Immunohistochemical Studies of Atypical Conjunctival Melanocytic Nevi

Home , Balloon cell nevus, Nevi and melanomas

CLINICAL SCIENCES Immunohistochemical Studies of Atypical Conjunctival Melanocytic Nevi

Frederick A. Jakobiec, MD, DSc; Kathryn Colby, MD, PhD; Ann M. Bajart, MD; S. John Saragas, MD; Alexandre Moulin, MD

Objective: To evaluate with immunohistochemical tical immunohistochemical profile. Only the balloon cell methods 5 atypical melanocytic conjunctival lesions. nevus was MART-1–positive and HMB-45–negative. The granular cell and blue nevi immunoreacted negligibly with Methods: This was a retrospective clinicoimmuno- Ki-67 (approximately 1% of cells). pathologic study. Routine histochemical staining was performed with multiparametric immunohistochemical Conclusions: S100 and MART-1 reliably immuno- analysis with monoclonal antibodies immunoreacted on stained all nevocytic morphologic variants. HMB-45 im- paraffin sections to identify the following cell antigens: munoreactivity of the granular, epithelioid/clonal, and S-100, MART-1, HMB-45, CD45, CD68, CD1a, lyso- blue nevi did not indicate a more active or proliferative zyme, and Ki-67 (nuclear proliferation protein). lesion but instead suggested abnormal melanogenesis. Ki-67 was the most valuable immunohistochemical ad- Results: A unique granular cell nevus contained peri- junct to morphology for the diagnosis of these benign odic acid–Schiff–positive, diastase-resistant granules and variant conjunctival nevi, because melanomas display a immunoreacted with monoclonal antibodies against S-100 much higher proliferation index (Ͼ10% nuclear posi- protein and melanocytic-associated antigens MART-1 and tivity among all cells counted) than the current nevi (ap- HMB-45. Results for CD45, CD1a, CD68, and lysozyme proximately 1%). immunostaining of the granular cells were negative. Two epithelioid cell (clonal or inverted) nevi exhibited an iden- Arch Ophthalmol. 2009;127(8):970-980

HE TRADITIONAL METHOD FOR nant melanoma, if one lacked familiarity diagnosing clinically suspi- with the entities. Their anomalous fea- cious pigmented conjuncti- tures are absent in common acquired val lesions has been to re- nevomelanocytic junctional, compound, or move them surgically and subepithelial nevi. The epibulbar conjunc- carefully evaluate their architectural and cy- tival nevus variants that we more fully char- T 1-5 tologic features with light microscopy and acterize herein were immunohistochemi- less frequently with electron microscopy.6 cally stained with monoclonal antibodies With the advent of immunohistochemical directed against S100 protein, the melano- stains for the detection of specific cellular cytic markers MART-1 and HMB-45,25,26 and antigens expressed by melanocytes, the di- Ki-67 nuclear proliferation protein that re- agnosis of cutaneous melanocytic prolifera- flects S-phase cycling cells.9-15 The current tions has achieved a higher level of sophis- lesions include the first reported examples tication and accuracy.7-15 Similar studies on of a granular cell and 2 epithelioid cell conjunctival lesions, which continue to pro- (clonal or inverted) nevi; an extremely rare Author Affiliations: 1,2,5 Department of Ophthalmology, vide diagnostic challenges, have fo- balloon cell nevus; and a somewhat more David G. Cogan Laboratory of cused on 1 or 2 markers at a time rather than frequent but still uncommon blue nevus. Ophthalmic Pathology a multiparametric analysis.16-24 Further- (Dr Jakobiec), and the Cornea more, they have failed to generate a coher- METHODS Service (Dr Colby), ent database that establishes these mark- Massachusetts Eye and Ear ers’ value in distinguishing benign from Infirmary and Harvard Medical malignant proliferations. From the regular and consultation files of the School, Boston; Ophthalmic We report for the first time immuno- David G. Cogan Laboratory of Ophthalmic Pa- Consultants of Boston, Boston pathologic findings derived from a panel of thology at the Massachusetts Eye and Ear In- (Dr Bajart); Saragas Eye Center, firmary, we retrieved conjunctival lesions that Somerville, Massachusetts probes applied to 4 types of rare conjunc- had been diagnosed as atypical nevus, nevus (Dr Saragas); and the Hoˆpital tival nevi that displayed exceptional cyto- with unusual features, borderline nevus, bal- Ophtalmique Jules Gonin, logic compositions or architectural fea- loon cell nevus, or blue nevus from 2005 to Lausanne, Switzerland tures. These unusual aspects could lead to 2008. Hematoxylin-eosin–stained slides were (Dr Moulin). a misdiagnosis, most seriously of malig- critically reexamined. Five lesions were judged

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 appropriate for inclusion in this study and further evaluation. While all were small specimens, only 1 case (blue nevus) had Table 1. Summary of Immunologic Probes insufficient archival tissue embedded in paraffin blocks for ad- ditional sectioning and staining. Routine histochemical stain- Antigen for ing included periodic acid–Schiff with and without diastase, Mas- Antibody Specificity Source Dilution son trichrome, and reticulin. Immunohistochemical staining S100 Cytoplasm of Mouse monoclonal, Prediluted with the immunoperoxidase method was conducted using the melanocyte, IgG2aa monoclonal antibodies and their targeted antigens listed in Schwann cell, glial Table 1. cell, among other The staining was done on BenchMark XT automated tissue cell types staining systems (Ventana Medical Systems, Tucson, Arizona) MART-1 Melanocytic Mouse monoclonal, 1:60 b using validated protocols. Endogenous peroxidase activity was cytoplasmic IgG2b premelanosomes blocked with H2O2 before antibody incubation. A combina- tion of EDTA and boric acid in Tris buffer (CC1 reagent; Ven- HMB-45 Melanocytic Mouse monoclonal, 1:100 cytoplasmic IgG1c tana Medical Systems) was applied to the tissue sections for an- premelanosomes tigen retrieval as needed, and the process was carried out prior PNL-2 Melanocytic and Mouse monoclonal, Prediluted to primary antibody incubations. The tissues sections were polymorphonuclear IgG1c washed and incubated with the primary antibodies indicated leukocytic cytoplasm above, followed by incubation with UltraView horseradish per- CD45 Lymphocytic, Mouse monoclonal, Prediluted oxidase–conjugated multimer antibody reagent. Antigen de- histiocytic, IgG1a tection was performed using UltraView and diaminobenzidine hematopoietic cell as the chromogen, which yields a brown/black reaction prod- membranes CD1a Langerhans cell Mouse monoclonal, Prediluted uct. In case 5, aminoethylcarbazole was used to produce a red a staining result. Tissues were counterstained with hematoxy- membrane IgG1 lin. Bleaching of melanin followed by immunoperoxidase stain- CD68 Monocyte/histiocytic Mouse monoclonal, Prediluted cytoplasm IgG1a ing was not conducted because of the frequent loss or partial Lysozyme Monocyte/histiocytic Mouse monoclonal, Prediluted dissolution of paraffin sections in our experience. cytoplasm IgG1a None of the lesions in this series was heavily pigmented and Ki-67 Nuclear proliferation Rabbit monoclonal, Prediluted all had a significant proportion of tumor cells that were either protein expressed in IgGa very lightly pigmented or wholly nonpigmented. Macro- S phase phages filled with phagocytosed melanin (melanophages) were present in all lesions in small to moderate numbers. They could aVentana Medical Systems, Tucson, Arizona. easily be distinguished from the lightly pigmented predomi- bSignet Laboratories, Dedham, Massachusetts. nant tumor cells by virtue of their coarsely clumped cytoplas- cDako Corporation, Carpinteria, California. mic melanin granules that totally obscured the nucleus. Con- sequently, it was easy to discern positive immunostaining, which lieved the lesion had increased in size but not in pig- was a deep brown/black with chromogen diaminobenzidine and ment. There was an eccentric darker brown region that evenly and diffusely distributed in the positive cells, com- approached the edge but no accompanying flat brown pig- pared with the fainter brown coloration conferred by the na- tive melanization of the tumor cells. In case 5 of the new blue mentation in the adjacent conjunctiva. There were sev- nevus, the red chromogen aminoethylcarbazole was used to eral freckles along the superior eyelid margins of both highlight positivity, obviating any possible confusion with mela- eyes. The patient had a strong family history of cutane- nin. The immunolabeling was evaluated impressionistically for ous melanoma. The conjunctival lesion was excised and the selected melanocytic antigens and graded. Counting of posi- has not recurred during 7 months of follow-up. tive nuclei labeled with Ki-67 was conducted in 2 high-power fields (ϫ400) using a 10ϫ10 reticule. This result was also stated Immunohistopathologic Findings as a proliferation index, that is, the percentage of positive cells in the 2 lesions in which any positivity was found (only the After fixation in formalin, the excised tissue measured 4 granular and blue nevi). mmϫ3mmϫ1 mm. The conjunctival epithelium con- Clinical information, including detailed ocular histories and clinical photographs when available, were reviewed and ana- tained many goblet cells and a small number of junc- lyzed in light of the histomorphologic immunohistochemical tional nests composed of cohesive, ovoid nevomelano- findings and the clinical follow-ups. The immunohistochemi- cytic cells that were also in the walls of shallow cal data were compared with those previously published in the evaginations of the surface epithelium. Two mildly sepa- ophthalmic and dermatologic literatures. rated junctional nests that were tightly organized were found beyond the subepithelial portion of the tumor on 1 side. In the subepithelial substantia propria under a thin RESULTS mantle of collagen was situated a sheetlike collection of unusual polyhedral eosinophilic to granular cells GRANULAR CELL NEVUS IN CASE 1 (Figure 1B) occupying the full thickness of the lesion. Common subepithelial nevocytes were not found. Fo- Clinical Findings cal collections of lymphocytes were found together with a light dispersion throughout the lesion. One year and a half before initial examination, a 22-year- The cells were either nonpigmented or faintly pig- old man had a right nasal epibulbar, elevated, movable, mented and exhibited a finely to coarsely granular ca- and exquisitely circumscribed tumor measuring 1.5 mm pacious cytoplasm, within which a relatively small non- in diameter near the plica (Figure 1A). The patient be- nucleolated nucleus with finely divided chromatin was

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Figure 1. A granular cell nevus (case 1). A, Exquisitely circumscribed, lightly pigmented, and freely movable epibulbar lesion. B, Large epithelioid, eosinophilic granular cells with small centrally located nuclei are associated with a lymphoid aggregate (left) and diffusely distributed lymphocytes (hematoxylin-eosin, original magnification ϫ160). C, The principal tumor cells contain large complex and small granules. Goblet cells are present within the epithelium (periodic acid–Schiff after diastase pretreatment, original magnification ϫ220). D, Subepithelial clusters and individual tumor cells are MART-1–positive. Intraepithelial dendritic melanocytes are also positively stained (immunoperoxidase reaction, diaminobenzidine chromogen, original magnification ϫ160). E, HMB-45 staining is unequivocal but less intense. No staining is apparent in the epithelium (immunoperoxidase reaction, original magnification ϫ160). F, Ki-67 staining for intranuclear proliferation protein fails to show a black immunohistochemical product in this typical high-power field. There is a faint cytoplasmic granularity as well as a light brown staining, the latter from the presence of finely dispersed melanin granules (immunoperoxidase reaction, original magnification ϫ400).

centrally or eccentrically placed, conferring a low nuclear bers were identified within conspicuous collections of lym- to cytoplasmic ratio; mitotic figures were absent. The deep phocytes. The periodic acid–Schiff stain highlighted the border was well defined, straight, and noninfiltrative. No cytoplasmic granularity, which was less dramatically mitotic figures were encountered. The reticulin stain re- brought out by the Masson trichrome stain; pretreat- vealed the sheathing of individual cells or small clusters ment with diastase failed to abolish the periodic acid– by delicate fibers; in contrast, haphazardly distributed fi- Schiff positivity. In some tumor cell clusters, the peri-

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 odic acid–Schiff–positive cytoplasmic granules were Immunohistopathologic Findings conglutinated (Figure 1C). Melanophages with coarsely clumped collections of pigment granules were widely scat- In the first of the 2 cases, the tissue received in the labo- tered beneath the epithelium and among the granular cells. ratory after formalin fixation measured 4 mmϫ4mmϫ1 S100 and MART-1 (Figure 1D) immunostained both mm. A pigmented cellular lesion occupied the vast ma- the intraepithelial dendritic melanocytes and the small jority of the conjunctival substantia propria (Figure 2B). nevocytic clusters within the well-defined junctional cavi- Within the goblet cell–rich epithelium were rare, well- ties. The subepithelial granular cells also stained posi- defined, lightly pigmented nests of conventional nevo- tively with these 2 probes; the lymphocytes stood out cytes. These cells were also in the walls of incipient shal- because of their negative staining, but they were CD45- low cysts derived from evaginations of the surface positive. HMB-45 staining was strongly manifested by the epithelium. Regular nevocytes were encountered in the granular cells (Figure 1E) but only faintly by the intra- superficial stroma admixed with lymphocytes, and a epithelial nevocytes within the junctional nests and the prominent collection was observed subepithelially to- intraepithelial dendritic melanocytes. The granular cells ward 1 edge of the lesion. were negative for lysozyme, CD45, and CD68 (macro- The largest part of the lesions in cases 2 and 3 was phage/histiocytes) as well as for CD1a (Langerhans cells). dominated by variably pigmented, eosinophilic epithe- Ki-67 immunoreaction revealed conspicuous nuclear lioid cells that were arranged in a dense sheet or else mod- staining within the epithelium and scattered lympho- erately separated from each other either individually or cytes, while only exceptionally among the granular cells in small clusters by a faintly fibrillar, lightly eosino- (Figure 1F). In 2 high-power fields (ϫ400), 259 cells were philic matrix. The epithelioid cells were endowed with counted, of which only 3 displayed nuclear immunohis- a generally fine dusting of cytoplasmic pigment of low tochemical product (proliferation index of 1.2%). Posi- density that never obliterated a clear view of the nucleus tively staining lymphocytic nuclei in S phase exhibited (Figure 2C). The latter could be central or eccentric, much smaller, rounder nuclei. nucleolated (basophilic but not acidophilic), binucleated, or multinucleated, and most characteristically pos- sessed small to extremely impressive intranuclear inclu- EPITHELIOID CELL (CLONAL OR INVERTED) sions of herniated cytoplasm. These imparted a bizarre NEVI IN CASES 2 AND 3 configuration to the nucleus, which often appeared hy- perchromatic owing to compression of the nuclear chro- Clinical Findings matinic material at the nuclear membrane. A small group of plump spindled cells was also present in the stroma, The first of 2 patients (case 2) with this lesion was a 19- and numerous melanophages were scattered about. No year-old man who had noted a spot 1 year earlier on the mitotic figures were discovered. nasal aspect of the surface of his right globe toward the The formalin-fixed tissue in the second, older patient plica. It had grown darker throughout several months. (case 3) measured 6 mmϫ4mmϫ1 mm. It failed to har- His vision was unaffected. The lesion was uniformly gray- bor any junctional nests; subepithelial foci of dystrophic ish black, 4 mm at the largest diameter, elegantly cir- calcification within elastotic material, however, were de- cumscribed, and freely moveable. The rest of the ocular tected. Beneath a prominent subepithelial population of examination results were unremarkable, and the lesion regular nevus cells (type B) were epithelioid cells that were was removed. There has been no recurrence during 6 virtually identical to those described in case 2. The stroma months of follow-up. between these latter cells was somewhat more sclerotic, The second patient (case 3) was a 56-year-old man who and the nuclei were often binucleated and vacuolated but became aware of a black spot on the mid-nasal aspect of generally less alarming in shape than those in the first his right globe approximately 30 years earlier. Through- case. The results of the immunostaining for the 2 cases out several years, the lesion slowly grew, though its color were identical. had not changed (Figure 2A). There were multiple small S100 was highly positive for the subepithelial epithe- feeding vessels. He had no other ocular complaints. The lioid cells, but less so for the conventional junctional and patient was a carpenter and had experienced consider- immediately subepithelial nevocytes in both lesions. able sun exposure in his life but had never developed any MART-1 was dramatically positive for the rare junctional skin cancers. He was awaiting kidney and liver trans- nevus cells, intraepithelial dendritic melanocytes, and sub- plantations as a consequence of long-standing diabetes epithelial epithelioid cells (Figure 2D), but more lightly mellitus and cirrhosis. Visual acuity was 20/20 OU with positive for the superficial subepithelial normal-appear- correction, and intraocular pressure was 12 mm Hg bi- ing nevocytes. HMB-45 stained most of the epithelioid cells laterally. The conjunctival lesion had a central, el- but not the small conventional nevocytes (Figure 2E). evated, dark to black area surrounded by nonpig- CD45 brought out many lymphocytes interspersed among mented, more translucent tissue exhibiting a fine intrinsic subepithelial nevocytes; far fewer histiocytes were iden- vascularity. A border of golden brown primary acquired tified with CD68 and lysozyme staining. Ki-67 displayed melanosis was not detected. The tumor measured 2.5 prominent intraepithelial nuclear staining, particularly mmϫ2.0 mm and was freely moveable on the globe. A along the basilar region that included basal germinal epi- simple excision with adjunctive cryotherapy was per- thelial cells. The small common subepithelial nevocytes formed. There was no recurrence during 11⁄2 years of fol- and the large epithelioid cells failed to exhibit staining of low-up, but the patient died of his systemic diseases. their nuclei with this marker (Figure 2F).

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Figure 2. Epithelioid cell (clonal or inverted) nevi (cases 2 and 3). A, Centrally pigmented lesion with outer rim of translucent tissue (case 3). B, Beneath the goblet cell–rich epithelium are superficial cysts with juxtaposed nevus cells and intermixed chronic inflammation. Eosinophilic epithelioid nevus cells without apparent pigmentation at this power and numerous heavily pigmented melanophages occupy the middle and lower portions of the substantia propria. The deep margin is sharp and noninfiltrating where it abuts the collagenous stroma (hematoxylin-eosin, original magnification ϫ100). C, The epithelioid nevus cells display a moderate dispersion of cytoplasmic melanin granules. Also conspicuous are bizarre single or multiple nuclei with herniations of eosinophilic cytoplasm. No mitotic figures are identified (hematoxylin-eosin, original magnification ϫ400). D, MART-1 highlights 2 intraepithelial nevus cell nests as well as basilar dendritic melanocytes. The cytoplasm of the subepithelial epithelioid cells is strikingly positive beyond the coloration conferred by intrinsic melanization. The scattered lymphocytes are negative (immunoperoxidase reaction, original magnification ϫ100). E, The cytoplasm of many but not all of the subepithelial nevus cells immunostained with HMB-45. Note that the intensity of the immunohistochemical product is greater than that of the discernible pigment in the hematoxylin- eosin–stained section in B, which has been photographed at the same magnification. An intraepithelial junctional nest on the upper left also labels (immunoperoxidase reaction, original magnification ϫ100). F, Ki-67 fails to stain the nuclei with their visible vacuoles. A positive reaction would obliterate the nuclear details with a black product. The brown pigment in the cytoplasm represents melanin (immunoperoxidase reaction, original magnification ϫ400).

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 BALLOON CELL NEVUS IN CASE 4 Immunostaining with S100 and MART-1 (Figure 3C) highlighted all populations of nevus cells, including the to- Clinical Findings tally clear cells (balloon cells); also positively reacting were abundant intraepithelial dendritic melanocytes and the ne- A 44-year-old woman who had worked outdoors for many vocytes in the rare junctional nests, particularly those lo- years as a park ranger was examined by her ophthalmolo- cated at the edge of the tumor. HMB-45 did not stain the gist in the fall of 2008. Ophthalmic examination revealed subepithelial balloon cells nor the conventional intraepi- a moderately brown lesion with foci of darker speckling. thelial and subepithelial nevocytes; the intraepithelial den- It was slightly elevated with an oval shape; movable, dritic cells stained faintly positive. MART-1 showed that smooth, and lustrous; and located on the right epibulbar the intraepithelial dendritic cells, while individually dis- surface near the plica. It measured 6 mmϫ4 mm. A smaller posed along the basal epithelial region, were obviously hy- contiguous inferior satellite situated in the lower portion perplastic; this feature was not seen in hematoxylin-eosin– of the plica semilunaris was somewhat darker and 1.5 mm stained sections. Ki-67 highlighted the nuclei of many at its greatest diameter. Earlier evaluations in 1994 and 2004 positively stained basilar epithelial cells but none in the cells had not documented either of the 2 neighboring lesions. constituting the junctional nests (Figure 3D). Neither the There were no discernible cysts, and a fine vascularity was balloon cells nor the conventional subepithelial nevo- only seen using a slitlamp. There was no flat surrounding cytes stained positively. The balloon cells were negative for pigmentation of primary acquired melanosis. Because of lysozyme, CD45 antigen, and CD68 antigen used for the the 2 components of the lesion, it was judged to be sus- identification of histiocytes/macrophages. picious and was removed. There has not been a recurrence during 4 months of follow-up. BLUE NEVUS IN CASE 5 Immunohistopathologic Findings Clinical Findings The 2 excised fragments of tissue measured 2 mm and 1 mm in greatest diameters. In hematoxylin-eosin–stained A 54-year-old man became aware of a left conjunctival sections, the conjunctival epithelium overlying the main pigmented lesion 21⁄2 years earlier on the superonasal sur- component of the lesion did not possess any apparent junc- face of his right globe. It had recently darkened over sev- tional activity nor dendritic melanocytic hyperplasia; epi- eral months and also doubled in size with the acquisi- thelial inclusion cysts were not found. There was a thin col- tion of a minimal but perceptible thickness (Figure 4A). lagenous grenz zone containing a moderate number of It measured 3 mmϫ4 mm; was flat and uniformly pig- melanophages that separated the epithelium from vari- mented; was situated several millimeters away from the ably sized collections of small standard nevus cells occu- limbus; had irregular feathery edges; and lacked cysts, a pying the superficial subepithelial substantia propria feeder vessel, and any associated surrounding flat, golden (Figure 3A). The latter cells were superficially arranged brown pigmentation. Fine blood vessels in the conjunc- in a sheet, while the deepest layers were composed of spindle tival substantia propria and episclera were outlined by cells that were lightly stromatized and incompletely ex- the pigmentation. The conjunctiva moved freely over the cised. Melanophages were minimally dispersed through- pigmented spot, while the latter was fixed to the sclera out the lesion, which was for the most part nonpigmented. and immovable. Vision was 20/20 OU, and the tension The most arresting feature of the lesion was a popu- by applanation tonometry was within normal levels in lation of polygonal, granular, clear, or ballooned cells lo- both eyes. Ultrasound biomicroscopy and gonioscopy cated in the middle and central subepithelial zones failed to demonstrate an underlying mass in the ciliary (Figure 3B). A generally centrally located, small nucleus body or chamber angle. A small, finely vascularized, non- contained an inconspicuous nucleolus; the nuclear chro- pigmented stromal thickening at the root of the blue iris matin was delicate and finely divided. Many of the nu- was diagnosed as an ephelis or small nevus. Several small, clei had intranuclear sequestrations of cytoplasm and in- flat pigmented nevi of the right fundus were discovered dentations of the nuclear membrane; some cells displayed on dilated fundus examination. These uveal findings sug- larger nuclei or binucleation. Periodic acid–Schiff stain- gested a forme fruste of ocular melanocytosis. The epi- ing of the clear cells failed to reveal any cytoplasmic granu- bulbar lesion was excised down to the scleral plane and larity. No mitotic figures were discovered. At the edges has not recurred during 11⁄2 years of follow-up. of the specimen near these balloon cells were conventional nevocytes that were densely packed with slightly Immunohistopathologic Findings angulated, small nuclei that occasionally exhibited intranuclear vacuoles but no nucleoli. Also interspersed After formalin fixation, 2 fragments of tissue measured among this population and at its outskirts were clear bal- 5mmϫ5mmϫ1mmand3mmϫ2mmϫ1 mm. There loon cells that resembled those in the center of the le- was no detectable intraepithelial melanocytic hyperpla- sion. The satellite, or plical portion, of the mass was com- sia. The superficial sclera and the deep substantia pro- posed of small subepithelial nevus cell nests or single cells pria contained loosely arranged spindled and occasion- exhibiting a perinuclear clear area and an outer submem- ally dendritiform cells set in a variably collagenized but branous rim of cytoplasmic melanin in the shape of a ring, generally loose stroma without a well-defined, tight fas- suggesting transitional cells. The overlying epithelium cicular pattern and with indistinct margins (Figure 4B). had 2 well-defined junctional nests. The cells endowed with less pigment displayed a deli-

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Figure 3. A balloon cell nevus (case 4). A, Beneath the uninvolved epithelium with a few goblet cells toward the upper left are common nevus cells that are beginning to acquire clear cytoplasm. This phenomenon is prominent in the middle of the field. A few melanophages are located in the upper right beneath the epithelium and among the balloon cells (hematoxylin-eosin, original magnification ϫ160). B, The balloon cells display some variability in the size and shape of their nuclei, which show vacuoles and occasional multinucleation. Note the melanophages (hematoxylin-eosin, original magnification ϫ400). C, MART-1 reacts with the intraepithelial dendritic melanocytes and the subepithelial balloon cells (immunoperoxidase reaction, original magnification ϫ200). D, Ki-67 vividly stains the nuclei of the basilar germinal cells of the squamous epithelium but none of the balloon cell nuclei. The inset demonstrates that the scattered dark staining material represents cytoplasmic melanin rather than intranuclear immunohistochemical product (immunoperoxidase reaction, original magnification ϫ200; inset, original magnification ϫ400).

cate diaphanous cytoplasm and were faintly pigmented liferation index of 0.6%). A summary of the preceding (Figure 4C). Those that were more superficially located immunohistochemical staining results of the 4 catego- contained a greater complement of cytoplasmic mela- ries of lesions comprising this series is presented in nin granules that did not obscure identification of their Table 2. banal nuclei. The latter tended to be small and widely separate from one another; punctate nucleoli and small intranuclear vacuoles (herniations of cytoplasm) were COMMENT readily apparent. Lymphocytes were absent; melanophages with large, coarsely clumped collections of mela- None of the lesions in this series was associated with pri- nin granules concealing the nucleus were present among mary acquired melanosis, the most common precursor the superficial, more heavily pigmented nevus cells. No of conjunctival melanoma.1-6 Four of 5 lesions were mor- mitotic figures were observed. Immunostaining for phologic variants of the common acquired conjunctival Melan-A (analogous to MART-1) (Figure 4D) and PNL2 nevus that begins in the epithelium as a nevomelano- (analogous to HMB-45), with only the red chromogen cytic proliferation of junctional nests (theques) and pro- aminoethylcarbazole used in this case, was positive. Ki-67 gressively evolves as the patient ages into compound or immunostaining demonstrated nuclear positivity in only completely subepithelial stages (as exemplified by our 1 of 176 cells counted in 2 high-power fields (ϫ400) (pro- oldest patient, aged 56 years, case 3), accompanied by

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Figure 4. A blue nevus (case 5). A, Juxtalimbal brown, immovable lesion with feathery edges. B, Superficial scleral lamellae are infiltrated by a banal and hypocellular population of lightly pigmented spindle cells. There is heavier pigmentation superficially (hematoxylin-eosin, original magnification ϫ100). C, The nuclei are delicate and frequently exhibit vacuoles. The stroma is loose and delicately fibrillar (hematoxylin-eosin, original magnification ϫ160). D, Melan-A–positive staining (same as MART-1) of the spindled melanocytes (immunoperoxidase reaction, aminoethylcarbazole chromogen; original magnification ϫ300).

Table 2. Results of Immunolabeling

Immunolabeling Grade Ki-67, % of Immunostained Nevus Type S-100 MART-1 HMB-45 CD1a CD68 Nuclei of Total Cellsa Granular cell nevus ϩϩϩ ϩϩϩ ϩϩϩ − − 1.2 Epithelioid cell nevib ϩϩϩ ϩϩϩ ϩϩ −− 0 Balloon cell nevusb ϩϩϩ ϩϩϩ −ND− 0 Blue nevus ND ϩϩ NDc ND ND 0.6

Abbreviations: ND, not done; −, negative; ϩ, light; ϩϩ, moderate, ϩϩϩ, heavy. a Proliferation index. b A subpopulation of common subepithelial nevocytes were also present and stained S-100 (ϩϩϩ), MART-1 (ϩϩϩ), HMB-45 (−), and Ki-67 (−). c PNL2 staining was performed instead and was moderately positive.

variably prominent epithelial inclusion cysts. Junc- to reside in the connective tissue and never reach the epi- tional nests did not extend radially far beyond the sub- thelium. In this series, the blue nevus was the only un- epithelial nevoid component. Subclinical microscopic epi- movable lesion owing to its location in the superficial thelial inclusion cysts were detected in 3 lesions. The blue sclera rather than the conjunctival substantia propria. nevus, on the other hand, represents an arrest in the mi- We describe for the first time the results obtained from gration of neural crest–derived melanocytes that come the simultaneous application of a panel of 3 probes (dis-

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 tinct from a single probe) to a subset of conjunctival nevi. keeping with the results described in the 2 cutaneous These markers are of proven value for the identification cases; HMB-45 is either negative or lightly positive only of cells of melanocytic origin, namely, antibodies against in the junctional cells of common compound nevome- S100 protein, MART-1, and HMB-45.7,8,25-27 Addition- lanocytic nevi.7,8,42 Ki-67 positivity was detected in only ally, immunolabeling for the Ki-67 nuclear prolifera- 3 of 259 cells counted in 2 high-power fields (ϫ400) of tion protein was undertaken.9-15 S100 stains both the our conjunctival lesion (unfortunately, it was not deter- nucleus and cytoplasm. This 21-kDa moiety was first dis- mined in the evaluation of the 2 cutaneous examples42). covered in glial cells and was termed S-protein owing to This is a proliferation index of around 1% and highly sup- its solubility in a supersaturated solution of ammonia sul- portive of a benign diagnosis.9,10,12,13,43 fate. A role has been imputed to it in guiding cytoplas- The 2 epithelioid cell nevi44,45 in this series are the most mic calcium fluxes or microtubular assembly. The alpha/ likely to be misdiagnosed as melanoma and are the first alpha dimer of S100 protein is preferentially expressed examples to be fully characterized in the conjunctiva. The in melanocytes. S100 protein is less specific than pre- first of our 2 patients’ lesions displayed rare junctional melanosome-associated antigens, since many nonmela- nests but was predominantly subepithelial, while the other nocytic cell types can express it. It retains a special role tumor in an older patient was completely subepithelial. in diagnosing desmoplastic melanomas, in which MART-1 These tumors are also referred to as clonal nevi because and MMB-45 antibodies are often nonreactive.25-27 of the striking population of subepithelial epithelioid cells MART-1, its close relative or twin Melan-A, and that coexists with conventional small nevus cells. Clonal- HMB-45 are intracytoplasmic epitopes of melanosome- ity in this context simply refers to a shared morphology related antigens that have been extensively used in stud- among the cells and not to any genetic identity. Inverted ies of cutaneous nevi and melanomas,7,8 but with less fruit- type A nevus is another term that has the virtue of high- ful current results in conjunctival investigations.16-21 lighting the location of the large epithelioid cells in the Antibodies against these antigens actually react with a middle or lower dermis of the skin or the same levels of premelanosomal group of glycoprotein antigens re- the conjunctival substantia propria, where small type B ferred to as gp100 (100 for kilodaltons). HMB-45 is at nevus cells are expected to be present. An earlier con- the gp10 (kilodalton) end of the complex and has been junctival study published prior to the report of this con- regarded as a cytoplasmic marker for melanocytic acti- dition in the skin probably described a related case.41 A vation8,16,18,20,28 that supposedly suggests a more suspect similar inverted phenomenon can occasionally be ob- cytologic composition when diffusely present within most served in juvenile conjunctival nevi.40 of the cells of a given lesion. Ki-67 immunostaining de- These epithelioid tumors displayed the most alarm- tects a nuclear proliferation protein that is preferen- ing cytologic features, consisting of an abundance of cy- tially expressed during late G1, S, M, and G2 phases of toplasm containing a fine dispersion of cytoplasmic pig- the cell cycle, while cells in the G0 (quiescent) phase are ment granules and possessing bizarre nuclei that often negative.9-13 It is far more sensitive for estimating prolif- displayed binucleation or multinucleation. These cells eration than counting mitotic figures. This marker has were juxtaposed to regular nevocytes and arranged in an accepted role when coupled with histopathologic evalu- either sheets or aggregated into small clusters separated ation in distinguishing benign from malignant cutane- by a lightly fibrillar or early sclerotic eosinophilic stroma. ous melanocytic lesions.9-15 To date, it has been sporadi- Their nuclei displayed multiform shapes and were often cally but not systematically used in the evaluation of dominated by large vacuoles (actually herniations of cy- conjunctival lesions,23,24 whether they are routine or atypi- toplasm); mitotic figures were not discovered. The deep cal, combined, spindle cell, dysplastic, inflamed, juve- margin was circumscribed and noninfiltrative. S100, nile, Spitz, or borderline nevi.29-41 MART-1, and HMB-45 immunostaining was positive in Case 1 is a granular cell nevus, the first ever recog- the cytoplasm of the epithelioid cells, which also showed nized in the conjunctiva. A single report of 2 cutaneous immunonegativity for CD45, CD68, and lysozyme; the examples has recently appeared in the dermatopathol- small collections of common subepithelial nevocytes in ogy literature.42 The initial pathologic impression was that both lesions were S-100– and MART-1–positive but HMB- the lesion represented an unusual histiocytic reaction, 45–negative. One lesion manifested a moderate subepi- which was reinforced by the presence of a prominent lym- thelial lymphocytic infiltrate. Ki-67 nuclear immunola- phocytic infiltrate. The constituent cells were polygonal beling was totally negative, which reinforced the or polyhedral and had a distinctive eosinophilic granu- interpretation of a benign lesion. lar cytoplasm that differed from the classically glassy cy- Benign and malignant balloon cell melanocytic neo- toplasm of epithelioid histiocytes and epithelioid mela- plasms are well recognized in the skin, are usually lightly noma cells. Their nuclei were generally small and centrally pigmented to nonpigmented, and generally occur in located and displayed punctate nucleoli. The granules younger individuals. They constitute 2.0% of cutaneous were sometimes conglutinated and were lightly positive nevi45 but are exceedingly rare in the conjunctiva. Shields on Masson trichrome staining but exhibited vivid peri- et al46 reported none among 410 consecutively excised nevi odic acid–Schiff–positive, diastase-resistant staining. Nega- at the Wills Eye Institute. At least 6 previous cases of con- tive CD45, CD68, CD1a, and lysozyme staining ruled out junctival balloon cell nevi have been described.47-51 Our the possibility that the epithelioid cells were of monocytic/ balloon cell nevus was composed of equal parts conven- histiocytic or Langerhans derivations. S100, MART-1, and tional nevocytes and polygonal, clear, vacuolated, and HMB-45 immunolabeling in the current case was uni- mostly mononucleated but rarely binucleated cells that were formly strongly positive throughout the entire lesion, in periodic acid–Schiff–negative. The nuclei were orthochro-

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 matic, small, centrally located and exhibited minute in the skin7,28 and conjunctiva16,18,20 and is expressed in nucleoli. The cells measured approximately 20 to 25 µm melanomas more often and more intensely than in in diameter; because of their large size and clear cyto- nevi.8,16,18,20,25-27 Common conjunctival nevus cells have plasm, they could be mistaken for xanthoma cells, adipo- been reported to be positive for HMB-45 in 8.5% to more cytes, or a metastasis from a renal cell carcinoma. than 50% of lesions.16,17 Steuhl and associates18 and Sharara Cutaneous and conjunctival balloon cells are created by and colleagues21 have accurately underscored the restric- collections of malformed vesicular premelanosomes51,52 and tion of this staining to the junctional region. MART-1 has not, as has been suggested by some, by the accumulation not been previously evaluated and contrasted with of cytoplasmic lipid. The conjunctival balloon cells in our HMB-45 in conjunctival lesions. Only 2 brief and incom- case were S100- and MART-1–positive but HMB-45– plete studies23,24 have described low counts of Ki-67 im- negative; CD45, CD68, and lysozyme negativity ruled out munoreactivity in conjunctival nevi, but did not clearly a histiocytic lineage; and Ki-67 immunostaining was en- state the immunostaining results with this marker in mela- tirely negative, indicating no proliferative tendency. In 2 nomas. In the skin, melanomas are frequently MART-1– earlier articles49,50 with immunohistochemical evalua- and HMB-45–positive, with a high proliferation index tions, the conjunctival balloon cells were S100-positive, (10%-70%) using Ki-67 antibodies7-15; nevi typically ex- though negative for HMB-45, lysozyme, and CD68, as ob- hibit MART-1 positivity, HMB-45 negativity, and a pro- served in our case. Based on their MART-1 positivity and liferation index of 2% or less.9,10,12,24,43 HMB-45 negativity, the balloon nevus cells parallel the stain- The 4 atypical types of conjunctival nevi delineated in ing results of conventional subepithelial nevus cells more this study yielded immunohistochemical findings support- closely than those comprising the granular and epitheli- ive of a benign diagnosis. Beyond the observations that the oid cell nevi described above, both of which were MART-1– granular and epithelioid cell nevi both stained strongly posi- and HMB-45–positive. tive for MART-1 and were aberrantly positive for HMB-45 When encountered in the conjunctiva, a “blue” ne- throughout all levels of the lesions, they more impor- vus is actually brown, unlike in the skin, where the Tyn- tantly displayed negligible to no Ki-67 positivity among the dall effect preferentially reflects the blue wavelengths. cells in the substantia propria. We have inferred from our While rarer in the conjunctiva than in the skin,53 blue data that HMB-45 positivity among variant nevus cells rep- nevi are probably the second most common nevus type resents an abnormality in melanogenesis rather than a in the conjunctiva54-56 after common acquired nevome- marker of cellular activation and therefore does not nec- lanocytic nevi, accounting for 1% of 410 excised nevi in essarily signal a menacing biologic potential. Currently, the previously mentioned series.46 The blue-brown ne- Ki-67 nuclear immunostaining for determining the prolif- vus in this study is the first epibulbar example to be evalu- eration index supersedes the diagnostic value of the dif- ated with immunohistochemical markers. It was lo- ferential expression of melanocyte-specific antigens. A Ki-67 cated in the superficial sclera and deep substantia propria. proliferation index of less than 2% is most compatible with The lesion was microscopically hypocellular (thereby dif- a nevus, whereas a result greater than 10% strengthens the fering from extrascleral extensions of uveal melano- diagnosis of a melanoma.9,12,13,43 mas) due to a delicate enveloping stroma. It was consti- A final caveat should be offered: When counting Ki-67 tuted by fusiform to stellate or dendritiform cells that were immunolabeled nuclei in melanocytic lesions, care must lightly pigmented or nonpigmented at its center and more be taken not to enumerate nuclei belonging to lympho- pigmented at the superficial periphery; the pigment was cytes, histiocytes, vascular endothelial cells, or epithe- never so coarsely clumped that it obscured the nucleus lial germinal cells participating in inclusion cysts. CD45 except in a few melanophages. The nuclei were delicate positivity of aggregates or a light dispersion of S-100– or and occasionally displayed intranuclear cytoplasmic se- MART-1–negative cells for positive ascertainment of lym- questrations and punctate nucleoli; no mitotic figures were phohistiocytic cells; CD31 and CD34 for vascular endo- found. Immunostaining was positive for MART-1 and thelial cells; and cytokeratin cocktail for squamous epi- PNL2 (the latter is a newer antimelanocyte monoclonal thelium should defend against these pitfalls. antibody that shares many of the immunolabeling prop- erties of both MART-1 and HMB-45, including positivity for common nevocytes and blue nevus cells57). The Submitted for Publication: February 19, 2009; final re- Ki-67 proliferation index was less then 1%. In contrast, vision received April 8, 2009; accepted April 9, 2009. episcleral extensions of uveal melanomas are typically hy- Correspondence: Frederick A. Jakobiec, MD, DSc, David percellular and have a proliferation index of around 20%.58 G. Cogan Ophthalmic Pathology Laboratory, Massachu- When evaluating the relative merits of the various im- setts Eye and Ear Infirmary, 243 Charles St, Room 321, munohistochemical probes used in this investigation, one Boston, MA 02114 ([email protected]). should remember that S100 immunoreactivity, while Financial Disclosure: None reported. highly sensitive, unfortunately indiscriminately stains both benign and malignant melanocytic lesions as well as a REFERENCES handful of other cell types.7,8,25,26 Previously reported immunohistochemical studies of cutaneous melanocytic le- 1. Jakobiec FA, Folberg R, Iwamoto T. Clinicopathologic characteristics of prema- sions analyzed with MART-1 and HMB-45 have estab- lignant and malignant melanocytic lesions of the conjunctiva. Ophthalmology. 1989;96(2):147-166. lished that these probes also cannot decisively separate 2. 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