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

SUMMARY

Nine halo nevi in various stages of regression wereexamined by electron microscopy for fine structural evidence of an immunological mechanism of tumor celldestruction and halo formation. Early regressing lesions(Stage I) showed nevus cells associated with infiltratinglymphocytes, monocytes, and plasma cells, but withoutnevus cell destruction. In later lesions (Stages II and III),vacuolar cytolysis was commonly observed in nevus cells. InStage I II lesions, portions of nevuscells are found withinmacrophages.The electron microscopic findings of lymphocyte, monocyte, and plasma cell infiltration of the tumorfollowed by vacuolar cytolysis support the concept of animmune reaction in regressing halo nevi.

INTRODUCTION

Halo nevus is a spontaneously regressing mole whichdevelops an enlarging area of depigmentation about thedisappearing lesion. The presence of an accompanyinginflammatory response in the lesion is taken by someinvestigators as morphological evidence for an immunological mechanism of tumor cell death and macroscopic haloformation (I 7). Recently, circulating antibodies against thecytoplasm of melanoma cells have been demonstrated inpatients with regressing halo nevi (3, 18). The authors ofthese papers suggest that halo nevus represents the successful early rejection of melanoma. A cell-mediated response inmelanoma patients with regressing halo nevi has also beenreported (5).

The mechanism of halo formation is not known, but itmay be secondary to the inflammatory response ( I5) or to acytotoxic antibody against melanocytes (14). Halos, inaddition to being present around benign moles (21), may befound about spontaneously regressing melanoma (6), aboutmoles and metastases in patients treated surgically formelanoma (26), or about primary and secondary melanomain patients who respond to either nonspecific immunestimulation (2) or adoptive immunization (26). The work ofLewis and Copeman (18) and of Copeman et a!. (3) hasprovided information on which to base a hypothesis for animmune mechanism of tumor cell destruction in halo nevussyndrome. We have examined 9 halo nevi for fine structuralevidence of such an immunological mechanism.

Received March 18, 1974;accepted October 7, 1974.

MATERIALS AND METHODS

Seventy-five halo nevi from 6 patients were examinedclinically (including Wood's light scan). Nine of the 75 halonevi in different stages of regression were removed from 4patients, and a compound nevus was excised frvm each of 2patients, neither of whom revealed evidence of past orpresent halo nevi or malignant melanoma.

Small portions of epidermis and dermis including thenevus, surrounding halo, and a narrow rim of normal skinwere obtained by punch biopsy from midback, scapular,thigh, or deltoid regions. Biopsy was done under localanesthesia by a peripheral nerve-block technique. Excisedspecimens were immediately immersed in 2% glutaraldehyde in 0. 1 M phosphate buffer. Fixed specimens were cut inhalf, and one half was placed in formalin and processedforroutine histology. The other half was dissected into 3portions: normal skin, halo, and mole. Extraneous fattytissue was removed and the specimens were minced intoI-cu mm blocks. Specimens were rinsed several times in 0.1M phosphate buffer followed by postfixation in I % osmiumtetroxide in 0. 1 M phosphate buffer. The fixed specimenswere dehydrated in a graded series of alcohols and embedded in Luft's 1:1 epon mixture (19). Thick and thinsections were cut on a Sorvall MT2 ultramicrotome. Thinsections were poststained with uranyl acetate and leadcitrate and examined in either a Philips 300 electron microscope1ora Zeiss EM 9A electron microscope.

RESULTS

Clinical Examination

Each of the 75 halo nevi was classified in I of thefollowing 4 main stages of clinically apparent regression.

In Stage I were inflammed nevi that showed erythemaand edema. In Stage II were erythematous and slightlyraised nevi with faint halo formation. The halo was morepronounced under 3600-A light (Wood's lamp). In StageIII were flat and less pigmented nevi without erythema butwith halo formation evident under natural light. In Stage IVwere oval white depigmented areas of epidermis withoutclinical evidence of nevi.

Light Microscopy

Stage I. Nevus melanization appeared intact in theepidermis. The papillary dermis was edematous with some

352 CANCER RESEARCH VOL. 35

Ultrastructural Evidence for Destruction in the Halo Nevus

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, Universityof Massachusetts Medical School, Worcester, Massachusetts 01610

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Ce!! Destruction in Halo Nevus

vascular dilation, but nevus cell nests were intact. In thereticular dermis there was an early infiltration of mononuclear cells at the margins of the tumor.

Stage II. Patchy demelanization was evident in the epidermal basal layer overlying the tumor with more markedloss of melanin in the epidermal basal layer peripheral to thetumor. There was minimal infiltration of the junctionalnests of nevus cells by mononuclear cells in the papillarydermis. The reticular dermis showed marked infiltration ofthe nevus cell nests by these mononuclear cells associatedwith degeneration of the nevus cells. Occasional nevus cellshad pyknotic nuclei and a finely vacuolated cytoplasm.

Stage III. Complete demelanization of the peripheralepidermal layer was present; it was present to a lesser extentin the epidermal basal layer overlying the tumor. Fewernevus cells, mononuclear cells, and melanophages werepresent in the papillary dermis. Similarly, in the reticulardermis fewer histiocytes, mononuclear cells, and nevuscellsand an occasional melanophage were present. In Stages I toIII, mononuclear cells were seen adjacent to some nevuscells (oil immersion), as well as clustered about and withinnevuscell nests.

Stage IV. The remaining halo was histologically identicalto vitiligo.

Electron Microscopy

Stage I. The inflammatory cell response was made up ofboth lymphocytes and monocytes. Both types of cells werefound between the widened endothelial cell junctions of thereticular and papillary dermal vessels. These cells werepresent in the dermis adjacent to nevuscell nests.Occasionally, plasma cells were found in these extravascular locations (Fig. 1). When present they were found sometimes incontact with nevus cells. At plasma cell-nevus cell junctions,the cell membranes of both cells were thickened andelectron-dense material was found between the 2 opposingmembranes (Fig. I).

Lymphocytes and monocytes were also within the nevuscell nests,surrounding the nevuscells, and separatingthem.When lymphocytes and nevus cells were in contact (Fig. 2),the involved cells had thickened cell membranes, and smallruptures were present along the points of contact. I@lectrondense flocculent material was found between the 2 membranes (Fig. 3). Vacuoles were not present in the nevus cellsin either Stage I or the control nevi.

Stage II. Small cytoplasmic vacuoleswere found in someisolated nevus cells (Fig. 4). These vacuoles appeared to bederived from the endoplasmic reticulum (Fig. 4) and werefilled with medium electron-dense granular material. Othernevus cells contained large, electron-lucid vacuoles. Thesecells had small amounts of melanin (Fig. 5) in theircytoplasm.

Stage III. Nevus cell destruction was extensive. Nucleiwere somewhat separated from cytoplasm which containedelongated vacuoles (Fig. 6), and macrophages containedphagocytized portions of nevus cells. The phagocytizednevus cell cytoplasm contained electron-lucid vacuoles in

addition to degenerated organelles (Fig. 7). In Stage II andIII halo nevi, lymphocytes were found in the epidermis (Fig.8). Nevus cells in junctional nests also contained vacuolesand appeared necrotic. Dendritic basal melanocytes wererounded with vacuolar change of the cytoplasm (Fig. 9).Langerhans cells were not affected by the degenerationprocess. Keratinocytes exhibited the same vacuolar changeas nevus cells and melanocytes, but only around themembrane-bound complex melanin granules (Fig. 10).Keratinocytes with large vacuoles had a complete absenceof melanin.

Stage IV. In the well-developed halo, keratinocytes werevirtually free of vacuoles and there was a total absence ofmelanin (Fig. 11). In the dermis ofthe halo area there weredisrupted mast cells, lymphocytes, pigment laden macrophages, and occasional nevus cells.

DISCUSSION

The nature of the inflammatory response and the tissuenecrosis in halo nevus have been examined by manyinvestigators (7, 8, 10, 13, 25, 29). The mononuclearinfiltrate and the mechanism of depigmentation have beendiscussed but not resolved. Fine structural studies (I , 4, 27)have focused on the depigmented epidermis, the absence of

5 melanocytes and melanin, and the replacement of melano

cytes by Langerhans cells. Abnormal nevus cells withvacuolatedmitochondria werereported by Epsteinet a!. (5),but these authors found little or no inflammatory responseabout the examined halo nevi. Stegmaier et a!. (25) notedinflammatory cells surrounding nevus cells and suggestedthat vacuolatedcells lacking melanin in the dermis might bedegenerated nevus cells.

It is well known that malignant melanoma can arise injunctional or compound nevi (24). The recent evidence ofLewis and Copeman (I 8) and Copeman et a!. (3) suggeststhat the halo nevusmay representthe host rejection of anearly malignant melanoma.

Evident from the present study is the structural delineation of a progressive involution of a melanocytic nevus(including melanocyte and nevus cell death) and the subsequent evolution of the depigmented halo.

From the morphology of the tissue obtained from halonevi in different stages of regression, a scheme of events canbe suggested that seems to correlate with available published immunological data. The earliest change in the lesionis a migration of lymphocytes and monocytes from localvessels into the tumor mass. Nevus cells that are tightlynested in both the dermis and epidermis are separated andsurrounded by the inflammatory cells. Attachment ofinflammatory cells to nevus cells causes focal nevus cellmembrane destruction.

At this point a diffusible cytotoxic substance(lymphotoxin) may be released by the lymphocytes, causing necrosisof basal melanocytes and nevus cells through osmoregulatory disruption (cytoplasmic vacuolarization). It has beenshownby Russellet al. (23) that lymphotoxin, whenappliedas a purified compound to cultured target cells, does

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J. B. Jacobs et al.

produce a vacuolar degeneration. This type of cytolysis isidentical to that observed between target cells and sensitized

. lymphocytes (22). Stegmaier et a!. (25) have suggested the

presence of a diffusible cytotoxic substance in developinghalo nevi, becausethe halo developswhile melanocytesare3-(3,4-dihydroxyphenyl)-L-alanine-positive and only a fewlymphocytes have invaded the epidermis.

Langhof et al. (16) reported the presenceof an antimelanin antibody in patients with vitiligo. A critical reviewof this work by Wasserman and Van Der Walt (28)concludesthat melanin is not antigenic and that the melaninpreparation of Langhof's group contained melanocyte cellular debris. We also believe that melanin is not antigenicbecausethe cytoplasmic vacuolesin nevuscells and melanocytes appear to originate from the endoplasmic reticulum,not premelanosomes or melanosomes. This belief is furtherstrengthened becausekeratinocytes in Stage III halo neviundergo cytolysis only around the membrane-boundcomplex melanin granules which contain small bits of phagocytized melanocyte cytoplasm (9, 20). The vacuoles in keratinocytes from Stage III halo nevi disappearby Stage IV, anddead and dying keratinocytes are not found in the epidermis. Keratinocytes also are not sloughed, because hyperkeratosis and scaling are absent in the depigmented halo andthe epidermis overlying the regressing nevus.

Dr. William Terry and hiscolleagues(personal communication) have demonstrated that cultured lymphocytes fromhalo nevuspatients quickly attach to severaldifferent linesof cultured human melanoma cells. The tumor cells swell,becomevacuolated,and die. If sheeperythrocytes are addedto the culture mixture and the supernatant is removed, thelymphocytes attach to the sheep erythrocytes and fewlymphocytes remain attached to melanoma cells. Thisevidence points to a possible T-cell-mediated response, inaddition to the humoral response (3, 18). Melanomapatients with regressing halo nevi have intact or increaseddelayed hypersensivity reactions (5). A strongly positivedelayed hypersensitivity reaction has been noted in one ofour halo nevus patients (1 1). Induced delayed hypersensitivity reactions to synthetic haptenesand microbial antigenshave been shown to result in regression of a number ofhuman skin tumors when reactions are induced at tumorsites (1 2). Klein and his associates have observed thatcutaneous metastases of melanoma convert to “benignhalonevi―with dense mononuclear infiltrates following nonspecific immunotherapy with dinitrochlorobenzene. Biopsiesfrom theselesionsprior to the immune challengeshowedanabsence of cellular infiltrate.

Although the data to date are insufficient to answer thequestion pertaining to the possibility that halo nevusrepresents an early host-rejected melanoma, further investigation of this lesion should be of value as a human model forstudying the mechanism of host destruction of a neoplasticpopulation of melanocytes.

ACKNOWLEDGMENTS

The technicalassistanceofJean Underwoodisgratefullyacknowledged.

REFERENCES

1. Berger, R. S., and Voorhees, J. J. Multiple Congenital GiantNevocullar Nevi with Halos. Arch. Dermatol., 104. 515-521, 1971.

2. Burdick, K. H., and Hdwk, W. A. Vitiligo in a Case of VacciniaVirus-Treated Melanoma. Cancer, 17: 708-712. 1964.

3. Copeman, P. W. M., Lewis, M. G., and Elliott, P. G. ImmunologicalAssociations of the Halo Naevus with Cutaneous Malignant Melanoma. Brit. J. Dermatol., 88: 127-137, 1973.

4. Ebner, V. H., and Niebauer, G. Elektronenoptische Befunde zumPigmentuerlust bein Naevus Sutton. Dermatologica, 137: 345@347,1968.

5. Epstein, W. L., Sagebeil, R., Spitler, L., Wybran, J., Reed, W. B., andBlois, M. S. Halo Nevi and Melanoma. J. Am. Med. Assoc., 225:373-377, 1973.

6. Everson,T. C., and Cole, W. H. Spontaneous Regressionof Cancer.Philadelphia: W. B. Saunders Co., 1966.

7. Feldman, S., and Lashinsky. I. M. Halo nevus-Leukoderma Centrifugum Acquisitium (Sutton): Leukopigmentary Nevus. Arch. Dermatol. Syphilis, 34: 590-604, 1963.

8. Findlay, G. H. The Histology of Sutton's Naevus. Brit. J. Dermatol.,69: 389-394, 1957. S

9. Fitzpatrick, T. B., and Breathnach, A. S. Das epidermale MelaninEinheit-System. Dermatol. Wochschr., 147: 481 -489. 1963.

10. Frank, S. B., and Cohen, J. H. The Halo Nevus. Arch. Dermatol., 89:367-373,1964.

11. Jhung, J. W., Edelstein, L. M., Church, A., Snyder, L. M., Bain, R.W., Castro, S., and Cariglia, N. Multiple Halo Nevi Developing afterOxymetholone Treatment of “AplasticAnemia.―Cutis, 12: 56-61,1973.

12. Klein, E., and Holtermann, 0. A. Immunotherapeutic Approaches tothe Management of Neoplasms. NatI. Cancer Inst. Monograph, 35:379-402, 1972.

13. Kopf, A. W., Morrill, S. D., and Silberberg, I. Broad Spectrum ofLeukoderma Acquisitum Centrifugum. Arch. Dermatol., 92: 14-35,1965.

14. Lancet. Leading Article. Aetiology of Vitiligo. Lancet, 2: 1298- 1299,1971.

15. Lancet. Leading Article. The Halo Nevus and Malignant Melanoma.Lancet, 2: 982, 1973.

16. Langhof, H., Fenerstein, M.. and Schabinski, F. Melanin AntibodyFormation in Vitiligo. Hautarzt, 16: 209-212, 1965.

17. Lerner, A. B. On the Etiology ofVitiligo and Gray Hair. Am. J. Med.,51:141-147,1971.

18. Lewis, M. G., and Copeman, P. W. M. Halo Naevus: A FrustratedMalignant Melanoma? Brit. Med. J., 2: 47-48, 1972.

19. Luft, J. I-I. Improvements in Epoxy Resin Embedding Methods. J.Biophys. Biochem. Cytol., 9: 409-414, 1961.

20. Mottaz, J. H., and Zelickson, A. S. Melanin Transfer: A PossiblePhagocytic Process. J. Invest. Dermatol., 49: 605-610, 1967.

21. Nicholls, E. M. Development and Elimination of Pigmented Molesand the Anatomical Distribution of Primary Malignant Melanoma.Cancer,32:191-195,1973.

22. Russell, S. W. Purification and Some Properties of Human Lymphotoxin. Ph.D. Dissertation, University of California, Davis, 1972.

23. Russell, S. W., Rosenau, W., and Lee, V. C. Cytolysis Induced byHuman Lymphotoxin. Cinemicrographic and Electron MicroscopicObservations. Am. J. Pathol., 69: 103-1 17, 1972.

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.

25. Stegmaier, 0. D., Becker, S. W., and Medenica, M. Multiple HaloNevi. Histopathological Findings in a 14-year-old Boy. Arch. Dermatol., 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 againstHuman 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). x10,000.

Fig. 3. High-power view of Fig. 2, showing increased electron density of cytoplasm of both cells and electron-dense material between contacting cellmembranes (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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1975;35:352-357. Cancer Res   J. B. Jacobs, L. M. Edelstein, L. M. Snyder, et al.   Ultrastructural Evidence for Destruction in the Halo Nevus

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