Download - Melanoacanthoma. Ultrastructural and Immunological Studies

Journal of Cutatteous Pathology 1978: 5: 127-141

MelanoacanthomaUltrastructural and Immunological Studies

O. L. A. SCHLAPPNER,' G. ROWDEN,' T. M. PHILLIPS' AND Z. RAHIM'

'Department of Dermatology, The Montreal General Hospital and McGill University, Montreal,Canada and^ Department of Pathology, Georgetown University Medical School, Washington, D.C., U.S.A.

Electronmieroscopic studies confirmed that melanoacanthoma is a non-nevoid elevated epithelialtumor composed of keratinocytes of botli basaloid and spinous differentiation and of large den-tritic melanocytes. The block in transfer of pigment from melanocytes to keratinocytes was foundnot to be complete. Langerhans cells, present in all the malpighian layers were normal in mor-phology. Immunofluorescent studies and an immunoprecipitin assay also showed our patient'smelanoacanthoma not to be related to malignant melanoma.

(Received for publieation February 13, 1978)

Melanoacanthoma is a rare pigmented be-nign mixed epithelial tumor of both melan-ocytes and keratinocytes. It may resemblesuch pigmented tumors as seborrheic kera-tosis, melanoma, nevus, benign or malignantlentigo and pigmented basal cell carcinoma.First described by Bloch in 1926 as a Melano-epithelioma, Mishima & Pinkus in 1960 gaveit clarity by introducing the term melano-acanthoma. The 17 cases reported in theliterature to date attest to the rarity of thislesion which may test the acuity of bothclinician and pathologist (Traub 1950, Spottet aL 1972, Reaves 1974, Delacr^taz, 1975).

Our study of a melanoacanthoma on theback of a 74-year-old white male patientprimarily concerned itself with the hithertoundescribed electronmicroscopy of thisunusual tumor as well as immunologicialassays for melanoma antigens.

Material and Methods

Case ReportA white male patient, still working at the ageof 74, on hospital admission for pneumoniawas incidentally noted to have a "mole" onhis back. The lesion had been present for 8years and had been asymptomatic except forits slow growth.

Examination revealed a 3 x 3 cm brown-ish plaque in the L2-3 area of the left sideof the back. The border of the lesion wasvariously irregular and arcuate. The colorvaried from black to dark brown to lightbrown. Some punctate black papular stip-pling was present at the superior aspect ofthe lesion (Fig. 1). Inguinal lymphadeno-pathy was absent.

Initially the clinical diagnosis was super-ficial spreading melanoma but the pathologyreport on a 3 mm punch biopsy was pig-mented seborrheic keratosis. Further 3 mmpunch biopsies as well as a review of theoriginal biopsy confirmed the impression ofmelanoacanthoma. This diagnosis wasfurther corroborated on sectioning of thewhole lesion which was subsequently excisedwith a 2 mm margin of normal skin.

The excised tumor was divided so thatadequate sampling was achieved. A cuff ofapparently normal skin was included forcontrol studies.

Several specimens of seborrheic keratosiswere similarly processed as controls.

Histology:Specimens were processed for conventionalparaffin histology, frozen sectioning, dopastaining and electron microscopy.

128 SCHLAPPNER ET AL.

Fig. 1. Gross appearance of specimen. Arrowhead indicates site of initial punch biopsy.

Five micron paraffin sections werestained with H & E , and by Masson'sammoniated silver nitrate method.

Cytoehemistry:1) A modified dopa reaction (Rodriguez &McGavran 1969) was carried out as follows:

a) Frozen Sections.Fresh tissue was sectioned at lO/nm, fixed

for 1 h at 4° C in 4% formaldehyde (bufferedat pH7.4 with O.IM cacodylate buffer 10min incubated in dopa medium for 5 h at37° C with changes of medium, washed indistilled water, counterstained with nuclearfast red, rinsed, dehydrated, cleared andmounted.

b) Paraffin Embedding.Tissue blocks 1 X 1 X 0.2 cm were fixed

for 3 h at 4° C as above, rinsed in buffer for10 min, incubated at 37° C for 16h in dopamedium with changes every 3h, washed inbuffer for 30 min, postfixed in neutral form-alin for 4h, dehydrated, cleared andembedded in paraffin. Five micron sectionswere then stained with nuclear fast red. The

dopa medium was made fresh from stocksolutions mixed in equal parts of 0.2M so-dium cacodylate buffer at pH7.4 and 0.2%aqueous solution of Dl-3, 4-dehydroxy-phenylalanine (dopa).2) Combined acid phosphatase-dopa reaction.Barka & Anderson's (1962) Naphthol AS-Blphosphate, hexazonium pararosanalinmethod for acid phosphatase was carried outon frozen sections and followed by the dopamethod as described by Mishima (1966).

Electron Microscopy:One mm^ blocks were fixed for 1 h at roomtemperature in half strength Karnovsky fixa-tive, osmicated, stained en bloc with aqueousuranyl acetate, dehydrated and embedded inSpurr resin. One micron sections cut with adiamond knife on a Reichert 0MU3 micro-tome were stained with alkaline toluidineblue. Thin sections were stained with leadcitrate and examined at 60 KV on a PhilipsEM301 electronmicroscope.

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* m

F f̂. 2. Histological appearance showing basaloid and spinous cell differentiation, together with melano-cytes present at all levels (arrowheads). H & E X 560.

Immunofluorescen ce:Tests were performed to determine whethermelanoma related antigens were present in thelesion (autologous test). Also, the patient'sserum was tested against melanoma eyto-plasmic antigen smears (allogeneic) todemonstrate any circulating anti-melanomaantibodies (anti-cytoplasmic).

The immunofluorescence was carried outby the indirect technique of Phillips & Lewis(1970).

The slides were examined under a LeitzOrthoplan microscope equipped with anincident light illuminator, an HBO 200 mer-cury lamp and 490 nm interference filter.


Fig. 3. Histological section. Papillomatous pattern with spinous eell and basaloid differentiation. Melano-cytes at all levels (arrowheads). Endokeratinization present. H & E X 560.

The secondary filtration was achieved with Resultsa 525-530 nm 2mm glass filter.

Both the punch biopsies and the excisionalImmunoprecipitin Tests: specimen showed a similar histology.The patient's serum was also assayed for anti- The irregular epidermis showed hyper-melanoma antibodies by a micro-immuno- keratosis, papOlomatosis, acanthosis andprecipitin test. pseudo-horn cyst formation. At higher

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Fig. 4. Higher magnification to show melanocytes in suprabasal location (arrowheads). H & E X 2240.

magnification, the keratinocytes were ofeither a small basaloid or spinous type. Thespinous cells were arranged in some areas innests (endokeratinization) and pseudo-horncysts were present. Scattered throughout theepidermis were large, richly dendritic melano-cytes (Figs. 2, 3 & 4). No nevus cells werepresent. The dermis was normal except for

some areas of mononuclear infiltrate in thepapillary regions. Some melanin was presentin dermal macrophages.

The dopa staining on frozen sections andon the paraffin sections confirmed the pres-ence of strongly positive melanocytes at alllevels of the epidermis (Fig. 5). All standardcontrols (Rodriquez & McGavran 1969) for


Fig. 5. Dopa reaction. Heavily stained melanoeytes in the hyperplastic epthelium. X 560.

the dopa stain were negative. Similarly, thesilver stain showed strong activity present inmelanoeytes (Fig. 6).

Control seborrheic keratosis specimensstained for either dopa or by the Massonmethod showed confinement of staining tothe basal layer (Fig. 7).

The combined acid phosphatase-dopamethod confirmed the presence of phago-

eytic cells in the dermis, i.e. melanophages.Little or no acid hydrolase acitivity wasevident in the epidermal melanoeytes.

Electron Microscopy:The presence of melanoeytes at all levels ofthe hyperplastic epidermis was confirmed.The melanoeytes showed large perikaryia

MELANOACANTHOMA

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133

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Fig. 6. Masson's ammoniated silver nitrate method. Heavily stained eells in the basal (arrowheads) andmore superfieial layers (arrow). X 560.

and extensively aborized dendritic processes(Fig. 8). Cells in mitosis were quite common(Fig. 9).

Melanosomes of all stages were present inthe melanoeytes and were especially rich inthe dendrites (Fig. 10). Melanin granuleswere present within the cytoplasm of ker-tinocytes, but these were sparse and in themain were found in areas of basal cell dif̂

ferentiation (Figs. 8, 9, 10). In comparison,the melanoeyte distribution and dispositionof pigment in seborrheic keratosis werestrikingly different. The melanoeytes weresmall and melanin granules were present inthe cytoplasm of the proliferating basaloidkeratinocytes in large numbers both singlyand in complexed forms (Fig. 11).

The Langerhans cells were present


Fig. 7. Seborrheic keratosis. Masson's ammoniated silver nitrate method. Staining confined to the basaland immediate suprabasal layers. X 140.

throughout the epidermis of the melano-acanthoma except in the basal layer andthey did not appear to be significantly dif-ferent in structure from normal epidermalLangerhans cells (Fig, 12).

Immunofluorescence and ImmunoprecipitinAssays:The patient's serum gave negative immuno-

fluoreseent staining against both the auto-logous lesion and the allogeneic tumor cells.

The micro-immunoprecipitin assay wassimilarly negative.

Discussion

Morphologically, melanoacanthoma is a pig-mented tumor with such variable character-

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Fig. 8. Electron micrograph. Large melanoeyte in the suprabasal layers (star). Melanin granules in theperikaryon and dendritic processes (arrows). Sparse melanin granules in surrounding keratinocytes (arrow-heads).

istics that diagnoses of such pigmented clinically not only had features of super-lesions as seborrheic keratosis, malignant ficial spreading melanoma, but also ofmelanoma, nevus, benign and malignant seborrheic keratosis and of lentigo malignalentigo or basal cell carcinoma are usually (Fig, 1), fii previous reports, melanoacan-entertained. In our patient the lesion thoma occurred on the head, neck, trunk

136 SCHLAPPNER ET AL,

Fig. 9. Electron micrograph. Melanoeyte in late telophase of mitosis (arrow). Surrounding spinous kera-tinocytes (star), X 9500,

or extremities. There was no obvious pre-dilection for sex or race, the average age ofonset being over 55 years and the averageduration before removal being 8—10 years(Mishima & Pinkus 1960, Reaves 1974).Our case happened to fit all these criteria

as our patient was 74-years-old and thelesion had been present for 8 years.

The two types of Bloch's (1926) originalbenign non-nevoid melanoepithelioma arenow recognized as separate entities. Histype I melanoepithelioma as melanoacan-

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Fj?. 70, Election micrograph. Dendritic processes (stars) lying between spinous keratinocytes, Melano-somes of varying degrees of development present, X 18,200,

thoma and his type II melanoepitheliomaas a type of pigmented seborrheic keratosis(Mishima & Pinkus 1960), It is the melano-acanthoma which is characterized histolo-gically by its hyperplastie epidermis with anadmixture of large dendritic melanoeytes,small dark-staining keratinocytes and large

non-pigmented prickle cells. Furthermore,this non-invasive cutaneous papOloma has atendency to central keratinization (endo-keratinization) and pseudo-horn cyst forma-tion (Mishima & Pinkus 1960),

Our histological studies confirmed thetumor as being of an elevated epidermal


Fig. 11. Electron micrograph. Seborrheic keratosis. Basal layer of the epidermis showing attenuatedmelanoeyte (star). Clumps of melanin in adjacent basaloid keratinocytes (arrowheads). X 3800.

type with no involvement of the corium.The predominant cell type was the kera-tinocyte with both a basaloid and a spinousdifferentiation pattern in different areas.Scattered throughout this background was apopulation of large, richly dendritic melano-

eytes. Their presence was confirmed by bothcytochemical and ultrastructural means. Thebulk of the melanin pigment was confinedto this cell type, however, it was evidentthat the block in transfer of pigment tokeratinocytes was not complete when viewed

MELANOACANTHOMA 139

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Fig. 12. Electron micrograph. Keratinocytes in a spinous pattern showing minimal melanin, Melanoeyte(*) and Langerhans cell (star) are present in the superfieial layers. X 3800.Detail of a Langerhans cell cytoplasm with characteristic granules (arrows). X 14,200,

at the ultrastructural level. Sparse pigmentgranules within keratinocytes were noted inareas where the basaloid pattern was the pre-dominant type.

A block in transfer of melanin frommelanoeytes to keratinocytes has primarily

been reported in inflamed Negro epidermis.In these studies, an expansion of dendriteswas noted (Rappaport 1956, Staricco &Pinkus 1957). As had been noted by others,and also here, the presence of inflammatoryinfiltrates appears to be common in many


melanoacanthomas. The nature of the blockin melanin transfer, however, is not clear,but may be related to alteration in the nor-mal pattern and speed of keratinocyte dif-ferentiation. Such changes might alter thecell surface characteristics of keratinocytesand thereby prevent or inhibit pigmentdonation.

The Langerhans cells that were present inall the malpighian layers, except the basallayers were apparently normal in morpho-logy. It was not possible at this stage to com-ment on any quantitative variations in num-bers in the melanoacanthoma versus normalepidermis. Since the nature and functionsof this cell type have recently come underrenewed scrutiny with respect to its possiblerole in allergen trapping (Silberberg et al.1976a, b, Rowden et al. 1977, Shelley &Juhlin 1977) it may be of interest to pursuesuch studies on future acquisitions. Asindicated in recent reviews (Wolff 1974,Rowden & Lewis 1976), there is still somesupport for the suggestion concerningLangerhans cell involvement in control ofproliferation of keratinocytes (Potten &Allen 1976). Clearly, in such a situation asexists in a tumor with derangements ofcontrol of proliferation of keratinocytes,some changes in the Langerhans cell/kera-tinocyte ratio might be predicted.

Since, however, the melanoacanthoma isclearly a mixed tumor, of keratinocytes andmelanoeytes, such investigation of Langer-hans cell numbers will be even more inter-esting if and when more material is available.

The negative results obtained with bothimmunofluorescence and the precipitin assayagainst a known system, i.e., the melanomacytoplasmic antigen, coupled with a goodresult in the positive control, indicates thatthe patient's lesion is not related to cu-taneous malignant melanoma.

In conclusion, the tumor was shown to bea non-nevoid type, not a malignant mela-noma, and consisting of a mixed populationof keratinocytes and melanoeytes.

References

Abulafia, J. & Grinspan, D. (1968) Atypical

melanoacanthoma. Medieina Cutanea Ibero-Latino-Americana 3, 125-130.

Barka, T. & Anderson, P. J. (1962) Histoehemiealmethods for acid phosphatase using hexa-zonium pararosanalin as coupler. Jourtial ofHistoehemistry and Cytochemistry 10, 741 —753.

Bloeh, B. (1926) Ueber benigne, nicht naevoideMelanoepitheliome der Haut nebst Bemer-kungen ueber das Wesen und die Genese derDendritenzellen. Arehiv fuer Dertnatologie undSyphilis 153, 20-40.

Delaeretaz, J. (1975) Melano-aeanthome. Derma-tologica 151, 236-240.

Mishima, Y. (1966) Enzymic differentiation ofmelanin phagoeytosis and synthesis, ActaDermato-Venereologiea 46, 307-309,

Mishima, Y. & Pinkus, H. (1960) Benign mixedtumor of melanoeytes and malpighian cells.Archives of Dermatology 81, 539-550.

Phillips, T. M. & Lewis, M. G. (:i970) A system ofimmunofluoreseenee in the study of tumorcells. Revue Europeentie d'Etudes Cliniques etBiologiques 15,1016- 1020.

Potten, C. S. & Allen, T. D. (1976) Model impli-cating Langerhans cell in keratinocyte pro-liferation control. Differentiation 5, 43-47,

Rappaport, B. Z. (1956) Studies on atopic derma-titis. III. The effeet of eorticotropin, cortisoneand hydroeortisone on storage of melanin inbasal cells and on dopa oxidase reaction. Ar-chives of Pathology 61, 395^-400.

Reaves, C. E, (1974) Melanoacanthoma: A casereport and review of the literature. Bulletinof the Association of Military Dermatologists22,55-58.

Rodriguez, H. A. & McGavran, M. H. (1969) AModified dopa reaction for the diagnosisand investigation of pigment cells. AmericanJournal of ClineialPathology 52, 219-227.

Rowden, G. & Lewis, M. G. (1976) Langerhanscells: Involvement in the pathogenesis ofmycosis fungoides. British Journal of Derma-tology 95, 66S-670.

Rowden, G., Lewis, M. G. & Sullivan, A, K, (1977)la antigen expression on human Langerhanseells. Nature 268, 247-248.

SheUey, W. B. & Juhlin, L. (1977) Selective up-take of contact allergens by the Langerhanscell. Archives of Dermatology 113, 187-192.

Silberberg, I., Baer, R. L. & Rosenthal, S. A.(1976a) The role of Langerhans cells in allergiccontact hypersensitivity. A review of findingsin man and %mma.iii%s. Journal of InvestigativeDermatology 66, 210-217.

Silberberg-Sinakin, I., Thorbeeke, G. J, Baer, R, L,Rosenthal, S. A. & Berezowsky, V. (1976b)Antigen-binding Langerhans cells in skin, der-mal lymphatics and in lymph nodes. CellularImmunology 25, 137-151.

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Spott, D. A. Wood, M. G. & Heaton, C. L. (1972)Melanoacanthoma of the eyelid, Arehives ofDermatology 105, 898-899,

Stariceo, R. J. & Pinkus, H, (1957) Quantitativeand qualitative data on pigment cells of adulthuman epidermis. Jourtial of InvestigativeDermatology 28, 33-45.

Traub, E. F. (1950) Benign non-nevoid melano-epithelioma of the skin (Bloch). Archives ofDermatology and Syphilology 61, 1025-1038.

Wolff, K. (1972) The Langerhans eeU. In Currentprobletns in Dertnatology, ed. Mali, J. W. H,,Vol, 4, p, 79- 145, Karger: Basel,

Address:a L. A. Sehlappner, M.D., F.R.C.P.(C)Clin ical A ssistan t ProfessorDivision of Dertnatology, Dept. of MedicineThe Utiiversity of British Columbia865 West 10th Ave. V5Z IL 7Vancouver, B.C., Catmda