immunohistochemical studies of atypical conjunctival

CLINICAL SCIENCES

Immunohistochemical Studies of AtypicalConjunctival Melanocytic NeviFrederick A. Jakobiec, MD, DSc; Kathryn Colby, MD, PhD; Ann M. Bajart, MD;S. John Saragas, MD; Alexandre Moulin, MD

Objective: To evaluate with immunohistochemicalmethods 5 atypical melanocytic conjunctival lesions.

Methods: This was a retrospective clinicoimmuno-pathologic study. Routine histochemical staining was per-formed with multiparametric immunohistochemicalanalysis with monoclonal antibodies immunoreacted onparaffin sections to identify the following cell antigens:S-100, MART-1, HMB-45, CD45, CD68, CD1a, lyso-zyme, and Ki-67 (nuclear proliferation protein).

Results: A unique granular cell nevus contained peri-odic acid–Schiff–positive, diastase-resistant granules andimmunoreacted with monoclonal antibodies against S-100protein and melanocytic-associated antigens MART-1 andHMB-45. Results for CD45, CD1a, CD68, and lysozymeimmunostaining of the granular cells were negative. Twoepithelioid cell (clonal or inverted) nevi exhibited an iden-

tical immunohistochemical profile. Only the balloon cellnevus was MART-1–positive and HMB-45–negative. Thegranular cell and blue nevi immunoreacted negligibly withKi-67 (approximately 1% of cells).

Conclusions: S100 and MART-1 reliably immuno-stained all nevocytic morphologic variants. HMB-45 im-munoreactivity of the granular, epithelioid/clonal, andblue nevi did not indicate a more active or proliferativelesion but instead suggested abnormal melanogenesis.Ki-67 was the most valuable immunohistochemical ad-junct to morphology for the diagnosis of these benignvariant conjunctival nevi, because melanomas display amuch higher proliferation index (�10% nuclear posi-tivity among all cells counted) than the current nevi (ap-proximately 1%).

Arch Ophthalmol. 2009;127(8):970-980

T HE TRADITIONAL METHOD FOR

diagnosing clinically suspi-cious pigmented conjuncti-val lesions has been to re-move them surgically and

carefully evaluate their architectural and cy-tologic features with light microscopy1-5 andless frequently with electron microscopy.6

With the advent of immunohistochemicalstains for the detection of specific cellularantigens expressed by melanocytes, the di-agnosis of cutaneous melanocytic prolifera-tions has achieved a higher level of sophis-tication and accuracy.7-15 Similar studies onconjunctival lesions, which continue to pro-vide diagnostic challenges,1,2,5 have fo-cused on 1 or 2 markers at a time rather thana multiparametric analysis.16-24 Further-more, they have failed to generate a coher-ent database that establishes these mark-ers’ value in distinguishing benign frommalignant proliferations.

We report for the first time immuno-pathologic findings derived from a panel ofprobes applied to 4 types of rare conjunc-tival nevi that displayed exceptional cyto-logic compositions or architectural fea-tures. These unusual aspects could lead toa misdiagnosis, most seriously of malig-

nant melanoma, if one lacked familiaritywith the entities. Their anomalous fea-tures are absent in common acquirednevomelanocytic junctional, compound, orsubepithelial nevi. The epibulbar conjunc-tival nevus variants that we more fully char-acterize herein were immunohistochemi-cally stained with monoclonal antibodiesdirected against S100 protein, the melano-cytic markers MART-1 and HMB-45,25,26 andKi-67 nuclear proliferation protein that re-flects S-phase cycling cells.9-15 The currentlesions include the first reported examplesof a granular cell and 2 epithelioid cell(clonal or inverted) nevi; an extremely rareballoon cell nevus; and a somewhat morefrequent but still uncommon blue nevus.

METHODS

From the regular and consultation files of theDavid G. Cogan Laboratory of Ophthalmic Pa-thology at the Massachusetts Eye and Ear In-firmary, we retrieved conjunctival lesions thathad been diagnosed as atypical nevus, nevuswith unusual features, borderline nevus, bal-loon cell nevus, or blue nevus from 2005 to2008. Hematoxylin-eosin–stained slides werecritically reexamined. Five lesions were judged

Author Affiliations:Department of Ophthalmology,David G. Cogan Laboratory ofOphthalmic Pathology(Dr Jakobiec), and the CorneaService (Dr Colby),Massachusetts Eye and EarInfirmary and Harvard MedicalSchool, Boston; OphthalmicConsultants of Boston, Boston(Dr Bajart); Saragas Eye Center,Somerville, Massachusetts(Dr Saragas); and the HopitalOphtalmique Jules Gonin,Lausanne, Switzerland(Dr Moulin).

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appropriate for inclusion in this study and further evaluation.While all were small specimens, only 1 case (blue nevus) hadinsufficient archival tissue embedded in paraffin blocks for ad-ditional sectioning and staining. Routine histochemical stain-ing included periodic acid–Schiff with and without diastase, Mas-son trichrome, and reticulin. Immunohistochemical stainingwith the immunoperoxidase method was conducted using themonoclonal antibodies and their targeted antigens listed inTable 1.

The staining was done on BenchMark XT automated tissuestaining systems (Ventana Medical Systems, Tucson, Arizona)using validated protocols. Endogenous peroxidase activity wasblocked with H2O2 before antibody incubation. A combina-tion of EDTA and boric acid in Tris buffer (CC1 reagent; Ven-tana Medical Systems) was applied to the tissue sections for an-tigen retrieval as needed, and the process was carried out priorto primary antibody incubations. The tissues sections werewashed and incubated with the primary antibodies indicatedabove, followed by incubation with UltraView horseradish per-oxidase–conjugated multimer antibody reagent. Antigen de-tection was performed using UltraView and diaminobenzidineas the chromogen, which yields a brown/black reaction prod-uct. In case 5, aminoethylcarbazole was used to produce a redstaining result. Tissues were counterstained with hematoxy-lin. Bleaching of melanin followed by immunoperoxidase stain-ing was not conducted because of the frequent loss or partialdissolution of paraffin sections in our experience.

None of the lesions in this series was heavily pigmented andall had a significant proportion of tumor cells that were eithervery lightly pigmented or wholly nonpigmented. Macro-phages filled with phagocytosed melanin (melanophages) werepresent in all lesions in small to moderate numbers. They couldeasily be distinguished from the lightly pigmented predomi-nant tumor cells by virtue of their coarsely clumped cytoplas-mic melanin granules that totally obscured the nucleus. Con-sequently, it was easy to discern positive immunostaining, whichwas a deep brown/black with chromogen diaminobenzidine andevenly and diffusely distributed in the positive cells, com-pared with the fainter brown coloration conferred by the na-tive melanization of the tumor cells. In case 5 of the new bluenevus, the red chromogen aminoethylcarbazole was used tohighlight positivity, obviating any possible confusion with mela-nin. The immunolabeling was evaluated impressionistically forthe selected melanocytic antigens and graded. Counting of posi-tive nuclei labeled with Ki-67 was conducted in 2 high-powerfields (�400) using a 10�10 reticule. This result was also statedas a proliferation index, that is, the percentage of positive cellsin the 2 lesions in which any positivity was found (only thegranular and blue nevi).

Clinical information, including detailed ocular histories andclinical photographs when available, were reviewed and ana-lyzed in light of the histomorphologic immunohistochemicalfindings and the clinical follow-ups. The immunohistochemi-cal data were compared with those previously published in theophthalmic and dermatologic literatures.

RESULTS

GRANULAR CELL NEVUS IN CASE 1

Clinical Findings

One year and a half before initial examination, a 22-year-old man had a right nasal epibulbar, elevated, movable,and exquisitely circumscribed tumor measuring 1.5 mmin diameter near the plica (Figure 1A). The patient be-

lieved the lesion had increased in size but not in pig-ment. There was an eccentric darker brown region thatapproached the edge but no accompanying flat brown pig-mentation in the adjacent conjunctiva. There were sev-eral freckles along the superior eyelid margins of botheyes. The patient had a strong family history of cutane-ous melanoma. The conjunctival lesion was excised andhas not recurred during 7 months of follow-up.

Immunohistopathologic Findings

After fixation in formalin, the excised tissue measured 4mm�3 mm�1 mm. The conjunctival epithelium con-tained many goblet cells and a small number of junc-tional nests composed of cohesive, ovoid nevomelano-cytic cells that were also in the walls of shallowevaginations of the surface epithelium. Two mildly sepa-rated junctional nests that were tightly organized werefound beyond the subepithelial portion of the tumor on1 side. In the subepithelial substantia propria under a thinmantle of collagen was situated a sheetlike collection ofunusual polyhedral eosinophilic to granular cells(Figure 1B) occupying the full thickness of the lesion.Common subepithelial nevocytes were not found. Fo-cal collections of lymphocytes were found together witha light dispersion throughout the lesion.

The cells were either nonpigmented or faintly pig-mented and exhibited a finely to coarsely granular ca-pacious cytoplasm, within which a relatively small non-nucleolated nucleus with finely divided chromatin was

Table 1. Summary of Immunologic Probes

Antigen forAntibody Specificity Source Dilution

S100 Cytoplasm ofmelanocyte,Schwann cell, glialcell, among othercell types

Mouse monoclonal,IgG2aa

Prediluted

MART-1 Melanocyticcytoplasmicpremelanosomes

Mouse monoclonal,IgG2bb

1:60

HMB-45 Melanocyticcytoplasmicpremelanosomes

Mouse monoclonal,IgG1c

1:100

PNL-2 Melanocytic andpolymorphonuclearleukocytic cytoplasm

Mouse monoclonal,IgG1c

Prediluted

CD45 Lymphocytic,histiocytic,hematopoietic cellmembranes

Mouse monoclonal,IgG1a

Prediluted

CD1a Langerhans cellmembrane


Prediluted

CD68 Monocyte/histiocyticcytoplasm


Prediluted

Lysozyme Monocyte/histiocyticcytoplasm


Prediluted

Ki-67 Nuclear proliferationprotein expressed inS phase

Rabbit monoclonal,IgGa

Prediluted

aVentana Medical Systems, Tucson, Arizona.bSignet Laboratories, Dedham, Massachusetts.cDako Corporation, Carpinteria, California.




centrally or eccentrically placed, conferring a low nuclearto cytoplasmic ratio; mitotic figures were absent. The deepborder was well defined, straight, and noninfiltrative. Nomitotic figures were encountered. The reticulin stain re-vealed the sheathing of individual cells or small clustersby delicate fibers; in contrast, haphazardly distributed fi-

bers were identified within conspicuous collections of lym-phocytes. The periodic acid–Schiff stain highlighted thecytoplasmic granularity, which was less dramaticallybrought out by the Masson trichrome stain; pretreat-ment with diastase failed to abolish the periodic acid–Schiff positivity. In some tumor cell clusters, the peri-

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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, eosinophilicgranular cells with small centrally located nuclei are associated with a lymphoid aggregate (left) and diffusely distributed lymphocytes (hematoxylin-eosin, originalmagnification �160). C, The principal tumor cells contain large complex and small granules. Goblet cells are present within the epithelium (periodic acid–Schiffafter diastase pretreatment, original magnification �220). D, Subepithelial clusters and individual tumor cells are MART-1–positive. Intraepithelial dendriticmelanocytes are also positively stained (immunoperoxidase reaction, diaminobenzidine chromogen, original magnification �160). E, HMB-45 staining isunequivocal but less intense. No staining is apparent in the epithelium (immunoperoxidase reaction, original magnification �160). F, Ki-67 staining forintranuclear proliferation protein fails to show a black immunohistochemical product in this typical high-power field. There is a faint cytoplasmic granularity aswell as a light brown staining, the latter from the presence of finely dispersed melanin granules (immunoperoxidase reaction, original magnification �400).




odic acid–Schiff–positive cytoplasmic granules wereconglutinated (Figure 1C). Melanophages with coarselyclumped collections of pigment granules were widely scat-tered beneath the epithelium and among the granular cells.

S100 and MART-1 (Figure 1D) immunostained boththe intraepithelial dendritic melanocytes and the smallnevocytic clusters within the well-defined junctional cavi-ties. The subepithelial granular cells also stained posi-tively with these 2 probes; the lymphocytes stood outbecause of their negative staining, but they were CD45-positive. HMB-45 staining was strongly manifested by thegranular cells (Figure 1E) but only faintly by the intra-epithelial nevocytes within the junctional nests and theintraepithelial dendritic melanocytes. The granular cellswere negative for lysozyme, CD45, and CD68 (macro-phage/histiocytes) as well as for CD1a (Langerhans cells).Ki-67 immunoreaction revealed conspicuous nuclearstaining within the epithelium and scattered lympho-cytes, while only exceptionally among the granular cells(Figure 1F). In 2 high-power fields (�400), 259 cells werecounted, of which only 3 displayed nuclear immunohis-tochemical product (proliferation index of 1.2%). Posi-tively staining lymphocytic nuclei in S phase exhibitedmuch smaller, rounder nuclei.

EPITHELIOID CELL (CLONAL OR INVERTED)NEVI IN CASES 2 AND 3

Clinical Findings

The first of 2 patients (case 2) with this lesion was a 19-year-old man who had noted a spot 1 year earlier on thenasal aspect of the surface of his right globe toward theplica. It had grown darker throughout several months.His vision was unaffected. The lesion was uniformly gray-ish black, 4 mm at the largest diameter, elegantly cir-cumscribed, and freely moveable. The rest of the ocularexamination results were unremarkable, and the lesionwas removed. There has been no recurrence during 6months of follow-up.

The second patient (case 3) was a 56-year-old man whobecame aware of a black spot on the mid-nasal aspect ofhis right globe approximately 30 years earlier. Through-out several years, the lesion slowly grew, though its colorhad not changed (Figure2A). There were multiple smallfeeding vessels. He had no other ocular complaints. Thepatient was a carpenter and had experienced consider-able sun exposure in his life but had never developed anyskin cancers. He was awaiting kidney and liver trans-plantations as a consequence of long-standing diabetesmellitus and cirrhosis. Visual acuity was 20/20 OU withcorrection, and intraocular pressure was 12 mm Hg bi-laterally. The conjunctival lesion had a central, el-evated, dark to black area surrounded by nonpig-mented, more translucent tissue exhibiting a fine intrinsicvascularity. A border of golden brown primary acquiredmelanosis was not detected. The tumor measured 2.5mm�2.0 mm and was freely moveable on the globe. Asimple excision with adjunctive cryotherapy was per-formed. There was no recurrence during 11⁄2 years of fol-low-up, but the patient died of his systemic diseases.


In the first of the 2 cases, the tissue received in the labo-ratory after formalin fixation measured 4 mm�4 mm�1mm. A pigmented cellular lesion occupied the vast ma-jority of the conjunctival substantia propria (Figure 2B).Within the goblet cell–rich epithelium were rare, well-defined, lightly pigmented nests of conventional nevo-cytes. These cells were also in the walls of incipient shal-low cysts derived from evaginations of the surfaceepithelium. Regular nevocytes were encountered in thesuperficial stroma admixed with lymphocytes, and aprominent collection was observed subepithelially to-ward 1 edge of the lesion.

The largest part of the lesions in cases 2 and 3 wasdominated by variably pigmented, eosinophilic epithe-lioid cells that were arranged in a dense sheet or else mod-erately separated from each other either individually orin small clusters by a faintly fibrillar, lightly eosino-philic matrix. The epithelioid cells were endowed witha generally fine dusting of cytoplasmic pigment of lowdensity that never obliterated a clear view of the nucleus(Figure 2C). The latter could be central or eccentric,nucleolated (basophilic but not acidophilic), binucle-ated, or multinucleated, and most characteristically pos-sessed small to extremely impressive intranuclear inclu-sions of herniated cytoplasm. These imparted a bizarreconfiguration to the nucleus, which often appeared hy-perchromatic owing to compression of the nuclear chro-matinic material at the nuclear membrane. A small groupof plump spindled cells was also present in the stroma,and numerous melanophages were scattered about. Nomitotic figures were discovered.

The formalin-fixed tissue in the second, older patient(case 3) measured 6 mm�4 mm�1 mm. It failed to har-bor any junctional nests; subepithelial foci of dystrophiccalcification within elastotic material, however, were de-tected. Beneath a prominent subepithelial population ofregular nevus cells (type B) were epithelioid cells that werevirtually identical to those described in case 2. The stromabetween these latter cells was somewhat more sclerotic,and the nuclei were often binucleated and vacuolated butgenerally less alarming in shape than those in the firstcase. The results of the immunostaining for the 2 caseswere identical.

S100 was highly positive for the subepithelial epithe-lioid cells, but less so for the conventional junctional andimmediately subepithelial nevocytes in both lesions.MART-1 was dramatically positive for the rare junctionalnevus cells, intraepithelial dendritic melanocytes, and sub-epithelial epithelioid cells (Figure 2D), but more lightlypositive for the superficial subepithelial normal-appear-ing nevocytes. HMB-45 stained most of the epithelioid cellsbut not the small conventional nevocytes (Figure 2E).CD45 brought out many lymphocytes interspersed amongsubepithelial nevocytes; far fewer histiocytes were iden-tified with CD68 and lysozyme staining. Ki-67 displayedprominent intraepithelial nuclear staining, particularlyalong the basilar region that included basal germinal epi-thelial cells. The small common subepithelial nevocytesand the large epithelioid cells failed to exhibit staining oftheir 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 gobletcell–rich epithelium are superficial cysts with juxtaposed nevus cells and intermixed chronic inflammation. Eosinophilic epithelioid nevus cells without apparentpigmentation at this power and numerous heavily pigmented melanophages occupy the middle and lower portions of the substantia propria. The deep margin issharp and noninfiltrating where it abuts the collagenous stroma (hematoxylin-eosin, original magnification �100). C, The epithelioid nevus cells display amoderate dispersion of cytoplasmic melanin granules. Also conspicuous are bizarre single or multiple nuclei with herniations of eosinophilic cytoplasm. Nomitotic figures are identified (hematoxylin-eosin, original magnification �400). D, MART-1 highlights 2 intraepithelial nevus cell nests as well as basilar dendriticmelanocytes. The cytoplasm of the subepithelial epithelioid cells is strikingly positive beyond the coloration conferred by intrinsic melanization. The scatteredlymphocytes are negative (immunoperoxidase reaction, original magnification �100). E, The cytoplasm of many but not all of the subepithelial nevus cellsimmunostained 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 �100). F, Ki-67 fails to stain the nuclei with their visible vacuoles. A positive reaction would obliterate thenuclear details with a black product. The brown pigment in the cytoplasm represents melanin (immunoperoxidase reaction, original magnification �400).




BALLOON CELL NEVUS IN CASE 4

Clinical Findings

A 44-year-old woman who had worked outdoors for manyyears as a park ranger was examined by her ophthalmolo-gist in the fall of 2008. Ophthalmic examination revealeda moderately brown lesion with foci of darker speckling.It was slightly elevated with an oval shape; movable,smooth, and lustrous; and located on the right epibulbarsurface near the plica. It measured 6 mm�4 mm. A smallercontiguous inferior satellite situated in the lower portionof the plica semilunaris was somewhat darker and 1.5 mmat its greatest diameter. Earlier evaluations in 1994 and 2004had not documented either of the 2 neighboring lesions.There were no discernible cysts, and a fine vascularity wasonly seen using a slitlamp. There was no flat surroundingpigmentation of primary acquired melanosis. Because ofthe 2 components of the lesion, it was judged to be sus-picious and was removed. There has not been a recur-rence during 4 months of follow-up.


The 2 excised fragments of tissue measured 2 mm and 1mm in greatest diameters. In hematoxylin-eosin–stainedsections, the conjunctival epithelium overlying the maincomponent of the lesion did not possess any apparent junc-tional activity nor dendritic melanocytic hyperplasia; epi-thelial inclusion cysts were not found. There was a thin col-lagenous grenz zone containing a moderate number ofmelanophages that separated the epithelium from vari-ably sized collections of small standard nevus cells occu-pying the superficial subepithelial substantia propria(Figure 3A). The latter cells were superficially arrangedin a sheet, while the deepest layers were composed of spindlecells that were lightly stromatized and incompletely ex-cised. Melanophages were minimally dispersed through-out the lesion, which was for the most part nonpigmented.

The most arresting feature of the lesion was a popu-lation of polygonal, granular, clear, or ballooned cells lo-cated in the middle and central subepithelial zones(Figure 3B). A generally centrally located, small nucleuscontained an inconspicuous nucleolus; the nuclear chro-matin was delicate and finely divided. Many of the nu-clei had intranuclear sequestrations of cytoplasm and in-dentations of the nuclear membrane; some cells displayedlarger nuclei or binucleation. Periodic acid–Schiff stain-ing of the clear cells failed to reveal any cytoplasmic granu-larity. No mitotic figures were discovered. At the edgesof the specimen near these balloon cells were conven-tional nevocytes that were densely packed with slightlyangulated, small nuclei that occasionally exhibited in-tranuclear vacuoles but no nucleoli. Also interspersedamong this population and at its outskirts were clear bal-loon cells that resembled those in the center of the le-sion. The satellite, or plical portion, of the mass was com-posed of small subepithelial nevus cell nests or single cellsexhibiting a perinuclear clear area and an outer submem-branous rim of cytoplasmic melanin in the shape of a ring,suggesting transitional cells. The overlying epitheliumhad 2 well-defined junctional nests.

Immunostaining with S100 and MART-1 (Figure 3C)highlighted all populations of nevus cells, including the to-tally clear cells (balloon cells); also positively reacting wereabundant intraepithelial dendritic melanocytes and the ne-vocytes in the rare junctional nests, particularly those lo-cated at the edge of the tumor. HMB-45 did not stain thesubepithelial balloon cells nor the conventional intraepi-thelial and subepithelial nevocytes; the intraepithelial den-dritic cells stained faintly positive. MART-1 showed thatthe intraepithelial dendritic cells, while individually dis-posed along the basal epithelial region, were obviously hy-perplastic; this feature was not seen in hematoxylin-eosin–stained sections. Ki-67 highlighted the nuclei of manypositively stained basilar epithelial cells but none in the cellsconstituting the junctional nests (Figure 3D). Neither theballoon cells nor the conventional subepithelial nevo-cytes stained positively. The balloon cells were negative forlysozyme, CD45 antigen, and CD68 antigen used for theidentification of histiocytes/macrophages.

BLUE NEVUS IN CASE 5

Clinical Findings

A 54-year-old man became aware of a left conjunctivalpigmented lesion 21⁄2 years earlier on the superonasal sur-face of his right globe. It had recently darkened over sev-eral months and also doubled in size with the acquisi-tion of a minimal but perceptible thickness (Figure 4A).It measured 3 mm�4 mm; was flat and uniformly pig-mented; was situated several millimeters away from thelimbus; had irregular feathery edges; and lacked cysts, afeeder vessel, and any associated surrounding flat, goldenbrown pigmentation. Fine blood vessels in the conjunc-tival substantia propria and episclera were outlined bythe pigmentation. The conjunctiva moved freely over thepigmented spot, while the latter was fixed to the scleraand immovable. Vision was 20/20 OU, and the tensionby applanation tonometry was within normal levels inboth eyes. Ultrasound biomicroscopy and gonioscopyfailed to demonstrate an underlying mass in the ciliarybody or chamber angle. A small, finely vascularized, non-pigmented stromal thickening at the root of the blue iriswas diagnosed as an ephelis or small nevus. Several small,flat pigmented nevi of the right fundus were discoveredon dilated fundus examination. These uveal findings sug-gested a forme fruste of ocular melanocytosis. The epi-bulbar lesion was excised down to the scleral plane andhas not recurred during 11⁄2 years of follow-up.


After formalin fixation, 2 fragments of tissue measured5 mm�5 mm�1 mm and 3 mm�2 mm�1 mm. Therewas no detectable intraepithelial melanocytic hyperpla-sia. The superficial sclera and the deep substantia pro-pria contained loosely arranged spindled and occasion-ally dendritiform cells set in a variably collagenized butgenerally loose stroma without a well-defined, tight fas-cicular pattern and with indistinct margins (Figure 4B).The cells endowed with less pigment displayed a deli-




cate diaphanous cytoplasm and were faintly pigmented(Figure 4C). Those that were more superficially locatedcontained a greater complement of cytoplasmic mela-nin granules that did not obscure identification of theirbanal nuclei. The latter tended to be small and widelyseparate from one another; punctate nucleoli and smallintranuclear vacuoles (herniations of cytoplasm) werereadily apparent. Lymphocytes were absent; mela-nophages with large, coarsely clumped collections of mela-nin granules concealing the nucleus were present amongthe superficial, more heavily pigmented nevus cells. Nomitotic figures were observed. Immunostaining forMelan-A (analogous to MART-1) (Figure 4D) and PNL2(analogous to HMB-45), with only the red chromogenaminoethylcarbazole used in this case, was positive. Ki-67immunostaining demonstrated nuclear positivity in only1 of 176 cells counted in 2 high-power fields (�400) (pro-

liferation index of 0.6%). A summary of the precedingimmunohistochemical staining results of the 4 catego-ries of lesions comprising this series is presented inTable 2.

COMMENT

None of the lesions in this series was associated with pri-mary acquired melanosis, the most common precursorof conjunctival melanoma.1-6 Four of 5 lesions were mor-phologic variants of the common acquired conjunctivalnevus that begins in the epithelium as a nevomelano-cytic proliferation of junctional nests (theques) and pro-gressively evolves as the patient ages into compound orcompletely subepithelial stages (as exemplified by ouroldest patient, aged 56 years, case 3), accompanied by

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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 arebeginning to acquire clear cytoplasm. This phenomenon is prominent in the middle of the field. A few melanophages are located in the upper right beneath theepithelium and among the balloon cells (hematoxylin-eosin, original magnification �160). B, The balloon cells display some variability in the size and shape oftheir nuclei, which show vacuoles and occasional multinucleation. Note the melanophages (hematoxylin-eosin, original magnification �400). C, MART-1 reactswith the intraepithelial dendritic melanocytes and the subepithelial balloon cells (immunoperoxidase reaction, original magnification �200). D, Ki-67 vividly stainsthe nuclei of the basilar germinal cells of the squamous epithelium but none of the balloon cell nuclei. The inset demonstrates that the scattered dark stainingmaterial represents cytoplasmic melanin rather than intranuclear immunohistochemical product (immunoperoxidase reaction, original magnification �200;inset, original magnification �400).




variably prominent epithelial inclusion cysts. Junc-tional nests did not extend radially far beyond the sub-epithelial nevoid component. Subclinical microscopic epi-thelial inclusion cysts were detected in 3 lesions. The bluenevus, on the other hand, represents an arrest in the mi-gration of neural crest–derived melanocytes that come

to reside in the connective tissue and never reach the epi-thelium. In this series, the blue nevus was the only un-movable lesion owing to its location in the superficialsclera rather than the conjunctival substantia propria.

We describe for the first time the results obtained fromthe simultaneous application of a panel of 3 probes (dis-

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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 andhypocellular population of lightly pigmented spindle cells. There is heavier pigmentation superficially (hematoxylin-eosin, original magnification �100).C, The nuclei are delicate and frequently exhibit vacuoles. The stroma is loose and delicately fibrillar (hematoxylin-eosin, original magnification �160).D, Melan-A–positive staining (same as MART-1) of the spindled melanocytes (immunoperoxidase reaction, aminoethylcarbazole chromogen; originalmagnification �300).

Table 2. Results of Immunolabeling

Nevus Type

Immunolabeling Grade Ki-67, %of Immunostained

Nuclei of Total CellsaS-100 MART-1 HMB-45 CD1a CD68

Granular cell nevus �� − − 1.2Epithelioid cell nevib �� − − 0Balloon cell nevusb �� − ND − 0Blue nevus ND �� NDc ND ND 0.6

Abbreviations: ND, not done; −, negative; �, light; ��, moderate, ��, heavy.aProliferation index.bA subpopulation of common subepithelial nevocytes were also present and stained S-100 (��), MART-1 (��), HMB-45 (−), and Ki-67 (−).cPNL2 staining was performed instead and was moderately positive.




tinct from a single probe) to a subset of conjunctival nevi.These markers are of proven value for the identificationof cells of melanocytic origin, namely, antibodies againstS100 protein, MART-1, and HMB-45.7,8,25-27 Addition-ally, immunolabeling for the Ki-67 nuclear prolifera-tion protein was undertaken.9-15 S100 stains both thenucleus and cytoplasm. This 21-kDa moiety was first dis-covered in glial cells and was termed S-protein owing toits solubility in a supersaturated solution of ammonia sul-fate. A role has been imputed to it in guiding cytoplas-mic calcium fluxes or microtubular assembly. The alpha/alpha dimer of S100 protein is preferentially expressedin melanocytes. S100 protein is less specific than pre-melanosome-associated antigens, since many nonmela-nocytic cell types can express it. It retains a special rolein diagnosing desmoplastic melanomas, in which MART-1and MMB-45 antibodies are often nonreactive.25-27

MART-1, its close relative or twin Melan-A, andHMB-45 are intracytoplasmic epitopes of melanosome-related antigens that have been extensively used in stud-ies of cutaneous nevi and melanomas,7,8 but with less fruit-ful current results in conjunctival investigations.16-21

Antibodies against these antigens actually react with apremelanosomal group of glycoprotein antigens re-ferred to as gp100 (100 for kilodaltons). HMB-45 is atthe gp10 (kilodalton) end of the complex and has beenregarded as a cytoplasmic marker for melanocytic acti-vation8,16,18,20,28 that supposedly suggests a more suspectcytologic composition when diffusely present within mostof the cells of a given lesion. Ki-67 immunostaining de-tects a nuclear proliferation protein that is preferen-tially expressed during late G1, S, M, and G2 phases ofthe cell cycle, while cells in the G0 (quiescent) phase arenegative.9-13 It is far more sensitive for estimating prolif-eration than counting mitotic figures. This marker hasan accepted role when coupled with histopathologic evalu-ation in distinguishing benign from malignant cutane-ous melanocytic lesions.9-15 To date, it has been sporadi-cally but not systematically used in the evaluation ofconjunctival lesions,23,24 whether they are routine or atypi-cal, combined, spindle cell, dysplastic, inflamed, juve-nile, Spitz, or borderline nevi.29-41

Case 1 is a granular cell nevus, the first ever recog-nized in the conjunctiva. A single report of 2 cutaneousexamples has recently appeared in the dermatopathol-ogy literature.42 The initial pathologic impression was thatthe lesion represented an unusual histiocytic reaction,which was reinforced by the presence of a prominent lym-phocytic infiltrate. The constituent cells were polygonalor polyhedral and had a distinctive eosinophilic granu-lar cytoplasm that differed from the classically glassy cy-toplasm of epithelioid histiocytes and epithelioid mela-noma cells. Their nuclei were generally small and centrallylocated and displayed punctate nucleoli. The granuleswere sometimes conglutinated and were lightly positiveon Masson trichrome staining but exhibited vivid peri-odic acid–Schiff–positive, diastase-resistant staining. Nega-tive CD45, CD68, CD1a, and lysozyme staining ruled outthe possibility that the epithelioid cells were of monocytic/histiocytic or Langerhans derivations. S100, MART-1, andHMB-45 immunolabeling in the current case was uni-formly strongly positive throughout the entire lesion, in

keeping with the results described in the 2 cutaneouscases; HMB-45 is either negative or lightly positive onlyin the junctional cells of common compound nevome-lanocytic nevi.7,8,42 Ki-67 positivity was detected in only3 of 259 cells counted in 2 high-power fields (�400) ofour conjunctival lesion (unfortunately, it was not deter-mined in the evaluation of the 2 cutaneous examples42).This is a proliferation index of around 1% and highly sup-portive of a benign diagnosis.9,10,12,13,43

The 2 epithelioid cell nevi44,45 in this series are the mostlikely to be misdiagnosed as melanoma and are the firstexamples to be fully characterized in the conjunctiva. Thefirst of our 2 patients’ lesions displayed rare junctionalnests but was predominantly subepithelial, while the othertumor in an older patient was completely subepithelial.These tumors are also referred to as clonal nevi becauseof the striking population of subepithelial epithelioid cellsthat coexists with conventional small nevus cells. Clonal-ity in this context simply refers to a shared morphologyamong the cells and not to any genetic identity. Invertedtype A nevus is another term that has the virtue of high-lighting the location of the large epithelioid cells in themiddle or lower dermis of the skin or the same levels ofthe conjunctival substantia propria, where small type Bnevus cells are expected to be present. An earlier con-junctival study published prior to the report of this con-dition in the skin probably described a related case.41 Asimilar inverted phenomenon can occasionally be ob-served in juvenile conjunctival nevi.40

These epithelioid tumors displayed the most alarm-ing cytologic features, consisting of an abundance of cy-toplasm containing a fine dispersion of cytoplasmic pig-ment granules and possessing bizarre nuclei that oftendisplayed binucleation or multinucleation. These cellswere juxtaposed to regular nevocytes and arranged ineither sheets or aggregated into small clusters separatedby a lightly fibrillar or early sclerotic eosinophilic stroma.Their nuclei displayed multiform shapes and were oftendominated by large vacuoles (actually herniations of cy-toplasm); mitotic figures were not discovered. The deepmargin was circumscribed and noninfiltrative. S100,MART-1, and HMB-45 immunostaining was positive inthe cytoplasm of the epithelioid cells, which also showedimmunonegativity for CD45, CD68, and lysozyme; thesmall collections of common subepithelial nevocytes inboth lesions were S-100– and MART-1–positive but HMB-45–negative. One lesion manifested a moderate subepi-thelial lymphocytic infiltrate. Ki-67 nuclear immunola-beling was totally negative, which reinforced theinterpretation of a benign lesion.

Benign and malignant balloon cell melanocytic neo-plasms are well recognized in the skin, are usually lightlypigmented to nonpigmented, and generally occur inyounger individuals. They constitute 2.0% of cutaneousnevi45 but are exceedingly rare in the conjunctiva. Shieldset al46 reported none among 410 consecutively excised neviat the Wills Eye Institute. At least 6 previous cases of con-junctival balloon cell nevi have been described.47-51 Ourballoon cell nevus was composed of equal parts conven-tional nevocytes and polygonal, clear, vacuolated, andmostly mononucleated but rarely binucleated cells that wereperiodic acid–Schiff–negative. The nuclei were orthochro-




matic, small, centrally located and exhibited minutenucleoli. The cells measured approximately 20 to 25 µmin diameter; because of their large size and clear cyto-plasm, they could be mistaken for xanthoma cells, adipo-cytes, or a metastasis from a renal cell carcinoma.

Cutaneous and conjunctival balloon cells are created bycollections of malformed vesicular premelanosomes51,52 andnot, as has been suggested by some, by the accumulationof cytoplasmic lipid. The conjunctival balloon cells in ourcase were S100- and MART-1–positive but HMB-45–negative; CD45, CD68, and lysozyme negativity ruled outa histiocytic lineage; and Ki-67 immunostaining was en-tirely negative, indicating no proliferative tendency. In 2earlier articles49,50 with immunohistochemical evalua-tions, the conjunctival balloon cells were S100-positive,though negative for HMB-45, lysozyme, and CD68, as ob-served in our case. Based on their MART-1 positivity andHMB-45 negativity, the balloon nevus cells parallel the stain-ing results of conventional subepithelial nevus cells moreclosely than those comprising the granular and epitheli-oid cell nevi described above, both of which were MART-1–and HMB-45–positive.

When encountered in the conjunctiva, a “blue” ne-vus is actually brown, unlike in the skin, where the Tyn-dall effect preferentially reflects the blue wavelengths.While rarer in the conjunctiva than in the skin,53 bluenevi are probably the second most common nevus typein the conjunctiva54-56 after common acquired nevome-lanocytic nevi, accounting for 1% of 410 excised nevi inthe previously mentioned series.46 The blue-brown ne-vus in this study is the first epibulbar example to be evalu-ated with immunohistochemical markers. It was lo-cated in the superficial sclera and deep substantia propria.The lesion was microscopically hypocellular (thereby dif-fering from extrascleral extensions of uveal melano-mas) due to a delicate enveloping stroma. It was consti-tuted by fusiform to stellate or dendritiform cells that werelightly pigmented or nonpigmented at its center and morepigmented at the superficial periphery; the pigment wasnever so coarsely clumped that it obscured the nucleusexcept in a few melanophages. The nuclei were delicateand occasionally displayed intranuclear cytoplasmic se-questrations and punctate nucleoli; no mitotic figures werefound. Immunostaining was positive for MART-1 andPNL2 (the latter is a newer antimelanocyte monoclonalantibody that shares many of the immunolabeling prop-erties of both MART-1 and HMB-45, including positiv-ity for common nevocytes and blue nevus cells57). TheKi-67 proliferation index was less then 1%. In contrast,episcleral extensions of uveal melanomas are typically hy-percellular and have a proliferation index of around 20%.58

When evaluating the relative merits of the various im-munohistochemical probes used in this investigation, oneshould remember that S100 immunoreactivity, whilehighly sensitive, unfortunately indiscriminately stains bothbenign and malignant melanocytic lesions as well as ahandful of other cell types.7,8,25,26 Previously reported im-munohistochemical studies of cutaneous melanocytic le-sions analyzed with MART-1 and HMB-45 have estab-lished that these probes also cannot decisively separatebenign from malignant tumors.8,16,18,20,27 HMB-45 has beenadvanced as a marker of cellular activity or proliferation

in the skin7,28 and conjunctiva16,18,20 and is expressed inmelanomas more often and more intensely than innevi.8,16,18,20,25-27 Common conjunctival nevus cells havebeen reported to be positive for HMB-45 in 8.5% to morethan 50% of lesions.16,17 Steuhl and associates18 and Shararaand colleagues21 have accurately underscored the restric-tion of this staining to the junctional region. MART-1 hasnot been previously evaluated and contrasted withHMB-45 in conjunctival lesions. Only 2 brief and incom-plete studies23,24 have described low counts of Ki-67 im-munoreactivity in conjunctival nevi, but did not clearlystate the immunostaining results with this marker in mela-nomas. In the skin, melanomas are frequently MART-1–and HMB-45–positive, with a high proliferation index(10%-70%) using Ki-67 antibodies7-15; nevi typically ex-hibit MART-1 positivity, HMB-45 negativity, and a pro-liferation index of 2% or less.9,10,12,24,43

The 4 atypical types of conjunctival nevi delineated inthis study yielded immunohistochemical findings support-ive of a benign diagnosis. Beyond the observations that thegranular and epithelioid cell nevi both stained strongly posi-tive for MART-1 and were aberrantly positive for HMB-45throughout all levels of the lesions, they more impor-tantly displayed negligible to no Ki-67 positivity among thecells in the substantia propria. We have inferred from ourdata that HMB-45 positivity among variant nevus cells rep-resents an abnormality in melanogenesis rather than amarker of cellular activation and therefore does not nec-essarily signal a menacing biologic potential. Currently,Ki-67 nuclear immunostaining for determining the prolif-eration index supersedes the diagnostic value of the dif-ferential expression of melanocyte-specific antigens. A Ki-67proliferation index of less than 2% is most compatible witha nevus, whereas a result greater than 10% strengthens thediagnosis of a melanoma.9,12,13,43

A final caveat should be offered: When counting Ki-67immunolabeled nuclei in melanocytic lesions, care mustbe taken not to enumerate nuclei belonging to lympho-cytes, histiocytes, vascular endothelial cells, or epithe-lial germinal cells participating in inclusion cysts. CD45positivity of aggregates or a light dispersion of S-100– orMART-1–negative cells for positive ascertainment of lym-phohistiocytic cells; CD31 and CD34 for vascular endo-thelial cells; and cytokeratin cocktail for squamous epi-thelium should defend against these pitfalls.

Submitted for Publication: February 19, 2009; final re-vision received April 8, 2009; accepted April 9, 2009.Correspondence: Frederick A. Jakobiec, MD, DSc, DavidG. Cogan Ophthalmic Pathology Laboratory, Massachu-setts Eye and Ear Infirmary, 243 Charles St, Room 321,Boston, MA 02114 ([email protected]).Financial Disclosure: None reported.

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