Ultrastructural Evidence for Destruction in the Halo Nevus

[CANCER RESEARCH 35, 352-357, February 1975]

J. B. Jacobs, L M. Edelstein, L. M. Snyder, and N. Fortier The Departments of Pathology and Hematology, The Saint Vincent Hospital, and the Departments of Pathology and Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01610

SUMMARY MATERIALS AND METHODS

Nine halo nevi in various stages of regression were Seventy-five halo nevi from 6 patients were examined examined by electron microscopy for fine structural evi clinically (including Wood's light scan). Nine of the 75 halo dence of an immunological mechanism of tumor cell nevi in different stages of regression were removed from 4 destruction and halo formation. Early regressing lesions patients, and a compound nevus was excised frvm each of 2 (Stage I) showed nevus cells associated with infiltrating patients, neither of whom revealed evidence of past or lymphocytes, monocytes, and plasma cells, but without present halo nevi or malignant melanoma. nevus cell destruction. In later lesions (Stages II and III), Small portions of epidermis and dermis including the vacuolar cytolysis was commonly observed in nevus cells. In nevus, surrounding halo, and a narrow rim of normal skin Stage I II lesions, portions of nevuscells are found within were obtained by punch biopsy from midback, scapular, macrophages.The electron microscopicfindings of lympho thigh, or deltoid regions. Biopsy was done under local cyte, monocyte, and plasma cell infiltration of the tumor anesthesia by a peripheral nerve-block technique. Excised followed by vacuolar cytolysis support the concept of an specimens were immediately immersed in 2% glutaralde immune reaction in regressing halo nevi. hyde in 0. 1 M phosphate buffer. Fixed specimens were cut in half, and one half was placed in formalin and processedfor routine histology. The other half was dissected into 3 INTRODUCTION portions: normal skin, halo, and mole. Extraneous fatty tissue was removed and the specimens were minced into I-cu mm blocks. Specimens were rinsed several times in 0.1 Halo nevus is a spontaneously regressing mole which M phosphate buffer followed by postfixation in I % osmium develops an enlarging area of depigmentation about the tetroxide in 0. 1 M phosphate buffer. The fixed specimens disappearing lesion. The presence of an accompanying were dehydrated in a graded series of alcohols and em inflammatory response in the lesion is taken by some bedded in Luft's 1:1 epon mixture (19). Thick and thin investigators as morphological evidence for an immunologi sections were cut on a Sorvall MT2 ultramicrotome. Thin cal mechanism of tumor cell death and macroscopic halo sections were poststained with uranyl acetate and lead formation (I 7). Recently, circulating antibodies against the citrate and examined in either a Philips 300 electron mi cytoplasm of melanoma cells have been demonstrated in croscope1ora Zeiss EM 9A electron microscope. patients with regressing halo nevi (3, 18). The authors of these papers suggest that halo nevus represents the success ful early rejection of melanoma. A cell-mediated response in RESULTS melanoma patients with regressing halo nevi has also been Clinical Examination reported (5). The mechanism of halo formation is not known, but it Each of the 75 halo nevi was classified in I of the may be secondary to the inflammatory response ( I5) or to a following 4 main stages of clinically apparent regression. cytotoxic antibody against melanocytes (14). Halos, in In Stage I were inflammed nevi that showed erythema addition to being present around benign moles (21), may be and edema. In Stage II were erythematous and slightly found about spontaneously regressing melanoma (6), about raised nevi with faint halo formation. The halo was more moles and metastases in patients treated surgically for pronounced under 3600-A light (Wood's lamp). In Stage melanoma (26), or about primary and secondary melanoma III were flat and less pigmented nevi without erythema but in patients who respond to either nonspecific immune with halo formation evident under natural light. In Stage IV stimulation (2) or adoptive immunization (26). The work of were oval white depigmented areas of epidermis without Lewis and Copeman (18) and of Copeman et a!. (3) has clinical evidence of nevi. provided information on which to base a hypothesis for an immune mechanism of tumor cell destruction in halo nevus Light Microscopy syndrome. We have examined 9 halo nevi for fine structural evidence of such an immunological mechanism. Stage I. Nevus melanization appeared intact in the Received March 18, 1974;accepted October 7, 1974. epidermis. The papillary dermis was edematous with some

352 CANCER RESEARCH VOL. 35

vascular dilation, but nevus cell nests were intact. In the addition to degenerated organelles (Fig. 7). In Stage II and reticular dermis there was an early infiltration of mononu III halo nevi, lymphocytes were found in the epidermis (Fig. clear cells at the margins of the tumor. 8). Nevus cells in junctional nests also contained vacuoles Stage II. Patchy demelanization was evident in the epi and appeared necrotic. Dendritic basal melanocytes were dermal basal layer overlying the tumor with more marked rounded with vacuolar change of the cytoplasm (Fig. 9). loss of melanin in the epidermal basal layer peripheral to the Langerhans cells were not affected by the degeneration tumor. There was minimal infiltration of the junctional process. Keratinocytes exhibited the same vacuolar change nests of nevus cells by mononuclear cells in the papillary as nevus cells and melanocytes, but only around the dermis. The reticular dermis showed marked infiltration of membrane-bound complex melanin granules (Fig. 10). the nevus cell nests by these mononuclear cells associated Keratinocytes with large vacuoles had a complete absence with degeneration of the nevus cells. Occasional nevus cells of melanin. had pyknotic nuclei and a finely vacuolated cytoplasm. Stage IV. In the well-developed halo, keratinocytes were Stage III. Complete demelanization of the peripheral virtually free of vacuoles and there was a total absence of epidermal layer was present; it was present to a lesser extent melanin (Fig. 11). In the dermis ofthe halo area there were in the epidermal basal layer overlying the tumor. Fewer disrupted mast cells, lymphocytes, pigment laden macro nevus cells, mononuclear cells, and melanophages were phages, and occasional nevus cells. present in the papillary dermis. Similarly, in the reticular dermis fewer histiocytes, mononuclear cells, and nevuscells and an occasional melanophage were present. In Stages I to DISCUSSION III, mononuclear cells were seen adjacent to some nevus cells (oil immersion), as well as clustered about and within The nature of the inflammatory response and the tissue nevuscell nests. necrosis in halo nevus have been examined by many Stage IV. The remaining halo was histologically identical investigators (7, 8, 10, 13, 25, 29). The mononuclear to vitiligo. infiltrate and the mechanism of depigmentation have been discussed but not resolved. Fine structural studies (I , 4, 27) have focused on the depigmented epidermis, the absence of

Electron Microscopy 5 melanocytes and melanin, and the replacement of melano cytes by Langerhans cells. Abnormal nevus cells with Stage I. The inflammatory cell response was made up of vacuolatedmitochondria werereported by Epsteinet a!. (5), both lymphocytes and monocytes. Both types of cells were but these authors found little or no inflammatory response found between the widened endothelial cell junctions of the about the examined halo nevi. Stegmaier et a!. (25) noted reticular and papillary dermal vessels. These cells were inflammatory cells surrounding nevus cells and suggested present in the dermis adjacent to nevuscell nests.Occasion that vacuolatedcells lacking melanin in the dermis might be ally, plasma cells were found in these extravascular loca degenerated nevus cells. tions (Fig. 1). When present they were found sometimes in It is well known that malignant melanoma can arise in contact with nevus cells. At plasma cell-nevus cell junctions, junctional or compound nevi (24). The recent evidence of the cell membranes of both cells were thickened and Lewis and Copeman (I 8) and Copeman et a!. (3) suggests electron-dense material was found between the 2 opposing that the halo nevusmay representthe host rejection of an membranes (Fig. I). early malignant melanoma. Lymphocytes and monocytes were also within the nevus Evident from the present study is the structural delinea cell nests,surrounding the nevuscells, and separatingthem. tion of a progressive involution of a melanocytic nevus When lymphocytes and nevus cells were in contact (Fig. 2), (including melanocyte and nevus cell death) and the subse the involved cells had thickened cell membranes, and small quent evolution of the depigmented halo. ruptures were present along the points of contact. I@lectron From the morphology of the tissue obtained from halo dense flocculent material was found between the 2 mem nevi in different stages of regression, a scheme of events can branes (Fig. 3). Vacuoles were not present in the nevus cells be suggested that seems to correlate with available pub in either Stage I or the control nevi. lished immunological data. The earliest change in the lesion Stage II. Small cytoplasmic vacuoleswere found in some is a migration of lymphocytes and monocytes from local isolated nevus cells (Fig. 4). These vacuoles appeared to be vessels into the tumor mass. Nevus cells that are tightly derived from the endoplasmic reticulum (Fig. 4) and were nested in both the dermis and epidermis are separated and filled with medium electron-dense granular material. Other surrounded by the inflammatory cells. Attachment of nevus cells contained large, electron-lucid vacuoles. These inflammatory cells to nevus cells causes focal nevus cell cells had small amounts of melanin (Fig. 5) in their membrane destruction. cytoplasm. At this point a diffusible cytotoxic substance(lympho Stage III. Nevus cell destruction was extensive. Nuclei toxin) may be released by the lymphocytes, causing necrosis were somewhat separated from cytoplasm which contained of basal melanocytes and nevus cells through osmoregula elongated vacuoles (Fig. 6), and macrophages contained tory disruption (cytoplasmic vacuolarization). It has been phagocytized portions of nevus cells. The phagocytized shown by Russellet al. (23) that lymphotoxin, whenapplied nevus cell cytoplasm contained electron-lucid vacuoles in as a purified compound to cultured target cells, does

FEBRUARY 1975 353

produce a vacuolar degeneration. This type of cytolysis is REFERENCES identical to that observed between target cells and sensitized . lymphocytes (22). Stegmaier et a!. (25) have suggested the 1. Berger, R. S., and Voorhees, J. J. Multiple Congenital Giant presence of a diffusible cytotoxic substance in developing Nevocullar Nevi with Halos. Arch. Dermatol., 104. 515-521, 1971. halo nevi, becausethe halo developswhile melanocytesare 2. Burdick, K. H., and Hdwk, W. A. Vitiligo in a Case of Vaccinia 3-(3,4-dihydroxyphenyl)-L-alanine-positive and only a few Virus-Treated Melanoma. Cancer, 17: 708-712. 1964. lymphocytes have invaded the epidermis. 3. Copeman, P. W. M., Lewis, M. G., and Elliott, P. G. Immunological Langhof et al. (16) reported the presenceof an anti Associations of the Halo Naevus with Cutaneous Malignant Mela melanin antibody in patients with vitiligo. A critical review noma. Brit. J. Dermatol., 88: 127-137, 1973. of this work by Wasserman and Van Der Walt (28) 4. Ebner, V. H., and Niebauer, G. Elektronenoptische Befunde zum concludesthat melanin is not antigenic and that the melanin Pigmentuerlust bein Naevus Sutton. Dermatologica, 137: 345@347, 1968. preparation of Langhof's group contained melanocyte cellu 5. Epstein, W. L., Sagebeil, R., Spitler, L., Wybran, J., Reed, W. B., and lar debris. We also believe that melanin is not antigenic Blois, M. S. Halo Nevi and Melanoma. J. Am. Med. Assoc., 225: becausethecytoplasmic vacuolesin nevuscells and melano 373-377, 1973. cytes appear to originate from the endoplasmic reticulum, 6. Everson,T. C., and Cole, W. H. Spontaneous Regressionof Cancer. not premelanosomes or melanosomes. This belief is further Philadelphia: W. B. Saunders Co., 1966. strengthened becausekeratinocytes in Stage III halo nevi 7. Feldman, S., and Lashinsky. I. M. Halo nevus-Leukoderma Cen undergo cytolysis only around the membrane-boundcom trifugum Acquisitium (Sutton): Leukopigmentary Nevus. Arch. Der plex melanin granules which contain small bits of phagocy matol. Syphilis, 34: 590-604, 1963. tized melanocyte cytoplasm (9, 20). The vacuoles in keratin 8. Findlay, G. H. The Histology of Sutton's Naevus. Brit. J. Dermatol., ocytes from Stage III halo nevi disappearby Stage IV, and 69: 389-394, 1957. S 9. Fitzpatrick, T. B., and Breathnach, A. S. Das epidermale Melanin dead and dying keratinocytes are not found in the epider Einheit-System. Dermatol. Wochschr., 147: 481 -489. 1963. mis. Keratinocytes also are not sloughed, because hyperker 10. Frank, S. B., and Cohen, J. H. The Halo Nevus. Arch. Dermatol., 89: atosis and scaling are absent in the depigmented halo and 367-373,1964. the epidermis overlying the regressing nevus. 11. Jhung, J. W., Edelstein, L. M., Church, A., Snyder, L. M., Bain, R. Dr. William Terry and hiscolleagues(personal communi W., Castro, S., and Cariglia, N. Multiple Halo Nevi Developing after cation) have demonstrated that cultured lymphocytes from Oxymetholone Treatment of â€œAplasticAnemia.â€•Cutis, 12: 56-61, halo nevuspatients quickly attach to severaldifferent lines 1973. of cultured human melanoma cells. The tumor cells swell, 12. Klein, E., and Holtermann, 0. A. Immunotherapeutic Approaches to becomevacuolated,and die. If sheeperythrocytesare added the Management of Neoplasms. NatI. Cancer Inst. Monograph, 35: to the culture mixture and the supernatant is removed, the 379-402, 1972. 13. Kopf, A. W., Morrill, S. D., and Silberberg, I. Broad Spectrum of lymphocytes attach to the sheep erythrocytes and few Leukoderma Acquisitum Centrifugum. Arch. Dermatol., 92: 14-35, lymphocytes remain attached to melanoma cells. This 1965. evidence points to a possible T-cell-mediated response, in 14. Lancet. Leading Article. Aetiology of Vitiligo. Lancet, 2: 1298- 1299, addition to the humoral response (3, 18). Melanoma 1971. patients with regressing halo nevi have intact or increased 15. Lancet. Leading Article. The Halo Nevus and Malignant Melanoma. delayed hypersensivity reactions (5). A strongly positive Lancet, 2: 982, 1973. delayed hypersensitivity reaction has been noted in one of 16. Langhof, H., Fenerstein, M.. and Schabinski, F. Melanin Antibody our halo nevus patients (1 1). Induced delayed hypersensitiv Formation in Vitiligo. Hautarzt, 16: 209-212, 1965. ity reactions to synthetic haptenesand microbial antigens 17. Lerner, A. B. On the Etiology ofVitiligo and Gray Hair. Am. J. Med., have been shown to result in regression of a number of 51:141-147,1971. 18. Lewis, M. G., and Copeman, P. W. M. Halo Naevus: A Frustrated human skin tumors when reactions are induced at tumor Malignant Melanoma? Brit. Med. J., 2: 47-48, 1972. sites (12). Klein and his associates have observed that 19. Luft, J. I-I. Improvements in Epoxy Resin Embedding Methods. J. cutaneous metastases of melanoma convert to â€œbenignhalo Biophys. Biochem. Cytol., 9: 409-414, 1961. neviâ€•with dense mononuclear infiltrates following nonspe 20. Mottaz, J. H., and Zelickson, A. S. Melanin Transfer: A Possible cific immunotherapy with dinitrochlorobenzene. Biopsies Phagocytic Process. J. Invest. Dermatol., 49: 605-610, 1967. from theselesionsprior to the immune challengeshowedan 21. Nicholls, E. M. Development and Elimination of Pigmented Moles absence of cellular infiltrate. and the Anatomical Distribution of Primary Malignant Melanoma. Although the data to date are insufficient to answer the Cancer,32: 191-195,1973. question pertaining to the possibility that halo nevus 22. Russell, S. W. Purification and Some Properties of Human Lympho represents an early host-rejected melanoma, further investi toxin. Ph.D. Dissertation, University of California, Davis, 1972. 23. Russell, S. W., Rosenau, W., and Lee, V. C. Cytolysis Induced by gation of this lesion should be of value as a human model for Human Lymphotoxin. Cinemicrographic and Electron Microscopic studying the mechanism of host destruction of a neoplastic Observations. Am. J. Pathol., 69: 103-1 17, 1972. population of melanocytes. 24. Sanderson, K. V. Melanocytic Naevi. in: A. J. Rook, D. S. Wilkinson, and F. J. G. Ebling (eds.), Textbook of Dermatology, pp. 101- I I I. Oxford: BlackwellScientificPublications, 1968. ACKNOWLEDGMENTS 25. Stegmaier, 0. D., Becker, S. W., and Medenica, M. Multiple Halo Nevi. Histopathological Findings in a 14-year-old Boy. Arch. Der The technicalassistanceofJean Underwoodisgratefullyacknowledged. matol., 99: 180-189, 1969.

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26. Sumner, W. C., and Foraker, A. G. Spontaneous Regression of 28. Wassermann, H. P., and Van Der Walt, J. J. Antibodies against Human Melanoma. Clinical and Experimental Studies. Cancer, /3: Melanin. The Significance ofNegative Results. S. African Med. J., 47: 79-81, 1960. 7-9, 1973. 27. Swanson, L. L., Wayte, D. M., and Helwig, E. B. Ultrastructure of 29. Wayte, D. M., and Helwig, E. B. Halo Nevi. Cancer, 22: 69-90, 1968. Halo Nevi. J. Invest. Dermatol., 50: 434-450, 1968.

Figs. I to I I. Stain used: uranylacetate +lead citrate. Fig. I . Extravascular plasma cell (P) attached to nevus cell (N). Note electron-dense material between cells (arrows). x 14,000. Fig. 2. Lymphocyte (L) attached to nevus cells (N). Note the increase of electron density of both cells along contacting cell membranes (arrow). x 10,000. Fig. 3. High-power view of Fig. 2, showing increased electron density of cytoplasm of both cells and electron-dense material between contacting cell membranes (arrows). x 18,000. Fig. 4. A single nevus cell undergoing early vacuolar changes. The vacuoles are filled with medium electron-dense material (arrows). x 5,000. Fig. 5. High-power view of a nevus cell showing early vacuolar cytolysis from endoplasmic reticulum (Arrow 1) and later vacuole change showing fission of vacuoles (Arrow 2). x 15,000. Fig. 6. A late stage of nevus cell degeneration. The cytoplasmic vaculoes have coalesced to form channels in the cytoplasm separating cellular components (arrows). A lymphocyte (L) is attached to the degenerating nevus cell. x 7,600. Fig. 7. A macrophage with 2 phagocytized bits ofnevus cells (Arrow 1). Each phagocytized portion ofnevus cell contains a single melanosome (Arrow 2). x 8,000. Fig. 8. A lymphocyte (L) within the epidermis is adjacent to a necrotic keratinocyte and the epidermal basement membrane (BM). x 12,00. Fig. 9. A condensed melanocyte (M) undergoing vacuolar cytolysis ( V). Note the vacuoles in and about the complex melanin granules of the keratinocytes (arrow). x 8,000. Fig. 10. In keratinocytes, vacuoles are present in or about the complex melanin granules (arrows). x 7,400. Fig. I I . Keratinocytes in the well-developed halo are devoid of melanin. Note the desmosome (arrow). x 7,000.

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Cancer Res 1975;35:352-357.

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