSITIVITY REACTIONS TO AUTOLOGOUS EXTRACTS OF MALIGNANT MELANOMA CELLS
116
CUTANEOUS HYPERSENSITIVITY before its removal but on each occasion no arcuate or the had been included: had REACTIONS TO AUTOLOGOUS EXTRACTS they been, larger arteries morphological diagnosis of severe chronic rejection OF MALIGNANT MELANOMA CELLS would have been apparent. This discrepancy between L. FASS R. B. HERBERMAN biopsy appearances and those in the graft as a whole is of J. L. ZIEGLER J. W. M. KIRYABWIRE great importance serving to emphasise not only the Lymphoma Treatment Centre, Makerere University College patchy nature of the morphological rejection process Medical School, Kampala, Uganda; and National Cancer but also the need to continue investigation of the anuric Institute, Bethesda, Maryland patient in the face of an uninformative biopsy. Arteriography had demonstrated this vascular narrowing. patients with malignant melaThe greatest number of biopsies were done as a part Summary Eight noma in Uganda were tested for of the investigation of postoperative anuria. It is delayed-hypersensitivity reactions to antigens on the during this period that the clinician is faced with the autologous tumour cells. The three patients with dilemma of distinguishing between anuria due to localised tumours gave positive skin reactions, and continuing tubular necrosis, anuria due to an immune the patients with metastatic disease all gave negative rejection episode, and anuria from other causes. In reactions. The negative responses could not be recipients of cadaver kidneys the biopsy served to attributed to general anergy, since all patients gave demonstrate an immune rejection process occurring positive reactions to one or more bacterial or other during this postoperative anuric phase but, despite test antigens. The results indicated that some patients increased immunosuppression, no patient showed any with malignant melanoma have detectable cellular in renal function, probably due striking improvement immunity to autologous tumour antigens and that to continuing tubular necrosis. However, it is possible positive reactions were correlated with the clinical that without this therapy the rejection process might status of the patients. have increased in intensity and irreversible changes occurred in the graft. Equally important at this time Introduction and after a diuresis has occurred is the knowledge that THE clinical and epidemiological features of maligno substantial rejection process is taking place. nant melanoma in Uganda have been described by Increased immunosuppression with all its attendant Lewis and co-workers.1-5 From the clinical presentadangers is then avoided and possible obstruction of the tion of the patient3 and in-vitro evidence of serum allograft ureter or of the vascular anastomoses should be investigated. In this series the presence of fibrinoid cytotoxicity,5 these investigators have postulated the existence of host-defence mechanisms against the necrosis of arterioles has always signified a severe and tumour. They distinguished three clinical groups: irreversible rejection process. This change may only be apparent in a single vessel in the biopsy emphasising (1) Patients with a long clinical history of a localised the importance of examining the entire specimen by malignant melanoma, usually on the sole of the foot and serial sections. Once fibrinoid necrosis is recognised with no metastasis. the graft should probably be removed rather than (2) Patients presenting with a primary tumour and attempting to reverse the process by increasing the local or distant metastasis, with a relatively short clinical course. immunosuppressive therapy. The significance of glomerular lesions in long term renal allografts has been discussed by Porter et al.14,15 and any observations about these appearances without also considering DR. MILLARD AND OTHERS: REFERENCES 1. Kincaid-Smith, P., Morris, P. J., Saker, B. M., Ting, A., Marshall; electronmicroscopy and immunofluorescent features V. C. Lancet, 1968, ii, 748. would be highly speculative. However, bearing in mind 2. Lee, H. M., Hume, D. M., Vredevoe, D. L., Mickey, M. R., the electron-microscopy studies of Muehrcke et al.16 on Terasaki, P. I. Transplantation, 1967, 5, 1040. 3. Calne, R. Y., Evans, D. B., Herbertson, B. M., Joysey, V., McMillan, untransplanted diseased kidneys, it is possible that R., Maginn, R. R., Millard, P. R., Pena, J. R., Salaman, J. R., similar studies on transplanted kidneys may well be of White, H. J. O., Withycombe, J. F. R., Yoffa, D. E. Br. med. J. little immediate importance. 1968, ii, 404. 4. Maginn, R. R., Bullimore, J. A. Br. J. Radiol. 1968, 41, 127. In conclusion, the light-microscopy features of 5. Hume, D. in The Kidney (edited by F. K. Mostofi and D. S. renal allograft biopsies are useful to the clinician, and Smith); p. 409. Baltimore, 1966. 6. Kolff, W. J. Ann. intern. Med. 1965, 63, 901. from the experience in this unit at the present time 7. Mathew, T. H., Kincaid-Smith, P., Eremin, J., Marshall, V. C. the main indications for biopsying the transplanted Med. J. Aust. 1968, i, 6. 8. Evans, D. B., Joysey, V. Unpublished. kidney are: (1) postoperative anuria of more than two 9. Salaman, J. R., Calne, R. Y., Pena, J., Sells, R. A., White, H. J. O., weeks in patients receiving cadaver kidneys; (2) Yoffa, D. E. Br. J. Surg. 1969, 56, 413. 10. Hill, R. B., Rowlands, D. T., Rifkind, D. Am. J. Med. 1967, following the diagnosis of a rejection episode in 42, 327. which increased immunosuppression is not associated 11. Evans, D. B. Proc. R. Soc. Med. 1969, 62, 597. with rapid improvement in renal function; or (3) the 12. Kissmeyer-Nielsen, F., Olsen, S., Petersen, V. P., Fjeldborg, O. Lancet 1966, ii, 662. presence of slowly decreasing renal function in a G.
previously functioning kidney. We thank Prof. R. Y. Calne for permission to publish details of his patients, the histology section of the John Bonnett Laboratories, Addenbrooke’s Hospital for processing and preparation of biopsy material, and Mrs. J. Small for preparing the typescript. Requests for reprints should be addressed to D. B. E.
References
at
foot of next column
13. Williams,
M., Hume, D. M., Hudson, R. P., Morris, P. J., Kano, K., Milgrom, F. New Engl. J. Med. 1968, 279, 611. 14. Porter, K. A., Dossetor, J. B., Marchioro, T. L., Peart, W. S., Rendall, J. M., Starzl, T. E., Terasaki, P. I. Lab. Invest. 1967, 16, 153. 15. Porter, J. A., Andres, G. A., Calder, M. W., Dossetor, J. B., Hsu, K. C., Rendall, J. M., Seegal, B. S., Starzl, T. E. ibid. 1968, 18, 159. 16. Muehrcke, R. C., Mandal, A. K., Gotoff, S. P., Isaacs, E. W., Volini, F. I. Ann. intern. Med. 1969, 124, 170.
117
(3) Patients presenting with without a detectable primary.3
metastatic
melanoma
histopathological differences suggested that a spread. In nineteen Ugandan patients with melanoma, Ziegler et al. found no gross impairment of humoral or cellular host immune mechanisms, regardless of the extent of disease. If host immunity plays a role in containment of tumour spread, subtle differences in specific tumour immunity or in tumour antigens of localised and metastatic melanoma cells may explain the postulated difference in host response to the These workers found
no
in the tumours of these groups, and host response may influence tumour
tumour.
To investigate possible tumour-specific cellular mechanisms in patients with localised or metastatic melanoma, we investigated the cutaneous delayed-
hypersensitivity response to autologous extracts in eight Ugandan patients.
tumour
Patients and Methods
Patient Selection We investigated eight patients admitted to Mulago Hospital or to the Lymphoma Treatment Centre, Makerere University College Medical School, Kampala, Uganda, with biopsy-proven malignant melanoma between October, 1968, and May, 1969. Each patient had a physical examination, complete blood-count, and blood-urea, serum enzymes, and bilirubin determinations. Chest X-rays and skeletal surveys for metastatic disease were also done. All patients were skin tested within a few days of
biopsy. Preparation of
Cell Extracts Sterile techniques were used
throughout. A tumour cell prepared by teasing the tissue with needles into 5 ml. of isotonic saline solution. The viability of the cell suspension was determined by dye exclusion with 0-2% trypan-blue in isotonic saline and varied between 30 and 80%(mean 60%). The cells were washed twice and suspended in 5 ml. of isotonic saline. Peripheral lymphocytes were used as control tissue. They were isolated by the procedure of Greenwalt et a1.7 20 ml. of blood was passed through a nylon-fibre column and the erythrocytes were then sedimented by addition of one-third volume of 3% gelatin in isotonic saline. After 30 minutes at 37°C, the supernatant fluid, containing the lymphocytes, was diluted twofold with isotonic saline and centrifuged at 500 g at 4°C for 10 minutes. Residual erythrocytes were removed by brief hypotonic lysis. The cells were resuspended in 5 ml. of isotonic saline. The concentration of tumour cells and lymphocytes varied suspension
was
between 106 and 107 cells per ml. Extracts of
prepared by
cells and lymphocytes were then modification of the methods of Davies8
tumour
a
and Mann et al. Extracts prepared in this manner contained cell membranes and were rich in transplantation antigens. The cell suspensions were quickly frozen to 70ùC for 30 minutes, then thawed in a 370C water bath and centrifuged at 500 g for 10 minutes. The supernatant fluid was placed in a sterile container and the cells were resuspended in 3 ml. of isotonic saline. The suspensions were then incubated at 4°C for 10 minutes and again centrifuged at 500 g for 10 minutes. The supernatant fluid was added to the first supernatant fluid. Cells were exposed twice to 0-45% saline, twice to 0-225% saline, and twice to distilled water. The pooled supernatant fluids were then concentrated over a period of 24-30 hours by pressure dialysis at 4°C to a final volume of 1 ml. If the original concentration of tumour cells was higher than that of the lymphocytes, enough isotonic saline was added to the tumour extract to equalise the two cell concentrations. Protein concentrations of the extracts were determined by the method of Lowry et al.1o The extracts were divided into 0.1 ml. volumes in tuberculin syringes and frozen at -70°C until -
testing. Skin Tests Skin tests were done before any chemotherapy. 0-11 ml. tumour and lymphocyte extracts was injected intradermally in the right forearm together with 0.1 ml. intradermal injections into the left forearm of:
Tuberculin.-Intermediate-strength purified-protein derivatuberculin, 0.0002 mg. P.P.D. (Parke Davis and Co., Detroit, Michigan). Mumps antigen (Eli Lilly and Company, Indianapolis, Indiana). Candida albicans extract 1/100.-’Dermatophytin 0’ (Hollister-Stier Laboratories, Spokane, Washington). Trichophyton 1/30.-(Hollister-Stier Laboratories). Brucella antigen.-’ Brucellergen Protein Nucleate’ (Mercke, Sharp and Dohme, West Point, Pennsylvania). tive of
interpreted by two of us (L. F. and of whom J. Z.) (J. Z.) had no knowledge of their source. Injection sites were examined at 15 minutes and at 4, 12, 24, 36, 40, 48, and 60 hours. No immediate skin reactions were noted. In all skin tests, maximum induration was observed at 40-48 hours. A positive skin test was defined as one having a diameter of greater than 5 mm. of induration. Skin-punch biopsies were performed on all positive tumour-extract skin tests and their lymphocyte controls. One patient with clinically negative skin tests was also biopsied. Biopsies were fixed in 10% formalin and stained with hasmatoxylin and eosin. One of us (R. H.) and Dr. Lewis Thomas and Dr. Costan Berard (pathological anatomy branch, National Cancer Institute), and Prof. M. S. R. Hutt (department of pathology, Makerere University Medical College) independently reviewed the histopathology of the skin-biopsy specimens. They did not know which slides represented tests with tumour extract or control extract or how the tests had been Skin
tests were
one
interpreted clinically.
DELAYED CUTANEOUS HYPERSENSITIVITY REACTIONS IN PATIENTS WITH MALIGNANT MELANOMA
118 Results
Clinical details of the eight patients tested and reactivity to skin tests administered are given in the table. Three patients had localised tumours of the foot, and five had more extensive disease. All of the patients responded to at least one of the bacteria or viral skin tests. The three patients with localised disease had positive reactions to the autologous tumour-cell extracts. None of the patients with generalised disease had positive reactions. All of the patients had negative reactions to the control lym-
phocyte extracts. Histologically, striking lymphocytic and histiocytic perivascular infiltrates compatible with delayedhypersensitivity reactions were observed in all of the clinically positive reactions. Biopsy specimens of the control lymphocyte extracts and of the clinically negative tumour extract showed little or no cellular infiltrates. In similar studies with patients with Burkitt’s lymphoma 1’ and other turnours/2 the protein con-
centration of the extracts seemed to have some effect clinical reactivity; and control extracts with high protein concentrations (>0-3 mg. per 0.1 ml.) produced some positive reactions. In our extracts, the protein concentrations of the tumour and control materials were generally quite low. The only tumour extract with high protein concentration (1-35 mg. per 0.1 ml.) gave a negative response. One of the tumour extracts gave a positive response at 0-002 mg. per 0.1 ml. The lymphocyte extracts had similar or higher protein concentrations than the tumour extracts. There was therefore no correlation between the protein concentrations of the different extracts and the skin reactivity.
on
Discussion
Cellular immune mechanisms are considered to be of major importance in host resistance to cancer.13 Hughes and Lytton 14 and Stewart 15 found that about a quarter of patients with carcinomas and sarcomas gave delayed skin reactions in response to intradermal inoculation of autologous tumour-cell extracts. Herberman and Oren 12 did skin tests with membrane extracts of leukxmia and lymphoma cells and obtained positive responses in 72% of patients. We found that eight of thirteen patients (61%) with Burkitt’s tumour gave positive reactions to extracts from their tumour cells. Positive reactions were only obtained in patients in remission or with small, localised tumours, and responsiveness was correlated with sustained remissions. In this investigation we found delayed-hypersensitivity reactions to autologous tumour extracts in patients with localised malignant melanoma but not in patients with metastatic disease. The difference in reactivity between the two groups could not be attributed to general anergy in the patients with All patients gave positive more advanced disease. reactions to one or more bacterial, fungal, or viral
antigens. This relationship of specific skin reactivity to clinical state might be due to qualitative or quanta-
tative differences in antigens on metastatic tumour cells. Alternatively, tumour-antigen excess in patients with disseminated disease could suppress the specific host immune response.16-18 These possibilities will be further explored by serial testing of individual patients with tumour extracts prepared from localised tumours and from subsequent metastatic
deposits.
i
Evidence for both humoral and cellular host immune reactions in malignant melanoma has been previously reported. Lewis 5,19 demonstrated a cytotoxic effect of autologous sera against melanoma cells in short-term tissue-culture. As in our series, positive sera were mainly obtained from patients with localised disease. Morton et al. 20 found antibodies by an immunofluorescent technique which reacted with melanoma cells in 61% of sera from melanoma patients and in 20% of sera from normal controls. In an attempt at serotherapy, Sumner and Foraker 21 achieved a clinical regression in a patient with disseminated melanoma by infusing serum from a patient with a spontaneous remission. Nadler and Moore,22 using sensitised lymphocytes from patients exposed to allogeneic melanoma cells, observed tumour regressions in six patients with malignant melanoma. Our finding that delayed-hypersensitivity reactions to tumour antigens can be identified in some patients with malignant melanoma should provide a useful technique for studying cellular immunity in patients with malignant melanoma and may aid in further clinical immunotherapeutic attempts. This work was supported by contract no. PH-43-67-1343 and
I
PH-43-67-47 from the National Cancer Institute, National Institutes of Health, U.S. Public Health Service. Requests for reprints should be addressed to R. B. H., Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. REFERENCES 1.
Lewis, M. G., Kiryabwire, J. W. M. Cancer, N.Y. 1968, 21,
2. 3. 4.
Lewis, M. G., Johnson, K. Br. J. Dermat. 1968, 80, 362. Lewis, M. G. Br. J. Cancer, 1967, 21, 483. Lewis, M. G., Kiryabwire, J. W. M. Br. J. Surg. 1968, 55,
876.
207. 5. Lewis, M. G. Lancet, 1967, ii, 921. 6. Ziegler, J. L., Lewis, M. G., Luyomba, J. M. S., Kiryabwire, J. W. M. Cancer, N.Y. (in the press). 7. Greenwalt, T. J., Gajewski, M., McKenna, J. L. Transfusion, 1962, 2, 221. 8. Davies, D. A. L. Immunology, 1966, 11, 115. 9. Mann, D. L., Rogentine, G. N., Fahey, J. L., Nathenson, S. G. Nature, Lond. 1968, 217, 1180. 10. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. J. biol. Chem. 1952, 193, 265. 11. Fass, L., Herberman, R. B., Ziegler, J. L. New Engl. J. Med. (in the press). 12. Herberman, R. B., Oren, M. E. Clin. Res. 1969, 17, 403. 13. Law, L. W. Cancer Res. 1969, 29, 1. 14. Hughes, L. E., Lytton, B. Br. med. J. 1964, i, 209. 15. Stewart, T. H. M. Cancer, N.Y. 1969, 23, 1368. 16. Mikulska, Z. B., Smith, C., Alexander, P. J. natn. Cancer Inst. 1966, 36, 29. 17. Wang, M. Int. J. Cancer, 1968, 3, 483. 18. Hoy, W. E., Nelson, D. B. Nature, Lond. 1969, 222, 1001. 19. Lewis, M. G., Ikonapisov, R. L., Nairn, R. C., Phillips, T. M., Hamilton-Fairley, G., Bodenham, D. C., Alexander, P. Br. med. J. 1969, iii, 547. 20. Morton, D. L., Malmgren, R. A., Holmes, E. C., Ketcham, A. S. Surgery, St. Louis, 1968, 64, 233. 21. Sumner, W. C., Foraker, A. G. Cancer, N.Y. 1960, 13, 79. 22. Nadler, S. H., Moore, G. E. Ann. Surg. 1966, 164, 482.
,
CUTANEOUS HYPERSENSITIVITY before its removal but on each occasion no arcuate or the had been included: had REACTIONS TO AUTOLOGOUS EXTRACTS they been, larger arteries morphological diagnosis of severe chronic rejection OF MALIGNANT MELANOMA CELLS would have been apparent. This discrepancy between L. FASS R. B. HERBERMAN biopsy appearances and those in the graft as a whole is of J. L. ZIEGLER J. W. M. KIRYABWIRE great importance serving to emphasise not only the Lymphoma Treatment Centre, Makerere University College patchy nature of the morphological rejection process Medical School, Kampala, Uganda; and National Cancer but also the need to continue investigation of the anuric Institute, Bethesda, Maryland patient in the face of an uninformative biopsy. Arteriography had demonstrated this vascular narrowing. patients with malignant melaThe greatest number of biopsies were done as a part Summary Eight noma in Uganda were tested for of the investigation of postoperative anuria. It is delayed-hypersensitivity reactions to antigens on the during this period that the clinician is faced with the autologous tumour cells. The three patients with dilemma of distinguishing between anuria due to localised tumours gave positive skin reactions, and continuing tubular necrosis, anuria due to an immune the patients with metastatic disease all gave negative rejection episode, and anuria from other causes. In reactions. The negative responses could not be recipients of cadaver kidneys the biopsy served to attributed to general anergy, since all patients gave demonstrate an immune rejection process occurring positive reactions to one or more bacterial or other during this postoperative anuric phase but, despite test antigens. The results indicated that some patients increased immunosuppression, no patient showed any with malignant melanoma have detectable cellular in renal function, probably due striking improvement immunity to autologous tumour antigens and that to continuing tubular necrosis. However, it is possible positive reactions were correlated with the clinical that without this therapy the rejection process might status of the patients. have increased in intensity and irreversible changes occurred in the graft. Equally important at this time Introduction and after a diuresis has occurred is the knowledge that THE clinical and epidemiological features of maligno substantial rejection process is taking place. nant melanoma in Uganda have been described by Increased immunosuppression with all its attendant Lewis and co-workers.1-5 From the clinical presentadangers is then avoided and possible obstruction of the tion of the patient3 and in-vitro evidence of serum allograft ureter or of the vascular anastomoses should be investigated. In this series the presence of fibrinoid cytotoxicity,5 these investigators have postulated the existence of host-defence mechanisms against the necrosis of arterioles has always signified a severe and tumour. They distinguished three clinical groups: irreversible rejection process. This change may only be apparent in a single vessel in the biopsy emphasising (1) Patients with a long clinical history of a localised the importance of examining the entire specimen by malignant melanoma, usually on the sole of the foot and serial sections. Once fibrinoid necrosis is recognised with no metastasis. the graft should probably be removed rather than (2) Patients presenting with a primary tumour and attempting to reverse the process by increasing the local or distant metastasis, with a relatively short clinical course. immunosuppressive therapy. The significance of glomerular lesions in long term renal allografts has been discussed by Porter et al.14,15 and any observations about these appearances without also considering DR. MILLARD AND OTHERS: REFERENCES 1. Kincaid-Smith, P., Morris, P. J., Saker, B. M., Ting, A., Marshall; electronmicroscopy and immunofluorescent features V. C. Lancet, 1968, ii, 748. would be highly speculative. However, bearing in mind 2. Lee, H. M., Hume, D. M., Vredevoe, D. L., Mickey, M. R., the electron-microscopy studies of Muehrcke et al.16 on Terasaki, P. I. Transplantation, 1967, 5, 1040. 3. Calne, R. Y., Evans, D. B., Herbertson, B. M., Joysey, V., McMillan, untransplanted diseased kidneys, it is possible that R., Maginn, R. R., Millard, P. R., Pena, J. R., Salaman, J. R., similar studies on transplanted kidneys may well be of White, H. J. O., Withycombe, J. F. R., Yoffa, D. E. Br. med. J. little immediate importance. 1968, ii, 404. 4. Maginn, R. R., Bullimore, J. A. Br. J. Radiol. 1968, 41, 127. In conclusion, the light-microscopy features of 5. Hume, D. in The Kidney (edited by F. K. Mostofi and D. S. renal allograft biopsies are useful to the clinician, and Smith); p. 409. Baltimore, 1966. 6. Kolff, W. J. Ann. intern. Med. 1965, 63, 901. from the experience in this unit at the present time 7. Mathew, T. H., Kincaid-Smith, P., Eremin, J., Marshall, V. C. the main indications for biopsying the transplanted Med. J. Aust. 1968, i, 6. 8. Evans, D. B., Joysey, V. Unpublished. kidney are: (1) postoperative anuria of more than two 9. Salaman, J. R., Calne, R. Y., Pena, J., Sells, R. A., White, H. J. O., weeks in patients receiving cadaver kidneys; (2) Yoffa, D. E. Br. J. Surg. 1969, 56, 413. 10. Hill, R. B., Rowlands, D. T., Rifkind, D. Am. J. Med. 1967, following the diagnosis of a rejection episode in 42, 327. which increased immunosuppression is not associated 11. Evans, D. B. Proc. R. Soc. Med. 1969, 62, 597. with rapid improvement in renal function; or (3) the 12. Kissmeyer-Nielsen, F., Olsen, S., Petersen, V. P., Fjeldborg, O. Lancet 1966, ii, 662. presence of slowly decreasing renal function in a G.
previously functioning kidney. We thank Prof. R. Y. Calne for permission to publish details of his patients, the histology section of the John Bonnett Laboratories, Addenbrooke’s Hospital for processing and preparation of biopsy material, and Mrs. J. Small for preparing the typescript. Requests for reprints should be addressed to D. B. E.
References
at
foot of next column
13. Williams,
M., Hume, D. M., Hudson, R. P., Morris, P. J., Kano, K., Milgrom, F. New Engl. J. Med. 1968, 279, 611. 14. Porter, K. A., Dossetor, J. B., Marchioro, T. L., Peart, W. S., Rendall, J. M., Starzl, T. E., Terasaki, P. I. Lab. Invest. 1967, 16, 153. 15. Porter, J. A., Andres, G. A., Calder, M. W., Dossetor, J. B., Hsu, K. C., Rendall, J. M., Seegal, B. S., Starzl, T. E. ibid. 1968, 18, 159. 16. Muehrcke, R. C., Mandal, A. K., Gotoff, S. P., Isaacs, E. W., Volini, F. I. Ann. intern. Med. 1969, 124, 170.
117
(3) Patients presenting with without a detectable primary.3
metastatic
melanoma
histopathological differences suggested that a spread. In nineteen Ugandan patients with melanoma, Ziegler et al. found no gross impairment of humoral or cellular host immune mechanisms, regardless of the extent of disease. If host immunity plays a role in containment of tumour spread, subtle differences in specific tumour immunity or in tumour antigens of localised and metastatic melanoma cells may explain the postulated difference in host response to the These workers found
no
in the tumours of these groups, and host response may influence tumour
tumour.
To investigate possible tumour-specific cellular mechanisms in patients with localised or metastatic melanoma, we investigated the cutaneous delayed-
hypersensitivity response to autologous extracts in eight Ugandan patients.
tumour
Patients and Methods
Patient Selection We investigated eight patients admitted to Mulago Hospital or to the Lymphoma Treatment Centre, Makerere University College Medical School, Kampala, Uganda, with biopsy-proven malignant melanoma between October, 1968, and May, 1969. Each patient had a physical examination, complete blood-count, and blood-urea, serum enzymes, and bilirubin determinations. Chest X-rays and skeletal surveys for metastatic disease were also done. All patients were skin tested within a few days of
biopsy. Preparation of
Cell Extracts Sterile techniques were used
throughout. A tumour cell prepared by teasing the tissue with needles into 5 ml. of isotonic saline solution. The viability of the cell suspension was determined by dye exclusion with 0-2% trypan-blue in isotonic saline and varied between 30 and 80%(mean 60%). The cells were washed twice and suspended in 5 ml. of isotonic saline. Peripheral lymphocytes were used as control tissue. They were isolated by the procedure of Greenwalt et a1.7 20 ml. of blood was passed through a nylon-fibre column and the erythrocytes were then sedimented by addition of one-third volume of 3% gelatin in isotonic saline. After 30 minutes at 37°C, the supernatant fluid, containing the lymphocytes, was diluted twofold with isotonic saline and centrifuged at 500 g at 4°C for 10 minutes. Residual erythrocytes were removed by brief hypotonic lysis. The cells were resuspended in 5 ml. of isotonic saline. The concentration of tumour cells and lymphocytes varied suspension
was
between 106 and 107 cells per ml. Extracts of
prepared by
cells and lymphocytes were then modification of the methods of Davies8
tumour
a
and Mann et al. Extracts prepared in this manner contained cell membranes and were rich in transplantation antigens. The cell suspensions were quickly frozen to 70ùC for 30 minutes, then thawed in a 370C water bath and centrifuged at 500 g for 10 minutes. The supernatant fluid was placed in a sterile container and the cells were resuspended in 3 ml. of isotonic saline. The suspensions were then incubated at 4°C for 10 minutes and again centrifuged at 500 g for 10 minutes. The supernatant fluid was added to the first supernatant fluid. Cells were exposed twice to 0-45% saline, twice to 0-225% saline, and twice to distilled water. The pooled supernatant fluids were then concentrated over a period of 24-30 hours by pressure dialysis at 4°C to a final volume of 1 ml. If the original concentration of tumour cells was higher than that of the lymphocytes, enough isotonic saline was added to the tumour extract to equalise the two cell concentrations. Protein concentrations of the extracts were determined by the method of Lowry et al.1o The extracts were divided into 0.1 ml. volumes in tuberculin syringes and frozen at -70°C until -
testing. Skin Tests Skin tests were done before any chemotherapy. 0-11 ml. tumour and lymphocyte extracts was injected intradermally in the right forearm together with 0.1 ml. intradermal injections into the left forearm of:
Tuberculin.-Intermediate-strength purified-protein derivatuberculin, 0.0002 mg. P.P.D. (Parke Davis and Co., Detroit, Michigan). Mumps antigen (Eli Lilly and Company, Indianapolis, Indiana). Candida albicans extract 1/100.-’Dermatophytin 0’ (Hollister-Stier Laboratories, Spokane, Washington). Trichophyton 1/30.-(Hollister-Stier Laboratories). Brucella antigen.-’ Brucellergen Protein Nucleate’ (Mercke, Sharp and Dohme, West Point, Pennsylvania). tive of
interpreted by two of us (L. F. and of whom J. Z.) (J. Z.) had no knowledge of their source. Injection sites were examined at 15 minutes and at 4, 12, 24, 36, 40, 48, and 60 hours. No immediate skin reactions were noted. In all skin tests, maximum induration was observed at 40-48 hours. A positive skin test was defined as one having a diameter of greater than 5 mm. of induration. Skin-punch biopsies were performed on all positive tumour-extract skin tests and their lymphocyte controls. One patient with clinically negative skin tests was also biopsied. Biopsies were fixed in 10% formalin and stained with hasmatoxylin and eosin. One of us (R. H.) and Dr. Lewis Thomas and Dr. Costan Berard (pathological anatomy branch, National Cancer Institute), and Prof. M. S. R. Hutt (department of pathology, Makerere University Medical College) independently reviewed the histopathology of the skin-biopsy specimens. They did not know which slides represented tests with tumour extract or control extract or how the tests had been Skin
tests were
one
interpreted clinically.
DELAYED CUTANEOUS HYPERSENSITIVITY REACTIONS IN PATIENTS WITH MALIGNANT MELANOMA
118 Results
Clinical details of the eight patients tested and reactivity to skin tests administered are given in the table. Three patients had localised tumours of the foot, and five had more extensive disease. All of the patients responded to at least one of the bacteria or viral skin tests. The three patients with localised disease had positive reactions to the autologous tumour-cell extracts. None of the patients with generalised disease had positive reactions. All of the patients had negative reactions to the control lym-
phocyte extracts. Histologically, striking lymphocytic and histiocytic perivascular infiltrates compatible with delayedhypersensitivity reactions were observed in all of the clinically positive reactions. Biopsy specimens of the control lymphocyte extracts and of the clinically negative tumour extract showed little or no cellular infiltrates. In similar studies with patients with Burkitt’s lymphoma 1’ and other turnours/2 the protein con-
centration of the extracts seemed to have some effect clinical reactivity; and control extracts with high protein concentrations (>0-3 mg. per 0.1 ml.) produced some positive reactions. In our extracts, the protein concentrations of the tumour and control materials were generally quite low. The only tumour extract with high protein concentration (1-35 mg. per 0.1 ml.) gave a negative response. One of the tumour extracts gave a positive response at 0-002 mg. per 0.1 ml. The lymphocyte extracts had similar or higher protein concentrations than the tumour extracts. There was therefore no correlation between the protein concentrations of the different extracts and the skin reactivity.
on
Discussion
Cellular immune mechanisms are considered to be of major importance in host resistance to cancer.13 Hughes and Lytton 14 and Stewart 15 found that about a quarter of patients with carcinomas and sarcomas gave delayed skin reactions in response to intradermal inoculation of autologous tumour-cell extracts. Herberman and Oren 12 did skin tests with membrane extracts of leukxmia and lymphoma cells and obtained positive responses in 72% of patients. We found that eight of thirteen patients (61%) with Burkitt’s tumour gave positive reactions to extracts from their tumour cells. Positive reactions were only obtained in patients in remission or with small, localised tumours, and responsiveness was correlated with sustained remissions. In this investigation we found delayed-hypersensitivity reactions to autologous tumour extracts in patients with localised malignant melanoma but not in patients with metastatic disease. The difference in reactivity between the two groups could not be attributed to general anergy in the patients with All patients gave positive more advanced disease. reactions to one or more bacterial, fungal, or viral
antigens. This relationship of specific skin reactivity to clinical state might be due to qualitative or quanta-
tative differences in antigens on metastatic tumour cells. Alternatively, tumour-antigen excess in patients with disseminated disease could suppress the specific host immune response.16-18 These possibilities will be further explored by serial testing of individual patients with tumour extracts prepared from localised tumours and from subsequent metastatic
deposits.
i
Evidence for both humoral and cellular host immune reactions in malignant melanoma has been previously reported. Lewis 5,19 demonstrated a cytotoxic effect of autologous sera against melanoma cells in short-term tissue-culture. As in our series, positive sera were mainly obtained from patients with localised disease. Morton et al. 20 found antibodies by an immunofluorescent technique which reacted with melanoma cells in 61% of sera from melanoma patients and in 20% of sera from normal controls. In an attempt at serotherapy, Sumner and Foraker 21 achieved a clinical regression in a patient with disseminated melanoma by infusing serum from a patient with a spontaneous remission. Nadler and Moore,22 using sensitised lymphocytes from patients exposed to allogeneic melanoma cells, observed tumour regressions in six patients with malignant melanoma. Our finding that delayed-hypersensitivity reactions to tumour antigens can be identified in some patients with malignant melanoma should provide a useful technique for studying cellular immunity in patients with malignant melanoma and may aid in further clinical immunotherapeutic attempts. This work was supported by contract no. PH-43-67-1343 and
I
PH-43-67-47 from the National Cancer Institute, National Institutes of Health, U.S. Public Health Service. Requests for reprints should be addressed to R. B. H., Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. REFERENCES 1.
Lewis, M. G., Kiryabwire, J. W. M. Cancer, N.Y. 1968, 21,
2. 3. 4.
Lewis, M. G., Johnson, K. Br. J. Dermat. 1968, 80, 362. Lewis, M. G. Br. J. Cancer, 1967, 21, 483. Lewis, M. G., Kiryabwire, J. W. M. Br. J. Surg. 1968, 55,
876.
207. 5. Lewis, M. G. Lancet, 1967, ii, 921. 6. Ziegler, J. L., Lewis, M. G., Luyomba, J. M. S., Kiryabwire, J. W. M. Cancer, N.Y. (in the press). 7. Greenwalt, T. J., Gajewski, M., McKenna, J. L. Transfusion, 1962, 2, 221. 8. Davies, D. A. L. Immunology, 1966, 11, 115. 9. Mann, D. L., Rogentine, G. N., Fahey, J. L., Nathenson, S. G. Nature, Lond. 1968, 217, 1180. 10. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. J. biol. Chem. 1952, 193, 265. 11. Fass, L., Herberman, R. B., Ziegler, J. L. New Engl. J. Med. (in the press). 12. Herberman, R. B., Oren, M. E. Clin. Res. 1969, 17, 403. 13. Law, L. W. Cancer Res. 1969, 29, 1. 14. Hughes, L. E., Lytton, B. Br. med. J. 1964, i, 209. 15. Stewart, T. H. M. Cancer, N.Y. 1969, 23, 1368. 16. Mikulska, Z. B., Smith, C., Alexander, P. J. natn. Cancer Inst. 1966, 36, 29. 17. Wang, M. Int. J. Cancer, 1968, 3, 483. 18. Hoy, W. E., Nelson, D. B. Nature, Lond. 1969, 222, 1001. 19. Lewis, M. G., Ikonapisov, R. L., Nairn, R. C., Phillips, T. M., Hamilton-Fairley, G., Bodenham, D. C., Alexander, P. Br. med. J. 1969, iii, 547. 20. Morton, D. L., Malmgren, R. A., Holmes, E. C., Ketcham, A. S. Surgery, St. Louis, 1968, 64, 233. 21. Sumner, W. C., Foraker, A. G. Cancer, N.Y. 1960, 13, 79. 22. Nadler, S. H., Moore, G. E. Ann. Surg. 1966, 164, 482.
,