Immunologic Factors in Melanoma CHERRIE K. DONAWHO, MD MARGARET L. KRIPKE, MD
C
utaneous melanoma is one of the most intensively investigated types of cancer from the viewpoint of immunology.1 In fact, melanoma is quite disproportionate among studies of the immunology of human cancers. The historical reasons for this stem from clinical observations on the pathogenesis of melanoma. Complete and partial regressions of cutaneous melanoma lesions are well documented and are associated histologically with mononuclear cell infiltrates. In addition, there are many reports of a long dormant period after excision of a primary melanoma, followed by a sudden onset of extensive metastatic disease. Such observations have led to speculation that immunologic mechanisms might be important in the control of cutaneous and metastatic melanoma. As we review below, this idea has been substantiated by recent studies of melanoma in both humans and animal models. In addition, two other lines of investigation have stimulated interest in the immunology of melanoma. The findings over the past decade concerning the considerable immunologic capabilities of the skin have raised questions concerning the role of cutaneous immunity in the development of all types of cutaneous cancers. Second, increasing evidence that ultraviolet (UV) radiation contributes somehow to the incidence of melanoma, coupled with the many demonstrations of the immunosuppressive activity of UV radiation, raises the possibility that the immunosuppressive as well as the carcinogenic action of UV radiation may contribute to the induction and pathogenesis of this disease.
From the Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, Texas. Address correspondence toMargaret L. Kripke, Department of Immunology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.
0 1992 by Elsevier Science Publishing Co., Inc.
l
0738-081x/92/$5.00
Melanoma
in Humans
Melanoma Antigens Serologic analyses2 have demonstrated that there are three classes of cell surface antigens on melanoma cells: unique antigens expressed only on individual melanomas, melanoma-specific antigens present on many melanomas but absent on other types of tumor and normal cells, and common antigens that are widely distributed on a variety of both normal and neoplastic cells. Because of their potential use in the therapy of melanoma, several of the melanoma-specific antigens have been characterized; foremost among these are the ganglioside antigens.3*4 Most melanomas express large quantities of a specific asialoganglioside (GD3) that is being used in various approaches for melanoma therapy. In addition, several melanoma-specific glycoproteins are being used in diagnostic assays and for the generation of monoclonal antibodies and vaccines for immunotherapy. It is clear from functional studies of T lymphocytes from melanoma patients that melanoma antigens recognized by T lymphocytes exist, and that there are both individually specific and melanoma-associated antigens that react with T lymphocytes. Because of the inherent difficulty involved in isolating T lymphocyte-defined antigens, however, these melanoma antigens have not yet been characterized, nor has their relationship to the serologically defined antigens been determined. Some melanomas also express class II molecules of the major histocompatibility complex (MHC). This is of interest because of the role that such molecules play in the presentation of antigens to T lymphocytes. Early cutaneous melanomas often express large amounts of class II molecules capable of presenting antigen to helper T lymphocytes, thereby initiating T cell activation. Tumor progression seems to be accompanied by the loss of antigen presenting function of the melanoma cells, suggesting that class II MHC expression and function may be impor-
69
70
DONAWHO AND KRIPKE
Clinics in Dermatology 1992;10:69-74
tant in determining the outcome of the host-tumor interaction.5 Furthermore, these molecules may contribute to the unusual immunogenic properties of melanomas.
Immune Responses
to Melanoma
Several types of analyses have been carried out in attempts to characterize the immune responses of melanoma patients to their tumors. Many studies have examined the numbers and types of cells associated with primary cutaneous melanomas using histologic and immunohistochemical approaches. The number and arrangement of epidermal Langerhans cells associated with melanoma lesions have been investigated extensively. Such studies have demonstrated that immune cells of various types are frequently associated with primary melanomas and less frequently with melanoma metastases. The functional significance of these associations is difficult to determine from this approach; however, there is a strong correlation between good prognosis and the presence of T lymphocytes in melanomas in the vertical growth phase.6 Many investigators have demonstrated that melanoma patients often have T lymphocytes and serum antibodies that react with autologous and/or allogeneic melanoma cells.z*7-9 Some of the antibodies have been used to help define the antigens expressed on human melanoma cells. Recently, T lymphocytes recovered from dissociated melanomas have been characterized in terms of their activity and specificity in conjunction with attempts to use these cells for adoptive immunotherapy. A significant proportion of melanomas contain T lymphocytes that are specifically cytotoxic for autologous melanoma cells in vitro. Nonspecifically cytotoxic T and natural killer (NK) cells are also present. It is intriguing that these cells are not cytotoxic when first recovered from the tumor but require in vitro activation with interleukin-2 (IL-2) to become cytolytic. Using this approach, it has become apparent that melanomas are unusually immunogenic compared with other solid tumors, in which it is difficult to find T lymphocytes with specific cytotoxicity for autologous tumor cells.*0
Immunotherapy
of Melanoma
Current directions in the immunotherapy of melanoma involve three basic approaches: monoclonal antibodies directed against melanoma antigens, melanoma vaccines, and in vim or in vitro activation of lymphocytes. Antibodies against GD3 melanoma antigen appear to be beneficial in retarding melanoma growth, both in humans and in animals.“J2 These and other antibodies are also being
explored as a means of targeting radioactive and toxic molecules to cancer cells in vi7m13~*4 Numerous clinical trials with various forms of melanoma vaccine preparations are being carried out. Most of these involve a mixture of allogeneic melanoma cells lines with or without an immunologic adjuvant. Antibody and delayed hypersensitivity responses to such vaccines have been documented, and this approach has proved to be beneficial for a significant fraction (20 to 40%) of melanoma patients.15 Another form of therapy has involved attempts to activate tumor-reactive lymphocytes in viva by infusing patients who have metastatic melanoma with the lymphocyte activator IL-2. Because of the toxicity of this treatment, subsequent attempts have been made to activate and expand lymphocytes in vitro with IL-2 and reinfuse them into the patients. Lymphocytes from peripheral blood, from the tumor tissue, and from the lymph nodes draining vaccination sites have all been used for this purpose.16*” It is not yet clear whether these approaches will improve the survival of patients with metastatic melanoma. Nevertheless, it is apparent from these studies that human melanomas have antigens that can serve as the target of various immune responses and that at least some melanoma patients can mount immune responses to these antigens. These findings indicate that continued effort and optimism are warranted in devising immunologic approaches for the treatment of melanoma. They also imply that the immunologic status of the host may play an important role in the development and progression of melanoma.
lmmunosuppression
and Melanoma
Because melanomas appear to be immunogenic, immunosuppression might be expected to be associated with a higher incidence of this neoplastic disease. Hodgkin’s disease, usually marked by lymphopenia and abnormalities in T-cell function, provides a test of this expectation. Patients with Hodgkin’s disease often have impaired cell-mediated immune responses’s and a high risk of developing second primary tumors.19 The most common of these is acute nonlymphocytic leukemia, but in addition, a significant increase has been noted in the incidence of melanoma in the fourth and fifth years after diagnosis of Hodgkin’s disease. Both the short interval between the occurrence of Hodgkin’s disease and the onset of the melanomas and the lack of a typical, intense mononuclear infiltrate in the tumor are suggestive of an association between immunosuppression and melanoma development in these patients.19
Clinics in Dermatology 1992;10:69-74 Another significant population of immunosuppressed individuals is transplant recipients, who are immunosuppressed with drugs to prevent graft rejection. In one study the transplant patients had a striking excess of nonHodgkin’s lymphoma and squamous cell carcinomas of the skin.20 In addition, a three- to six-fold excess of melanomas occurred in these patients. Their tumors were also characterized by a sparse inflammatory infiltrate. Two patients in this study had previously had a primary melanoma removed, but were disease-free at the time of transplantation. Unfortunately, both developed and subsequently died from melanoma metastases after the advent of immunosuppressive therapy. This observation led the authors to question the wisdom of renal transplantation for individuals with a history of previous melanoma if immunosuppression was required.*’ In patients with Hodgkin’s disease and renal transplants, the risk of cutaneous melanoma is significantly increased, especially for people with dysplastic nevi. 19s2*Such patients with dysplastic nevi should be monitored closely for the occurrence of melanoma. A condition of increasing concern and significance in this decade is the acquired immune deficiency syndrome (AIDS). This progressively degenerative infectious disease is associated with human immunodeficiency virus (HIV) infection. AIDS is characterized by a severe T cellmediated immune deficiency and decreases in NK cells, resulting in an unusual susceptibility to opportunistic infections and a high incidence of certain malignancies.22 There are reports of increased incidence of Kaposi’s sarcoma, malignant lymphoma, and squamous cell carcinoma in HIV-infected person.23 In addition, there have been several reports of the occurrence of melanoma with a particularly aggressive course in AIDS patients,21*22*24 but there is no definitive study of melanoma occurrence in HIV-positive individuals. Unfortunately, as time passes, the increased occurrence of additional neoplasms may become evident, particularly if improved treatment results in a longer life span for AIDS patients. Given the immunologic properties of melanomas, this form of cancer may increase in these individuals.
Studies in Animal Models Additional interest in the possible role of immunologic factors in the development of cutaneous melanoma has resulted from new information on the immunology of the skin and on the interactions between cutaneous immunity and UV radiation. The role of such factors in melanoma induction and progression is still largely undefined because, until recently, convenient animal models for in-
DONAWHO AND KRIPKE
71
IMMUNOLOGIC FACTORS IN MELANOMA
ducing primary melanomas did not exist, and only a few transplantable murine melanomas were available.
Immunologic Effects of Ultraviolet Radiation Recent studies have demonstrated that a population of immune cells residing in the skin is responsible for initiating immune responses. Epidermal Langerhans cells take up foreign substances and process them into antigens that can be recognized by T lymphocytes. Langerhans cells are the primary antigen presenting cells for the induction of contact allergy, and they may also be responsible for initiating immune responses to skin cancers and infectious organisms in the skin. Studies in mice demonstrated that exposing the skin to suberythemal doses of UV radiation reduces the number and alters the morphology of epiderma1 Langerhans cells. These changes are accompanied by an alteration in immune function that can be demonstrated by applying a contact-sensitizing agent onto the UV-irradiated skin. Instead of inducing a normal contact allergy reaction, the sensitizer induces antigen-specific suppressor T lymphocytes and a state of immunologic tolerance that inhibits a subsequent response to the same sensitizer.25*26 In addition, UV irradiation has been shown to alter the effector phase of some cutaneous immune reactions.27 UV radiation can also inhibit the induction and elicitation of immune responses at distant sites.28 Not all immune responses are affected, but contact allergy, delayed hypersensitivity, and the rejection of UV-induced skin cancers are all inhibited under certain conditions of UV irradiation and antigen sensitization. In all of these instances, immune suppression is associated with the formation of suppressor T lymphocytes. The mechanism by which UV irradiation of one site alters the immune response to antigens introduced at a distant site is not completely clear; however, recent evidence suggests that exposure of keratinocytes to UV radiation causes the release of soluble mediators that modify the immune response.29 One important aspect of UV-inducedimmunosuppression is the role it plays in UV carcinogenesis in experimental animals. Studies in mice demonstrate that exposure to UV radiation decreases the host’s ability to respond to antigens on UV-induced tumor cells.30 This allows the tumors to escape the normal immune surveillance mechanism, thereby increasing the probability of tumor development. This UV-induced alteration involves the generation of suppressor T cells that prevent the rejection of UV-induced tumors. Because these suppressor cells are present throughout the body, this effect of the UV radiation is systemic. Thus, UV radiation induces a systemic alteration in the immune system that contributes to the growth of UV-induced skin cancers.
72
DONAWHO AND KRIPKE
These studies raise the possibility that UV radiation might contribute to the pathogenesis of melanoma at stages other than the initial transformation event, by means of its immunosuppressive activity, regardless of any direct role it might play in carcinogenesis.
Role of Ultraviolet Radiation in the induction of Melanoma That UV radiation plays a role in melanoma induction has been substantiated in studies with a murine model. Using an approach developed by Berkelhammer and Oxenhandler,31 we investigated the role of UV radiation in the induction and pathogenesis of murine melanoma. Melanomas were induced by treating 4-day-old mice with DMBA, an initiating agent, and at 3 weeks of age painting with croton oil (a promoter) twice per week.31 We either substituted UV radiation for the initiator or the promoter or added UV radiation to the promotion protocol.32 We found that UV radiation could serve as a weak initiator or promoter in the induction of melanomas, In addition, irradiating the site of tumor initiation and promotion dramatically accelerated the appearance of melanoma. This effect of UV radiation on the development of melanomas was not a systemic effect, because UV irradiation of a distant site did not affect melanoma induction. Thus, UV irradiation exerted a direct effect on melanoma development at the site of melanoma induction.32 Our studies demonstrated that UV radiation can act directly to induce melanomas by serving as the initiator or promoter for the transformation of melanocytes. Additionally, it can act as a cofactor that promotes the outgrowth of melanomas after their induction by chemical agents. This diversity of roles for UV radiation seems to correlate well with the confusing picture of the etiology of melanoma that has arisen from epidemiologic studies.
Local Effect of Ultraviolet Irradiation on Melanoma Growth Recently, we have investigated how UV radiation is able to accelerate the development of primary melanomas, We used transplantable melanoma cell lines in a series of experiments to determine whether UV radiation directly affected the melanoma cells themselves or interacted with the surrounding host tissues.33 We found that transplanted melanomas appeared earlier and reached a higher incidence when the cells were injected into the ears of UV-irradiated mice. Six moderate (4.8 kJ/m*) exposures to UVB radiation affected host tissues in a way that enhanced the outgrowth of melanoma cells injected into the UV-irradiated site. The effect was localized to the site of tumor implantation and did not represent a systemic alteration of UV radiation. Because UV irradiation
Clinics in Dermatology 2992;10:69- 74 has been shown to have immunosuppressive activity in mice, we investigated whether the immunologic alterations caused by UV irradiation could be responsible for its effect on melanoma development.34 We first examined the specificity of the effect of UV irradiation on in vim tumor growth. We found the melanomas were not the only tumors affected by UV irradiation; however, only the growth of immunogenic tumors was accelerated in UV-irradiated mice. We also found an exact correlation between tumors that were immunogenic and tumors that were affected by UV irradiation.34 Next, we examined the effect of UV irradiation on tumor growth in immunosuppressed mice. We reasoned that if UV irradiation caused local immunosuppression, which was responsible for the effect of UV radiation on tumor outgrowth, then the effect would not be observed in immunodeficient mice. Indeed, we found no effect of UV radiation on the incidence of melanoma in mice immunosuppressed by X-irradiation, T cell depletion, or congenital athymia. These results strongly suggested that the effect of UV radiation is immunologic in nature and probably involves a reduction of T-lymphocyte function.34 Our third approach was to investigate the effects of UV radiation on the expression of systemic tumor immunity in mice. Exposing mice to UV radiation after immunization prevented rejection of the melanoma cells when they were injected into the UV-irradiated site; however, the mice challenged in a hair-protected site (the flank) were unaffected by exposure to UV radiation. These experiments demonstrated that the expression of an existing immune response to melanoma is perturbed in UV-irradiated skin (C. K. Donawho and M. L. Kripke, unpublished data). These results strengthen the possibility that the effect of UV irradiation on melanoma development is due, at least in part, to an immunosuppressive effect of the UV radiation. Also, it demonstrates that moderate exposures to UV irradiation can impair the expression of immunity to tumor cells within UV-irradiated skin.
Conclusions The etiology of human melanoma remains unclear, but it is likely to be multifactorial and to involve UV radiation. Melanoma is clearly a complex disease, with multiple histologic forms, and is subject to various genetic and environmental influences. The increasing exposure of the population to solar UV radiation resulting from changes in life-style and recreation makes UV radiation a logical candidate for contributing to the increasing incidence of melanoma. The exposure to chemicals in our environment may also be contributing to this increase in incidence, but to date, no such agents have been identified.
Clinics in Dermatology 1992;10:69- 74 With the expected decrease in the concentration of stratospheric ozone, it has been predicted that ambient UV-B radiation will increase and, therefore, the incidence of melanoma will also increase.35 Thus, it is important to understand the role played by UVB radiation in the induction and pathogenesis of melanoma. We found that UV radiation can play a variety of roles in this disease, namely, tumor initiator, tumor promoter, and inhibitor of local immune responsiveness. Our finding that UV irradiation has an indirect, local effect on melanoma growth implies that sun exposure after the diagnosis of melanoma may hasten the progress of disease by adversely affecting the local immunologic state of the host’s skin. Also, systemic immunosuppression may contribute to the occurrence and severity of cutaneous melanoma in humans. A comprehensive revieti6 reports that three of the ten recognized risk factors for melanoma are immunosuppression, sun sensitivity, and excessive sun exposure. Undeniably the most critical aspect in prevention of deaths from melanoma is the early detection and removal of the tumor. This emphasizes the need for close observation and early detection of the melanomas that might occur in susceptible individuals, While most of the other risk factors appear to be inherent in the genetics of susceptible individuals, these three factors suggest avenues for prevention in these persons. That immunosuppression is a significant risk factor for melanomas implies that melanomas are immunogenic and suggests that at least some melanomas may be amenable to immunologic therapy. In addition, it suggests that physicians who care for immunosuppressed patients should be vigilant for signs of cutaneous melanoma.
References 1. Halliday WJ. Specificity of cell-mediated immunoreactivity in melanoma and comments on the nature of serum blocking factors. In: Reisfeld RA, Ferrone S, editors. Melanoma antigens and antibodies. New York, Plenum Press, 1982;173-86.
2. Livingston PO, Michitisch R, Shiku H, et al. Antibody-dependent cell-mediated cytotoxicity for cultured autologous melanoma cells. Cell Immunol 1981;64:131-43.
3. Puke1 CS, Lloyd KO, Travassos LR, et al. GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody. J Exp Med 1982;155:1133-47.
DONAWHO AND KRIPKE IMMUNOLOGIC
ity to present antigens. 1991;32:238, Abstract.
73
FACTORS IN MELANOMA
Proc
Am Assoc
Cancer
Res
6. Clark WH Jr, Elder DE, van Horn M. The biologic forms of malignant melanoma. Hum Path01 1986;17:443-50. 7. Houghton AN, Real FX, Davis LJ, et al. Phenotypic heterogeneity of melanoma. J Exp Med 1987;164:812-29. 8. Herlyn M, Clark WH, Rodeck U, et al. Biology of tumor progression in human melanocytes. Lab Invest 1987; 56:461- 74. 9. Schulz G, Staffileno LK, Reisfeld RA, Dennert G. Eradication of established human melanoma tumors in nude mice by antibody-directed effector cells. J Exp Med 1985; 161:1315-25. 10. Balch CM, Riley LB, Bae YJ, et al. Patterns of human tumorinfiltrating lymphocytes in 120 human cancers. Arch Surg 1990;125:200-5. 11. Houghton AN, Mintzer D, Cordon-Cardo C, et al. Mouse monoclonal IgG3 antibody detecting GD3 ganglioside: A phase I trial in patients with malignant melanoma. Proc Nat1 Acad Sci USA 1985;82:1242-6. 12. Cheresh DA, Honsik CJ, Staffileno LK, et al. Disialoganglioside GD3 on human melanoma serves as a relevant target antigen for monoclonal antibody-mediated tumor cytolysis. Proc Nat1 Acad Sci USA 1985;82:5155-9. 13. Connett JM, Zhu X, O’Halloran LR, Philpott GW. Radioimmunotherapy of human colon carcinoma (GW-39) in hamsters with 1131anti-colon cancer monoclonal antibody (MAb) lA3. Proc Am Assoc Cancer Res 1991;32:263, Abstract. 14. LoRusso PM, Baker L, Vaitkevicius V, et al. Phase I study of the IgG2b anticolerectal antibody-ricin A immunotoxin xomazyme-791 in patients with metastatic colerectal carcinoma. Proc Am Assoc Cancer Res 1991;32:263, Abstract. 15. Morton DL, Hoon DS, Nizze JA, et al. Active specific immunotherapy with melanoma cell vaccine and immunomodulation in patients with metastic melanoma. In: Hersey P, editor. Biological agents in the treatment of cancer. Proceedings of the International Conference (Newscastle), 1990. 16. Topalian SL, Solomon D, Rosenberg SA. Tumor specific cytolysis by lymphocytes infiltrating human melanomas. J Immunol 1989;142:3714-25. 17. Itoh K, Platsoucas CD, Balch CM. Autologous tumor-specific cytotoxic T lymphocytes in the infiltrates of human metastic melanomas. Activation by interleukin 2 and autologous tumor cells, and the involvement of the T cell receptor. J Exp Med 1988;168:1419-41. 18. Kaplan HS. The nature of the immunologic defect. In: Kaplan HS, editor. Hodgkin’s disease. 2nd ed. Cambridge (MA): Harvard University Press, 1980: pp 236-79.
4. Cheresh DA, Reisfeld RA. 0-Acetylation
of diaganglioside CD3 by human melanoma cells creates a unique antigenic determinant. Science 1984;225:844-6.
19. Tucker MA, Misfeldt D, Coleman CN, et al. Cutaneous malignant melanoma after Hodgkin’s disease. Ann Inter Med 1985;102:37-41.
5. Alexander MA, Lee W, Guerry D. Retroviral vector trans-
20. Birkeland SA, Kemp E, Hauge M. Renal transplantation and cancer: The Scandia transplant material. Tissue Antigens 1975;6:28-36.
fection of a class II positive human metastatic melanoma cell line with a matched HLA-DR Bl gene restores its capac-
74
DONAWHO AND KRIPKE
21. Greene MH, Young TI, Clark WH Jr. Malignant melanoma in renal transplant recipients. Lancet 1981;1:1196-9.
Clinics in Dermatology 2992;10:69- 74 tive effects on the generation of contact and delayed hypersensitivity after exposure to UVA or UVB radiation. J Invest Dermatol 1990;94:26-32.
22. Gupta S, Imam A. Malignant melanoma in a homosexual man with HTLV-III/LAV exposure. Am J Med 1987; 82:1027-30.
30. Kripke ML. Immunologic mechanisms in UV radiation carcinogenesis. Adv Cancer Res 1981;34:69-106.
23. Tirelli U, Vaccher E, Zagonel V. Malignant tumors other than lymphoma and Kaposi’s sarcoma in association with HIV infection. Cancer Detect Prevent 1988;12:267-72.
31. Berkelhammer J, Oxenhandler RW. Evaluation of premalignant and malignant lesions during the induction of mouse melanomas. Cancer Res 1987;47:1251-4.
24. Krause W, Mittag H, Gieler U, et al. A case of malignant melanoma in AIDS-related complex. Arch Dermatol 1987;123:867-8. 25. Elmets CA, Bergstresser PR, Tigelaar RE, et al. Analysis of the mechanism of unresponsiveness produced by haptens painted on skin exposed to low dose ultraviolet radiation. J Exp Med 1983;158:781-94. 26. Toews GB, Bergstresser PR, Streilein JW. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J 1mmuno11980;124:445-53. 27. O’Dell BL, Jessen RT, Becker LE, et al. Diminished immune skin. Arch Dermatol in sun damaged response 1980;116:559-61. 28. Kripke ML. Immunological unresponsiveness induced by ultraviolet radiation. Immunol Rev 1984;80:87- 102. 29. Kim TY, Kripke ML, Ullrich SE. Immunosuppression by factors released from UV-irradiated epidermal cells: Selec-
32. Romerdahl CA, Stephens LC, Bucana C, Kripke ML. The role of ultraviolet radiation in the induction of melanocytic skin tumors in inbred mice. Cancer Commun 1989; 1:209-16. 33. Romerdahl CA, Donawho C, Fidler IJ, Kripke ML. Effect of ultraviolet B radiation on the in uivo growth of murine melanoma cells. Cancer Res 1988;48:400710. 34. Donawho C, Kripke ML. Evidence that the local effect of UV radiation on the growth of murine melanomas is immunologically mediated. Cancer Res 1991;51:4176-4181. 35. United States Environmental Protection Agency, Office of Air and Radiation. Ultraviolet Radiation and Melanoma with a Special Focus on Assessing the Risks of Ozone Depletion. EPA 400/l-87/001D, 1987. 36. Rhodes AR, Weinstock MA, Fitzpatrick TB, et al. Risk factors for cutaneous melanoma: A practical method of recognizing predisposed individuals. JAMA 1987;258:3146-54.
C
utaneous melanoma is one of the most intensively investigated types of cancer from the viewpoint of immunology.1 In fact, melanoma is quite disproportionate among studies of the immunology of human cancers. The historical reasons for this stem from clinical observations on the pathogenesis of melanoma. Complete and partial regressions of cutaneous melanoma lesions are well documented and are associated histologically with mononuclear cell infiltrates. In addition, there are many reports of a long dormant period after excision of a primary melanoma, followed by a sudden onset of extensive metastatic disease. Such observations have led to speculation that immunologic mechanisms might be important in the control of cutaneous and metastatic melanoma. As we review below, this idea has been substantiated by recent studies of melanoma in both humans and animal models. In addition, two other lines of investigation have stimulated interest in the immunology of melanoma. The findings over the past decade concerning the considerable immunologic capabilities of the skin have raised questions concerning the role of cutaneous immunity in the development of all types of cutaneous cancers. Second, increasing evidence that ultraviolet (UV) radiation contributes somehow to the incidence of melanoma, coupled with the many demonstrations of the immunosuppressive activity of UV radiation, raises the possibility that the immunosuppressive as well as the carcinogenic action of UV radiation may contribute to the induction and pathogenesis of this disease.
From the Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, Texas. Address correspondence toMargaret L. Kripke, Department of Immunology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.
0 1992 by Elsevier Science Publishing Co., Inc.
l
0738-081x/92/$5.00
Melanoma
in Humans
Melanoma Antigens Serologic analyses2 have demonstrated that there are three classes of cell surface antigens on melanoma cells: unique antigens expressed only on individual melanomas, melanoma-specific antigens present on many melanomas but absent on other types of tumor and normal cells, and common antigens that are widely distributed on a variety of both normal and neoplastic cells. Because of their potential use in the therapy of melanoma, several of the melanoma-specific antigens have been characterized; foremost among these are the ganglioside antigens.3*4 Most melanomas express large quantities of a specific asialoganglioside (GD3) that is being used in various approaches for melanoma therapy. In addition, several melanoma-specific glycoproteins are being used in diagnostic assays and for the generation of monoclonal antibodies and vaccines for immunotherapy. It is clear from functional studies of T lymphocytes from melanoma patients that melanoma antigens recognized by T lymphocytes exist, and that there are both individually specific and melanoma-associated antigens that react with T lymphocytes. Because of the inherent difficulty involved in isolating T lymphocyte-defined antigens, however, these melanoma antigens have not yet been characterized, nor has their relationship to the serologically defined antigens been determined. Some melanomas also express class II molecules of the major histocompatibility complex (MHC). This is of interest because of the role that such molecules play in the presentation of antigens to T lymphocytes. Early cutaneous melanomas often express large amounts of class II molecules capable of presenting antigen to helper T lymphocytes, thereby initiating T cell activation. Tumor progression seems to be accompanied by the loss of antigen presenting function of the melanoma cells, suggesting that class II MHC expression and function may be impor-
69
70
DONAWHO AND KRIPKE
Clinics in Dermatology 1992;10:69-74
tant in determining the outcome of the host-tumor interaction.5 Furthermore, these molecules may contribute to the unusual immunogenic properties of melanomas.
Immune Responses
to Melanoma
Several types of analyses have been carried out in attempts to characterize the immune responses of melanoma patients to their tumors. Many studies have examined the numbers and types of cells associated with primary cutaneous melanomas using histologic and immunohistochemical approaches. The number and arrangement of epidermal Langerhans cells associated with melanoma lesions have been investigated extensively. Such studies have demonstrated that immune cells of various types are frequently associated with primary melanomas and less frequently with melanoma metastases. The functional significance of these associations is difficult to determine from this approach; however, there is a strong correlation between good prognosis and the presence of T lymphocytes in melanomas in the vertical growth phase.6 Many investigators have demonstrated that melanoma patients often have T lymphocytes and serum antibodies that react with autologous and/or allogeneic melanoma cells.z*7-9 Some of the antibodies have been used to help define the antigens expressed on human melanoma cells. Recently, T lymphocytes recovered from dissociated melanomas have been characterized in terms of their activity and specificity in conjunction with attempts to use these cells for adoptive immunotherapy. A significant proportion of melanomas contain T lymphocytes that are specifically cytotoxic for autologous melanoma cells in vitro. Nonspecifically cytotoxic T and natural killer (NK) cells are also present. It is intriguing that these cells are not cytotoxic when first recovered from the tumor but require in vitro activation with interleukin-2 (IL-2) to become cytolytic. Using this approach, it has become apparent that melanomas are unusually immunogenic compared with other solid tumors, in which it is difficult to find T lymphocytes with specific cytotoxicity for autologous tumor cells.*0
Immunotherapy
of Melanoma
Current directions in the immunotherapy of melanoma involve three basic approaches: monoclonal antibodies directed against melanoma antigens, melanoma vaccines, and in vim or in vitro activation of lymphocytes. Antibodies against GD3 melanoma antigen appear to be beneficial in retarding melanoma growth, both in humans and in animals.“J2 These and other antibodies are also being
explored as a means of targeting radioactive and toxic molecules to cancer cells in vi7m13~*4 Numerous clinical trials with various forms of melanoma vaccine preparations are being carried out. Most of these involve a mixture of allogeneic melanoma cells lines with or without an immunologic adjuvant. Antibody and delayed hypersensitivity responses to such vaccines have been documented, and this approach has proved to be beneficial for a significant fraction (20 to 40%) of melanoma patients.15 Another form of therapy has involved attempts to activate tumor-reactive lymphocytes in viva by infusing patients who have metastatic melanoma with the lymphocyte activator IL-2. Because of the toxicity of this treatment, subsequent attempts have been made to activate and expand lymphocytes in vitro with IL-2 and reinfuse them into the patients. Lymphocytes from peripheral blood, from the tumor tissue, and from the lymph nodes draining vaccination sites have all been used for this purpose.16*” It is not yet clear whether these approaches will improve the survival of patients with metastatic melanoma. Nevertheless, it is apparent from these studies that human melanomas have antigens that can serve as the target of various immune responses and that at least some melanoma patients can mount immune responses to these antigens. These findings indicate that continued effort and optimism are warranted in devising immunologic approaches for the treatment of melanoma. They also imply that the immunologic status of the host may play an important role in the development and progression of melanoma.
lmmunosuppression
and Melanoma
Because melanomas appear to be immunogenic, immunosuppression might be expected to be associated with a higher incidence of this neoplastic disease. Hodgkin’s disease, usually marked by lymphopenia and abnormalities in T-cell function, provides a test of this expectation. Patients with Hodgkin’s disease often have impaired cell-mediated immune responses’s and a high risk of developing second primary tumors.19 The most common of these is acute nonlymphocytic leukemia, but in addition, a significant increase has been noted in the incidence of melanoma in the fourth and fifth years after diagnosis of Hodgkin’s disease. Both the short interval between the occurrence of Hodgkin’s disease and the onset of the melanomas and the lack of a typical, intense mononuclear infiltrate in the tumor are suggestive of an association between immunosuppression and melanoma development in these patients.19
Clinics in Dermatology 1992;10:69-74 Another significant population of immunosuppressed individuals is transplant recipients, who are immunosuppressed with drugs to prevent graft rejection. In one study the transplant patients had a striking excess of nonHodgkin’s lymphoma and squamous cell carcinomas of the skin.20 In addition, a three- to six-fold excess of melanomas occurred in these patients. Their tumors were also characterized by a sparse inflammatory infiltrate. Two patients in this study had previously had a primary melanoma removed, but were disease-free at the time of transplantation. Unfortunately, both developed and subsequently died from melanoma metastases after the advent of immunosuppressive therapy. This observation led the authors to question the wisdom of renal transplantation for individuals with a history of previous melanoma if immunosuppression was required.*’ In patients with Hodgkin’s disease and renal transplants, the risk of cutaneous melanoma is significantly increased, especially for people with dysplastic nevi. 19s2*Such patients with dysplastic nevi should be monitored closely for the occurrence of melanoma. A condition of increasing concern and significance in this decade is the acquired immune deficiency syndrome (AIDS). This progressively degenerative infectious disease is associated with human immunodeficiency virus (HIV) infection. AIDS is characterized by a severe T cellmediated immune deficiency and decreases in NK cells, resulting in an unusual susceptibility to opportunistic infections and a high incidence of certain malignancies.22 There are reports of increased incidence of Kaposi’s sarcoma, malignant lymphoma, and squamous cell carcinoma in HIV-infected person.23 In addition, there have been several reports of the occurrence of melanoma with a particularly aggressive course in AIDS patients,21*22*24 but there is no definitive study of melanoma occurrence in HIV-positive individuals. Unfortunately, as time passes, the increased occurrence of additional neoplasms may become evident, particularly if improved treatment results in a longer life span for AIDS patients. Given the immunologic properties of melanomas, this form of cancer may increase in these individuals.
Studies in Animal Models Additional interest in the possible role of immunologic factors in the development of cutaneous melanoma has resulted from new information on the immunology of the skin and on the interactions between cutaneous immunity and UV radiation. The role of such factors in melanoma induction and progression is still largely undefined because, until recently, convenient animal models for in-
DONAWHO AND KRIPKE
71
IMMUNOLOGIC FACTORS IN MELANOMA
ducing primary melanomas did not exist, and only a few transplantable murine melanomas were available.
Immunologic Effects of Ultraviolet Radiation Recent studies have demonstrated that a population of immune cells residing in the skin is responsible for initiating immune responses. Epidermal Langerhans cells take up foreign substances and process them into antigens that can be recognized by T lymphocytes. Langerhans cells are the primary antigen presenting cells for the induction of contact allergy, and they may also be responsible for initiating immune responses to skin cancers and infectious organisms in the skin. Studies in mice demonstrated that exposing the skin to suberythemal doses of UV radiation reduces the number and alters the morphology of epiderma1 Langerhans cells. These changes are accompanied by an alteration in immune function that can be demonstrated by applying a contact-sensitizing agent onto the UV-irradiated skin. Instead of inducing a normal contact allergy reaction, the sensitizer induces antigen-specific suppressor T lymphocytes and a state of immunologic tolerance that inhibits a subsequent response to the same sensitizer.25*26 In addition, UV irradiation has been shown to alter the effector phase of some cutaneous immune reactions.27 UV radiation can also inhibit the induction and elicitation of immune responses at distant sites.28 Not all immune responses are affected, but contact allergy, delayed hypersensitivity, and the rejection of UV-induced skin cancers are all inhibited under certain conditions of UV irradiation and antigen sensitization. In all of these instances, immune suppression is associated with the formation of suppressor T lymphocytes. The mechanism by which UV irradiation of one site alters the immune response to antigens introduced at a distant site is not completely clear; however, recent evidence suggests that exposure of keratinocytes to UV radiation causes the release of soluble mediators that modify the immune response.29 One important aspect of UV-inducedimmunosuppression is the role it plays in UV carcinogenesis in experimental animals. Studies in mice demonstrate that exposure to UV radiation decreases the host’s ability to respond to antigens on UV-induced tumor cells.30 This allows the tumors to escape the normal immune surveillance mechanism, thereby increasing the probability of tumor development. This UV-induced alteration involves the generation of suppressor T cells that prevent the rejection of UV-induced tumors. Because these suppressor cells are present throughout the body, this effect of the UV radiation is systemic. Thus, UV radiation induces a systemic alteration in the immune system that contributes to the growth of UV-induced skin cancers.
72
DONAWHO AND KRIPKE
These studies raise the possibility that UV radiation might contribute to the pathogenesis of melanoma at stages other than the initial transformation event, by means of its immunosuppressive activity, regardless of any direct role it might play in carcinogenesis.
Role of Ultraviolet Radiation in the induction of Melanoma That UV radiation plays a role in melanoma induction has been substantiated in studies with a murine model. Using an approach developed by Berkelhammer and Oxenhandler,31 we investigated the role of UV radiation in the induction and pathogenesis of murine melanoma. Melanomas were induced by treating 4-day-old mice with DMBA, an initiating agent, and at 3 weeks of age painting with croton oil (a promoter) twice per week.31 We either substituted UV radiation for the initiator or the promoter or added UV radiation to the promotion protocol.32 We found that UV radiation could serve as a weak initiator or promoter in the induction of melanomas, In addition, irradiating the site of tumor initiation and promotion dramatically accelerated the appearance of melanoma. This effect of UV radiation on the development of melanomas was not a systemic effect, because UV irradiation of a distant site did not affect melanoma induction. Thus, UV irradiation exerted a direct effect on melanoma development at the site of melanoma induction.32 Our studies demonstrated that UV radiation can act directly to induce melanomas by serving as the initiator or promoter for the transformation of melanocytes. Additionally, it can act as a cofactor that promotes the outgrowth of melanomas after their induction by chemical agents. This diversity of roles for UV radiation seems to correlate well with the confusing picture of the etiology of melanoma that has arisen from epidemiologic studies.
Local Effect of Ultraviolet Irradiation on Melanoma Growth Recently, we have investigated how UV radiation is able to accelerate the development of primary melanomas, We used transplantable melanoma cell lines in a series of experiments to determine whether UV radiation directly affected the melanoma cells themselves or interacted with the surrounding host tissues.33 We found that transplanted melanomas appeared earlier and reached a higher incidence when the cells were injected into the ears of UV-irradiated mice. Six moderate (4.8 kJ/m*) exposures to UVB radiation affected host tissues in a way that enhanced the outgrowth of melanoma cells injected into the UV-irradiated site. The effect was localized to the site of tumor implantation and did not represent a systemic alteration of UV radiation. Because UV irradiation
Clinics in Dermatology 2992;10:69- 74 has been shown to have immunosuppressive activity in mice, we investigated whether the immunologic alterations caused by UV irradiation could be responsible for its effect on melanoma development.34 We first examined the specificity of the effect of UV irradiation on in vim tumor growth. We found the melanomas were not the only tumors affected by UV irradiation; however, only the growth of immunogenic tumors was accelerated in UV-irradiated mice. We also found an exact correlation between tumors that were immunogenic and tumors that were affected by UV irradiation.34 Next, we examined the effect of UV irradiation on tumor growth in immunosuppressed mice. We reasoned that if UV irradiation caused local immunosuppression, which was responsible for the effect of UV radiation on tumor outgrowth, then the effect would not be observed in immunodeficient mice. Indeed, we found no effect of UV radiation on the incidence of melanoma in mice immunosuppressed by X-irradiation, T cell depletion, or congenital athymia. These results strongly suggested that the effect of UV radiation is immunologic in nature and probably involves a reduction of T-lymphocyte function.34 Our third approach was to investigate the effects of UV radiation on the expression of systemic tumor immunity in mice. Exposing mice to UV radiation after immunization prevented rejection of the melanoma cells when they were injected into the UV-irradiated site; however, the mice challenged in a hair-protected site (the flank) were unaffected by exposure to UV radiation. These experiments demonstrated that the expression of an existing immune response to melanoma is perturbed in UV-irradiated skin (C. K. Donawho and M. L. Kripke, unpublished data). These results strengthen the possibility that the effect of UV irradiation on melanoma development is due, at least in part, to an immunosuppressive effect of the UV radiation. Also, it demonstrates that moderate exposures to UV irradiation can impair the expression of immunity to tumor cells within UV-irradiated skin.
Conclusions The etiology of human melanoma remains unclear, but it is likely to be multifactorial and to involve UV radiation. Melanoma is clearly a complex disease, with multiple histologic forms, and is subject to various genetic and environmental influences. The increasing exposure of the population to solar UV radiation resulting from changes in life-style and recreation makes UV radiation a logical candidate for contributing to the increasing incidence of melanoma. The exposure to chemicals in our environment may also be contributing to this increase in incidence, but to date, no such agents have been identified.
Clinics in Dermatology 1992;10:69- 74 With the expected decrease in the concentration of stratospheric ozone, it has been predicted that ambient UV-B radiation will increase and, therefore, the incidence of melanoma will also increase.35 Thus, it is important to understand the role played by UVB radiation in the induction and pathogenesis of melanoma. We found that UV radiation can play a variety of roles in this disease, namely, tumor initiator, tumor promoter, and inhibitor of local immune responsiveness. Our finding that UV irradiation has an indirect, local effect on melanoma growth implies that sun exposure after the diagnosis of melanoma may hasten the progress of disease by adversely affecting the local immunologic state of the host’s skin. Also, systemic immunosuppression may contribute to the occurrence and severity of cutaneous melanoma in humans. A comprehensive revieti6 reports that three of the ten recognized risk factors for melanoma are immunosuppression, sun sensitivity, and excessive sun exposure. Undeniably the most critical aspect in prevention of deaths from melanoma is the early detection and removal of the tumor. This emphasizes the need for close observation and early detection of the melanomas that might occur in susceptible individuals, While most of the other risk factors appear to be inherent in the genetics of susceptible individuals, these three factors suggest avenues for prevention in these persons. That immunosuppression is a significant risk factor for melanomas implies that melanomas are immunogenic and suggests that at least some melanomas may be amenable to immunologic therapy. In addition, it suggests that physicians who care for immunosuppressed patients should be vigilant for signs of cutaneous melanoma.
References 1. Halliday WJ. Specificity of cell-mediated immunoreactivity in melanoma and comments on the nature of serum blocking factors. In: Reisfeld RA, Ferrone S, editors. Melanoma antigens and antibodies. New York, Plenum Press, 1982;173-86.
2. Livingston PO, Michitisch R, Shiku H, et al. Antibody-dependent cell-mediated cytotoxicity for cultured autologous melanoma cells. Cell Immunol 1981;64:131-43.
3. Puke1 CS, Lloyd KO, Travassos LR, et al. GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody. J Exp Med 1982;155:1133-47.
DONAWHO AND KRIPKE IMMUNOLOGIC
ity to present antigens. 1991;32:238, Abstract.
73
FACTORS IN MELANOMA
Proc
Am Assoc
Cancer
Res
6. Clark WH Jr, Elder DE, van Horn M. The biologic forms of malignant melanoma. Hum Path01 1986;17:443-50. 7. Houghton AN, Real FX, Davis LJ, et al. Phenotypic heterogeneity of melanoma. J Exp Med 1987;164:812-29. 8. Herlyn M, Clark WH, Rodeck U, et al. Biology of tumor progression in human melanocytes. Lab Invest 1987; 56:461- 74. 9. Schulz G, Staffileno LK, Reisfeld RA, Dennert G. Eradication of established human melanoma tumors in nude mice by antibody-directed effector cells. J Exp Med 1985; 161:1315-25. 10. Balch CM, Riley LB, Bae YJ, et al. Patterns of human tumorinfiltrating lymphocytes in 120 human cancers. Arch Surg 1990;125:200-5. 11. Houghton AN, Mintzer D, Cordon-Cardo C, et al. Mouse monoclonal IgG3 antibody detecting GD3 ganglioside: A phase I trial in patients with malignant melanoma. Proc Nat1 Acad Sci USA 1985;82:1242-6. 12. Cheresh DA, Honsik CJ, Staffileno LK, et al. Disialoganglioside GD3 on human melanoma serves as a relevant target antigen for monoclonal antibody-mediated tumor cytolysis. Proc Nat1 Acad Sci USA 1985;82:5155-9. 13. Connett JM, Zhu X, O’Halloran LR, Philpott GW. Radioimmunotherapy of human colon carcinoma (GW-39) in hamsters with 1131anti-colon cancer monoclonal antibody (MAb) lA3. Proc Am Assoc Cancer Res 1991;32:263, Abstract. 14. LoRusso PM, Baker L, Vaitkevicius V, et al. Phase I study of the IgG2b anticolerectal antibody-ricin A immunotoxin xomazyme-791 in patients with metastatic colerectal carcinoma. Proc Am Assoc Cancer Res 1991;32:263, Abstract. 15. Morton DL, Hoon DS, Nizze JA, et al. Active specific immunotherapy with melanoma cell vaccine and immunomodulation in patients with metastic melanoma. In: Hersey P, editor. Biological agents in the treatment of cancer. Proceedings of the International Conference (Newscastle), 1990. 16. Topalian SL, Solomon D, Rosenberg SA. Tumor specific cytolysis by lymphocytes infiltrating human melanomas. J Immunol 1989;142:3714-25. 17. Itoh K, Platsoucas CD, Balch CM. Autologous tumor-specific cytotoxic T lymphocytes in the infiltrates of human metastic melanomas. Activation by interleukin 2 and autologous tumor cells, and the involvement of the T cell receptor. J Exp Med 1988;168:1419-41. 18. Kaplan HS. The nature of the immunologic defect. In: Kaplan HS, editor. Hodgkin’s disease. 2nd ed. Cambridge (MA): Harvard University Press, 1980: pp 236-79.
4. Cheresh DA, Reisfeld RA. 0-Acetylation
of diaganglioside CD3 by human melanoma cells creates a unique antigenic determinant. Science 1984;225:844-6.
19. Tucker MA, Misfeldt D, Coleman CN, et al. Cutaneous malignant melanoma after Hodgkin’s disease. Ann Inter Med 1985;102:37-41.
5. Alexander MA, Lee W, Guerry D. Retroviral vector trans-
20. Birkeland SA, Kemp E, Hauge M. Renal transplantation and cancer: The Scandia transplant material. Tissue Antigens 1975;6:28-36.
fection of a class II positive human metastatic melanoma cell line with a matched HLA-DR Bl gene restores its capac-
74
DONAWHO AND KRIPKE
21. Greene MH, Young TI, Clark WH Jr. Malignant melanoma in renal transplant recipients. Lancet 1981;1:1196-9.
Clinics in Dermatology 2992;10:69- 74 tive effects on the generation of contact and delayed hypersensitivity after exposure to UVA or UVB radiation. J Invest Dermatol 1990;94:26-32.
22. Gupta S, Imam A. Malignant melanoma in a homosexual man with HTLV-III/LAV exposure. Am J Med 1987; 82:1027-30.
30. Kripke ML. Immunologic mechanisms in UV radiation carcinogenesis. Adv Cancer Res 1981;34:69-106.
23. Tirelli U, Vaccher E, Zagonel V. Malignant tumors other than lymphoma and Kaposi’s sarcoma in association with HIV infection. Cancer Detect Prevent 1988;12:267-72.
31. Berkelhammer J, Oxenhandler RW. Evaluation of premalignant and malignant lesions during the induction of mouse melanomas. Cancer Res 1987;47:1251-4.
24. Krause W, Mittag H, Gieler U, et al. A case of malignant melanoma in AIDS-related complex. Arch Dermatol 1987;123:867-8. 25. Elmets CA, Bergstresser PR, Tigelaar RE, et al. Analysis of the mechanism of unresponsiveness produced by haptens painted on skin exposed to low dose ultraviolet radiation. J Exp Med 1983;158:781-94. 26. Toews GB, Bergstresser PR, Streilein JW. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J 1mmuno11980;124:445-53. 27. O’Dell BL, Jessen RT, Becker LE, et al. Diminished immune skin. Arch Dermatol in sun damaged response 1980;116:559-61. 28. Kripke ML. Immunological unresponsiveness induced by ultraviolet radiation. Immunol Rev 1984;80:87- 102. 29. Kim TY, Kripke ML, Ullrich SE. Immunosuppression by factors released from UV-irradiated epidermal cells: Selec-
32. Romerdahl CA, Stephens LC, Bucana C, Kripke ML. The role of ultraviolet radiation in the induction of melanocytic skin tumors in inbred mice. Cancer Commun 1989; 1:209-16. 33. Romerdahl CA, Donawho C, Fidler IJ, Kripke ML. Effect of ultraviolet B radiation on the in uivo growth of murine melanoma cells. Cancer Res 1988;48:400710. 34. Donawho C, Kripke ML. Evidence that the local effect of UV radiation on the growth of murine melanomas is immunologically mediated. Cancer Res 1991;51:4176-4181. 35. United States Environmental Protection Agency, Office of Air and Radiation. Ultraviolet Radiation and Melanoma with a Special Focus on Assessing the Risks of Ozone Depletion. EPA 400/l-87/001D, 1987. 36. Rhodes AR, Weinstock MA, Fitzpatrick TB, et al. Risk factors for cutaneous melanoma: A practical method of recognizing predisposed individuals. JAMA 1987;258:3146-54.
