Europ. J. CancerVol. 12, pp. 585-594. Pergamon Press 1976. Printed in Great Britain

Perspectives in Cancer Research

The Bio]Logy and Treatment of Malignant Melanoma ALISTAIR J. COCHRAN The University of Glasgow Department of Pathology, The Western Infirmary, Glasgow G11 6NT, Great Britain.

SINCE ITS recognition as a clinical and pathological entity in the early 19th century, malignant melanoma has commanded interest disproportionate to its relatively low frequency. Clinical concern and unflagging enthusiasm among biologists for pigment cell research have ensured a massive literature on pigment cellrelated topics, a :~eries of highly successful International Pigment Cell Congresses, a new Journal, Pigment Cell, and current moves to establish a multidisciplinary International Pigment Cell Society. This paper summarizes some pertinent findings from recent studies of clinical, pathological and biological aspects of the melanocytic turaours. Melanoma has a particularly sinister reputation which is enti:cely justified in individual cases, but is excessive if one compares the fate of melanoma patie~ats as a whole with that of patients with, for instance, bronchogenic carcinoma or gastric carcinoma. The need for an understanding of the mechanisms of malignant transformation of melanocytes, the factors involved in metastasis formation and the development of improved treatment is underlined by a steady increase in the frequency of melanoma and of deaths from it. Reports of an increasing incidence come from areas as diverse as Norway [1], Australia [2], the United States of America, and England and Wales [3]. There :is some increase in all age groups, but adults under 65 seem maximally affected and the J[ncrease involves men and women equally. ]:he disease has tended to increase most in areas of affluence and in higher socio-economic groups. The reasons for this increase are complex but various factor,~ may have an aefiological role. Sunlight-derived radiant energy has been

much discussed. Black people seldom develop melanoma, the incidence in U.S. blacks being around 5 per 100,000. Conversely albinos and individuals of Northern European stock, who tan poorly or freckle, seem specially susceptible. The maximum incidence is seen in those of Northern European stock who live in sunny areas such as Australia and South Africa, the peak reported incidence in this group being 265 per 100,000 in Queensland. The frequency of melanoma and death rate from it increase progressively as the equator is approached, both in Australia [4, 5] and in the U.S. [6]. Involvement of solar radiation is further suggested by the observed differences of primary melanomas of different sites in men and women, which correlate with dress habits. The high frequency of primaries on the lower leg in women, for example, fits with the exposure of the leg associated with the wearing of skirts. The situation is complicated by the observation that unlike other skin cancers, the increased frequency of melanoma in Australia involves all areas of the body, not only those exposed to sunlight. Lee and Merrill [7], therefore, postulate that sun-damaged melanocytes may release materials (solar circulating factor) which circulate and m a y cause malignant transformation of melanocytes in areas remote from those exposed to the sun. There is as yet no evidence to support (or refute) this interesting speculation. A simpler possibility is that light clothing may not adequately screen out carcinogenic rays. This certainly appears to be the case with stockings and tights. Another paradox is that m a n y melanoma patients do not have especially "outdoor" occupations and analysis of leisure activities

585

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Alistair J. Cochran

has so far failed to show a totally convincing correlation with melanoma incidence. It is possible that acute exposure of the skin, especially that which tans poorly, by injudicious sunbathing is more dangerous than chronic exposure of conditioned skin which tans effectively [8]. The role of physical trauma in melanoma remains enigmatic. Specific traumatic events recorded by a minority of patients are probably examples of "traumatic determinism". However melanomas of the sole of the foot do occur frequently in populations who go barefooted [9]. Endocrine factors m a y also be important. Melanoma is more common in women of child-bearing age than in men of comparable age. However, in such women the disease appears somewhat less aggressive. The view that melanoma is more common during pregnancy has not been substantiated by recent studies, but in individual cases pregnancy is associated with an increased rate of tumour growth and extension. It seems likely that endocrine factors are not directly carcinogenic, but act as cofactors. Endocrine contraceptive agents may well influence melanocytes, and definitive studies of these materials in melanocyte biology and their effect on the incidence of melanomas are desirable. Recent reports of therapeutic successes with hormones are of interest, and it seems that this approach is due for re-examination [10]. The role of viruses in human carcinogenesis is under detailed scrutiny and studies of this possibility in melanoma have been reported. Birkmayer et al. [11] found viral particles in h u m a n melanomas which share characteristics with the oncorna viruses of animal melanomas. These workers also identified reverse transcriptase and 70S KNA in virus containing h u m a n melanomas, characteristics of oncogenic KNA viruses in more defined animal situations. T h e y also reported positive nucleic acid hybridization studies with virus-specific DNA and material from human melanomas, but not with other skin cancers. These exciting findings require confirmation and even if this is forthcoming the problem of proving a causal association remains. However reports of similar antigens on different melanomas (see below) do reflect the situation in animal tumours induced by viruses. Genetic factors are involved. I have discussed the susceptibility of certain ethnic groups to melanoma, and the persistence of high melanoma incidence in the descendents of immigrants from Northern Europe to Australia

indicates that melanoma susceptibility is heritable. Additionally there are families in which melanomas are specially frequent, develop relatively early in life and may be multiple. In a study in Queensland the risk of a first degree relative of a melanoma patient developing this tumour was roughly twice that of the general population. However, these individuals also have an increased likelihood of developing cancers other than melanoma. The nature of the genetic defect remains unknown. Wallace and Exton [12] merely state that it is polygenic, but Sutherland et al. [13] interpret their initial data on familial melanoma as in keeping with the effects of an autosomal gene with incomplete penetrance. Diagnosis remains a problem. In contrast with other skin malignancies, which are often suspected to be malignant and assigned to the correct diagnostic category, melanoma is correctly diagnosed in less than 50% of cases. This is no doubt due to a low frequency of heavily melanized melanomas and the absence of clinically detectable pigment in some (although less than 1% are amelanotic on cytochemical examination). Accuracy of diagnosis depends on a willingness to consider melanoma in the differential diagnosis of even totally non-pigmented skin lesions, a careful clinical history with emphasis on the nature and colour of any lesion which previously existed at the tumour site and careful examination of the tumour and the skin around it for any alterations of colour and not just for brown or black pigmentation. This is highlighted by Mihm et al. [14] in their excellent diagnostic atlas of melanoma and by the W H O pamphlet, "Clinical Diagnosis of Malignant Melanoma" [15]. In cases where, despite careful inspection, the diagnosis remains in doubt, magnification by a hand lens or operating microscope [16] may be of assistance. Careful clinical examination and history taking will indicate the diagnosis in all but a few melanomas, and any doubts are settled by histological examination of biopsy. The biopsy should, whenever possible, be an excision biopsy, but there is little convincing evidence that incision biopsy is harmful, if followed promptly by excisional surgery. Ingenious nonoperative aids to diagnosis have been introduced and in the hands of enthusiasts have had some success. These include the uptake of 32p [17 18], thermography [19] and the uptake of radio-labelled quinolines with affinity for melanin [20]. In the face of the diagnostic efficiency and simplicity of careful clinical observation, and the accessibility of this tumour

The Biology and Treatment of Malignant Melanoma

for biopsy, it is difficult to see the place of such technically complex procedures in melanoma diagnosis. Diagnosis by estimation of tumour cell products has attracted interest recently. Estimation of urinary melanogens and dopa oxidase or tyrosinase in the blood have insufficient specificity to be really useful, especially in patients with early tumours. A promising approach is the estimation of 5-S-cysteinyldopa, a catechol precursor of phaeomelanin, in the urine [21]. This is excreted by normal individuals, but lew~ls are raised in the urine of melanoma patients. Interpretational problems exist, the level of excretion being raised, for example, after sunbathing, but further study may well indicate a role for estimation of 5-S-cysteinyldopa, at least as a monitor for recurrent melanoma. Hopes for immunodiagnosis of this and other tumours have not yet been realized. However, studies of immunological aspects of melanoma have yielded much of interest. In 1967 Lewis [22] found that serum from patients with slowly evolving melanomas killed autologous tumour cells in vitro, an effect not seen with serum from patients with rapidly progressing melanomas. Apparently melanoma-specific antibodies were detected by Morton et al. [23], Lewis et al. [24], Muna et al. [25] and Romsdahl and Cox [26] and subsequently by numerous other authors. These were directed against antigens in two sites: the cytoplasm and the cell membrane. There is agreement that antibodies to cytopla,~mic components cross-react with allogeneic melanomas, however there is less unanimity about the specificity of membrane-reactive antibodies. Lewis et al. [24], and more recently Bodurtha et al. [27], claim that the antibodies to the membrane antigens are individual-specific while others find these too cross-reactive. This disagreement remains to be explained, but is at least partly due to differences in technique. A nucleolar antigen has also been described [28] against which a proportion of patients develop antibodies. The possession of this nucleolar antigen indicates a poor prognosis. Melanoma cell killing and cytostasis by specifically sensitized lymphocytes and non-sensitized lymphocytes and macrophages have been demonstrated in vitro, and such ,=ells seem likely to be active against tumour cells in vivo. Specifically sensitized lymphocytes ihave been demonstrated by in vitro cytotoxicity p9, 30], tumour-antigen mediated transformation [31,32] and tumour-antigen mediated inhibition of leucocyte migration

587

[33, 34]. Sensitization of a proportion of melanoma patients to melanoma-derived materials has also been demonstrated by skin reactivity studies [35-37]. The exact specificity of these reactions is as yet unclear and it is possible that some may be reactions against foetal antigens rather than tumour-associated antigens with a rejection potential. Studies of lymphocytes subpopulations in melanoma patients [38] have been reported and indicate variations in, for instance, T cells capable of forming rosettes with sheep erythrocytes under suboptimal conditions [39]. Tumour-directed antibodies and lymphoid cells have been most readily detected by most workers in early malignancy when the tumour volume is small but even in patients with advanced disease autoimmunisation with tumour cells will cause the transient reappearance of turnout-directed antibodies [40] and lymphoid cells [41]. The mechanisms for production of tumour immunity thus remain intact, though eclipsed, in advanced disease. In the case of tumour-directed antibodies Lewis et al. [42] advance the interesting suggestion that anti-idiotypic antibodies are produced in advanced melanoma which react with tumour-directed antibodies and neutralize them. With mounting evidence of immune reactions to melanoma-associated antigens the question has to be asked whether such reactions influence tumour progression or regression or are merely epiphenomena. There is as yet no clear answer to this. Indices of anti-tumour reactivity decline with advancing disease, but whether this is the cause of tumour progression or secondary to it remains unknown. Serial studies of patients may shed light on this problem, but although loss of tumour-immune reactivity may precede and indeed predict clinically apparent metastases in some patients the findings are not sufficiently clear cut to allow their routine clinical use. The situation is complicated by the observation that the longer a patient remains tumour-free after removal of a melanoma the less likely is it that tumour-directed reactivity will be detectable [43]. There is also much interest in means whereby tumour cells may evade cytotoxic immune mechanisms. Most studies have concerned themselves with "blocking" mechanisms. It was initially thought that non-cytotoxic tumourdirected antibodies might coat tumour ceils and exclude cytotoxic lymphocytes and possibly cytotoxic non-lymphocytic mononuclear ceils (Fig. l-A). There followed a period in which

588

A l i s t a i r J . Cochran

the role of antibodies was discounted in favour of the involvement of antigen-antibody complexes and free tumour antigen. Interest in tumour directed antibodies has been restimulated by the observations of Keisari and Witz [44] that lysosomal enzymes of tumour cells can cleave tumour directed antibodies leaving Fab fragments, which would not be detectable by most anti-Ig sera. However this may be, the majority of workers believe that complexes oftumour-derived antigen and tumour-directed antibody impede effector cells in a manner which varies with the proportions of antigen (A)

By non-cytotoxic anti-tumour antibodies.*

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cell excludedfrom tarnour

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.-antibody complex

* Note. h o w ev er , that in s o m e s y s t e m s t u m o u r c e l l s p r i m e d by antibody a r e r e n d e r e d s u s c e p t i b l e to K - c e l l k i l l i n g

Fig. 1. Possible mechanisms whereby effector cells may be denied access to tumour cells, or rendered inactive.

and antibody present (Fig. l-C). Currie et al. [45, 46] present strong evidence for their hypothesis that free tumour-derived antigens may also react with potential effector cells (Fig. l-B) and this hypothesis is made the more attractive by their report [46] that tumour ceils which readily and continually shed their antigens are those which are weakly immunogenic and are more likely to metastasize than those with more stable membrane antigens. Most studies of host response to tumours have concerned the role of the lymphocyte. However, attention is increasingly directed to the activities of macrophages and certainly tumours with a high content of macrophages are those which are relatively highly immunogenie and exhibit a lower tendency to metastasize [47]. The intimate relationship between lymphocytes and macrophages is indicated by

the finding that macrophage accumulation depends upon an intact T-cell system. Gauci [48] has shown that the macrophage content of early melanomas is higher than that in metastases. It is also possible that some of the cells which kill antibody-coated target cells [49] (K cells) are derived from the macrophage series. One of the earliest findings suggestive of a melanoma-associated immune response was the observation of cellular infiltrates in and around melanomas. These appear to consist, on light microscopy, of lymphocytes, macrophages and occasionally plasma cells. However, Cesarini and Roubin [50] have presented ultrastructural evidence that the great majority of such cells are monocytoid. These infiltrates are present maximally in the early stages of tumour development, decline as the vertically invasive tumour develops and are virtually absent from metastases. The limited studies of such infiltrates have not found them strongly cytotoxic to tumour cells and Carr and Underwood [78] thought that they were cells undergoing instruction rather than active effector cells. Unlike gastric cancer [51] and medullary carcinoma of the breast [52] infiltration of a melanoma by mononuclear cells does not, in the view of most authors, presage a good outcome. In a personally studied series, however, local recurrences were less frequent in patients with infiltrated tumours, perhaps indicating enhanced local immunity [53]. Interest in morphological aspects of malignant melanomas has remained high. The work of Clark [54] and McGovern [55] has produced a better understanding of the histogenesis of malignant melanoma. On the basis of the experience of Clark and his associates, Cochran [53], McGovern [55], Little [57] and others, an attempt was made in 1973 during the V I I I t h International Pigment Cell Conference in Sydney to produce guidelines for a clinicopathological classification of primary melanomas. The main points recommended for consideration are summarized in the Table. It is hoped that this will be exploited during the next decade when, on the basis of the experience gained, a new and better classification will then be produced. The pathologists of the World Health Organisation Melanoma Reference Centre have also produced a new classification based on their extensive histological material and it will be of interest to see how this compares with the "Sydney" classification. The significance of Mishima's interesting division of melanomas into melanocytic and naevocytic types [56] deserves more attention

The Biology and Treatment of Malignant Melanoma than it has yet received. Although there is an extensive and fascinating literature on ultrastructural studies of melanocytes and melanomas the main practical place of electron microscopy remains the identification of melanosomes in doubtful tumours. Those concerned with the histological diagnosis of pigmented tumours have had much to ponder with renewed interest in the halo naevus, the description of desmoplastic malignant melanoma [58], the separation of the Spitz juvenile melanoma into spindle cell and epithelioid cell naevi, the realization that these lesions occur in adults and the account of "delayed maturation naevi" [59]. There is an increasing, if grudging, acceptance that frozen section diagnosis of pigmented tumours is practical, although the real exponents and proponents of this difficult diagnostic service are, not surprisingly, those who see the greatest number of pigmented tumours [60, 61]. M y own very limited experience suggests that the separation of melanocytic from non-melanocytic lesions on frozen sections is practical and worthwhile. For the exact diagnosis of melanocytic lesions I much prefer conventionally fixed and stained sections. The optimal treatment of malignant melanoma remains debatable. The early and superficial tumours are well treated by surgery with survival rates of between 80 and 90 o/0 at 5 years, although it is true that some of the survivors at 5 years will subsequently develop metastases and die. Yet even at this stage there is debate about the margin of excision necessary and the extent to which an "adequate" excision necessitates grafting. The case for elective nodal dissection has weakened over the years and although enthusiasm for this technique remains in some quarters it seems that, in the face of clinically impalpable nodes, most surgeons choose to wait and see. The paper by Conrad [62] for instance, certainly suggests that prophylactic dissection of impalpable nodes was not helpful. It is to be hoped that the major study of this problem by the World Health Organization Melanoma Reference Centre, at present approaching a stage where definitive analysis will be possible, will finally settle this difficult problem. It is, of course, possible that while elective nodal dissection is not the treatment of choice for most patients, it may be applicable to those whose tumours, by virtue of site or pathological type, have a specially unfavourable prognosis. Treatment by surgery becomes dramatically inadequate in the face of overt node involvement or visceral metastases. In this situation

589

the available alternatives are chemotherapy, radiotherapy, and the recently introduced techniques of immune stimulation. Chemotherapy has never been satisfactory in melanoma, with most agents achieving, at best, a transient response in 8-10% of patients. The relatively new agent imidazole carboxamide (DTIC) has done better, producing remissions in up to 30% of patients [63], but even this seems little return for therapy which has of itself powerful and undesirable side effects. Combinations of up to five agents have not improved on D T I C alone and newer agents such as dibromodulcitol show no magical specificity in early trials. New and very specific chemotherapeutic agents or striking synergy with immunotherapy are necessary if chemotherapy is to achieve any lasting place in the therapy of melanomas. The technically difficult techniques of regional perfusion have produced some improvement in survival in the hands of experts but seem unlikely to find general application. Radiotherapy continues to be used in most Western countries primarily in palliation of the advanced patient, a function which it performs well in some cases. However, in occasional situations such as localized inoperable tumours, tumours in patients unfit for surgery or unwilling to accept such treatment and mucosal melanomas radiotherapy may achieve a good if transitory response [64]. Radiotherapy remains relatively more popular as a treatment for melanoma in the Eastern European Countries and, in the hands of experienced therapists, is reportedly effective. We have been impressed by apparent synergy between radiotherapy and non-specific immune stimulation by BCG in a small number of cases and this is worthy of further study. Endolymphatic radiolipiodol has been examined in a number of centres, but has been relatively disappointing. The demonstration of melanoma associated antigens (MAA) and immune responses to them allows one to ask whether treatment by immunological means, immunotherapy, is possible. Two main approaches exist: active immunotherapy, which endeavours to stimulate endogenous anti-tumour reactivity and passive immunotherapy which administers anti-tumour factors raised in animals or other humans. Based on the studies of Mathd [65] attempts are in progress to stimulate, in a non-specific way, the patient's immunological system by agents such as Bacille Calmette-Gudrin, Corynebacterium parvum, vaccinia virus and Levamisole. Some workers attempt to add an

590

Alistair 3". Cochran

element of specificity by giving autologous or allogeneic (usually sublethally irradiated) tumour cells. Passive immunotherapy has been attempted (or is being considered) by cross immunizing cancer patients and subsequently exchanging their sera [66], by transfusing sera from patients whose melanomas have regressed [67] by giving "transfer factor" from regressed cases or non-melanomatous family members [68], by the administration of " i m m u n e " R N A [69] and by the administration of non-sensitized lymphocytes or lymphocytes sensitized to MAA in vivo or in vitro. Golub [70] has certainly shown in vitro sensitization to be feasible. Interest has also focused on the use of tumourspecific antibodies as carriers of chemotherapeutic agents [71] and this theoretically attractive, though technically difficult approach is being actively investigated. Synergy of immunotherapy with chemotherapy is presently under investigation by Gutterman et al. [72] with initially promising results. Local immunotherapy, the application to accessible melanomas of materials which induce delayed hypersensitivity responses, has yielded interesting results, especially the use of intralesional BCG by Morton et al. [73] and of dinitrochlorobenzene by Malek-Mansour and her associates [74]. The regression of noninjected cutaneous secondaries and very occasionally of visceral metastases (Malek-Mansour, personal communication) suggests that this may in fact be more than "local" immunotherapy. In summary, much remains to be done to establish the place of immunotherapy in the treatment of melanoma (and other malignancies). Some patients have obtained significant clinical benefit, but we know little of the best agents to use, the optimum routes of administration, acceptable doseages of the various adjuvants, methods of monitoring adjuvant-induced effects and how to select those patients most likely to respond. The morbidity and mortality of immunotherapy has mainly been related to hypersensitivity to and infection [75] by living adjuvants, indicating the desirability of non-living adjuvants.

Information leading to the more scientific use of immunotherapy will most probably be obtained from properly designed trials and those contemplating such treatment should, in m y view, consider very seriously collaborating within the existing trials of E O R T C , W H O or the American Clinical Cooperative groups [76]. Table 1. The form of histological report of an invasive malignant melanoma recommended in the paper by McGovern et al. [77] A.

Diagnosis according to histogenetic pattern

1. Malignant intra-epidermal freckle type or, 2. Malignant intra-epidermal type, or 3. Malignant intra-epidermal 4. Malignant intra-epidermal B.

melanoma, invasive, with adjacent component of Hutchinson's melanotic melanoma, invasive, with adjacent component of superficial spreading melanoma, invasive, with adjacent component, unclassifiable, or, melanoma, invasive without adjacent component.

Cross-sectional profile of section on slide*

1. flat or uneven 2. convex or plateau of measurable height 3. polypoidal. C.

Levels of invasion

1. 2. 3. 4. 5. D. E.

intra-epidermal papillary-dermal papillary-reticular interface reticular-dermal subcutaneous fat. Ulceration, more than 6 mm in extent Vascularinvasion

1. lymphatics 2. blood vessels. F.

Mitotic Activity, assessed on at least 10 high power ( x 300) fields (h.p.f.)

1. fewer than 1/5 h.p.f. 2. between 1/5 h.p.f, and 1/h.p.f. 3. 1/h.p.f. and over. G. Other parameters such as cytology, pigmentation, "lymphocytic" infiltrates, evidence of spontaneous regression, associated naevi and solar changes in the dermis are mainly of research interest at present, and their recording is left to the individual histologist's discretion. . *Breslow's measurement of thickness [79] is clearly superior to this crude examination.

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K. MAGNUS,Incidence of malignant melanoma of the skin in Norway 19551970. Cancer (Philad.) 32, 1275 (1973). J . j . McGovERN and M. M. LANEBROWN, In The Nature of Melanoma, p. 113. Charles C. Thomas, Springfield, 1969. J . A . H . LEE and ANNEP. CARTER, Secular trends in mortality from malignant melanoma, or. nat. Cancer Inst. 45, 91 (1970).

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For information on current trials contact Dr. Dorothy Windhorst, Coordinator, International Registry of Tumor Immunotherapy, National Institutes of Health, Building 10, Room 4B15, Bethesda, Md. 20014, U.S.A. VINCENTJ. McGOVERN, MARTIN C. MIHM, JR., CHRISTIANEBAILLY,JOAN CI. BOOTH, WALLACEH. CLARK,JR., ALISTAIRJ. COCHRAN,t~. G. HARDY,J. D. HICKS, ARNOLDLEVENE, MARTIN G. LEWIS,J. H. LITTLE and G. W. MILTON, The classification of malignant melanoma and its histologic reporting. Cancer (Philad.) 32, 1446 (1973). I. CARR and J. C. E. UNDERWOOD,The ultra structure of the local cellular response to neoplasia. Int. Rev. Cytol. 37, 329 (1974). A. BRESLOW, Thickness, cross-sectional areas and depth of invasion in the prognosis of cutaneous melanoma. Ann. Surg. 172, 902 (1970).