Immunohistochemical Studies of Conjunctival Nevi and Melanomas

CLINICAL SCIENCES Immunohistochemical Studies of Conjunctival Nevi and Melanomas

Frederick A. Jakobiec, MD, DSc; Pooja Bhat, MD; Kathryn A. Colby, MD, PhD

Objective: To evaluate the role of immunohistochemi- Ki-67 proliferation indices were 1.89% for the nevi and cal methods in the diagnosis of benign and malignant con- 17.3% for the melanomas. CD-45 can help to highlight junctival melanocytic proliferations. lymphocytes that immunostain with Ki-67. Melanomas in situ and atypical primary acquired melanoses had more Design: Retrospective immunohistopathologic study. than twice the Ki-67 proliferation counts of intraepithelial junctional nevocytes (PϽ.001) and more intense Methods: Paraffin-embedded tissue sections from 20 con- HMB-45 cytoplasmic staining than junctional zone junctival nevi and 15 invasive melanomas were immu- nevocytes. noreacted with antibodies against cellular antigens S-100 protein, MART-1, HMB-45, CD-45, and Ki-67 nuclear pro- Conclusions: S-100 and MART-1 were not useful in sepa- liferation protein. rating benign from malignant lesions. Results for nevus cells beneath the junctional zone were overwhelmingly Results: All nevi immunostained moderately to strongly negative for HMB-45 and Ki-67. Two nevi and all mela- for S-100 protein and MART-1. Results for HMB-45 were nomatous nodules were positive for HMB-45 (PϽ.001). negative in the middle and lower subepithelial portions A higher Ki-67 proliferation index convincingly sepa- of 18 of 20 lesions; it was usually only weakly positive rated melanomas from nevi (PϽ.001). Immunostain- within the superficial junctional zone. Only 1 mela- ing for HMB-45 and Ki-67 are valuable adjuncts to care- noma did not stain positively for S-100; MART-1 and ful histopathologic evaluation in assessing benign and HMB-45 were positive in all lesions at some level of in- malignant conjunctival melanocytic tumors. tensity. Ki-67 positivity was restricted to the junctional zone of nevi and was diffuse in melanomas. The mean Arch Ophthalmol. 2010;128(2):174-183

MMUNOHISTOCHEMICAL STAIN- METHODS ing of common cutaneous nevomelanocytic nevi and mela- From the regular and consultation files of the nomas with antibodies directed David G. Cogan Laboratory of Ophthalmic Pa- against S-100, HMB-45, and thology at the Massachusetts Eye and Ear In- MART-1 antigens have yielded valuable di- firmary, lesions that carried the diagnoses of con- I 1-13 agnostic data. These markers have been junctival nevi and malignant melanoma were used with less profit in the analysis of con- retrieved from January 1, 2004, to February 28, junctival lesions.14-19 The comparison of the 2009. Hematoxylin-eosin–stained slides were immunoprofiles of uveal and conjuncti- critically reexamined without knowledge of the val melanocytic lesions, or extrapolating clinical history. Twenty nevocytic lesions in 20 results from one to the other,16,17 can be patients, 15 nodules of melanoma in 13 pa- misleading because of their fundamen- tients, and 2 melanomas in situ in 2 patients were judged appropriate for inclusion in this study tally different biological behaviors and im- and further evaluation. The Massachusetts Eye munostaining patterns. In this article, we and Ear Infirmary Institutional Review Board describe systematic findings derived from considered this study exempt under local guide- the immunostaining of multiple melano- lines. Health Insurance Portability and Account- cyte-related antigens (rather than a single ability Act compliance was maintained, and care antigen) of the cells that make up un- of the patients in this study was in accordance equivocal conjunctival nevi and invasive with the Declaration of Helsinki and all federal melanomas. Our objective was to demon- and state laws. Author Affiliations: David D. strate which are diagnostically most use- Immunohistochemical staining using mono- Cogan Laboratory of ful, either singly or in combination. We clonal antibodies with the immunoperoxi- Ophthalmic Pathology dase method was performed with appropriate (Drs Jakobiec and Bhat) and additionally assessed the potential value controls in the Immunodiagnostic Labora- Cornea Service (Dr Colby), of the nuclear proliferation marker tory of the Department of Pathology at the Mas- 5,8-13 Department of Ophthalmology, Ki-67 for the identification of S- sachusetts General Hospital. Staining was per- Massachusetts Eye and Ear cycling cells in separating the 2 classes of formed retrospectively on all benign and Infirmary, Boston. conjunctival tumors. malignant lesions with antibodies for the iden-

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 tification of S-100 protein (melanocytes, Schwann cells, and licles also stained for Ki-67, but little confusion was encoun- Langerhans cells), MART-1/Melan-A antigens (premelano- tered because of their architecture. somal epitopes), HMB-45 antigen (premelanosomal gp100 com- Ki-67 positivity was compared among the intraepithelial junc- plex), CD-45 (lymphopoietic and hematopoietic cells, includ- tional nests of nevi, the overlying or lateral (radial) primary ac- ing monocytes/macrophages), and Ki-67 nuclear proliferation quired melanoses within the epithelium associated with nod- protein. The characteristics and sources of the antibodies are ules of melanoma, and the melanomas in situ in 4 high-power as follows: S-100, mouse monoclonal antibody (IgG2a, predi- (ϫ400) microscopic fields. The nuclear outlines of positively luted; Ventana Medical Systems, Tucson, Arizona); MART-1, stained basal germinal epithelial cells could not be reliably ex- mouse monoclonal antibody (IgG2b, 1:60 predilution; Signet cluded from those of the melanocytes. The results were there- Laboratories Inc, Dedham, Massachusetts); HMB-45, mouse fore scored as the mean of the total number of immunoreac- monoclonal antibody (IgG1, 1:100 dilution, Dako Corpora- tive cells counted in 4 high-power fields, which commingled tion, Carpinteria, California); CD-45, mouse monoclonal an- the nuclei of melanocytes and a subpopulation of basal germi- tibody (IgG1, prediluted; Ventana Medical Systems); and Ki- nal epithelial cells. The latter were presumed to be similar in 67, rabbit monoclonal antibody (IgG, prediluted, Ventana number from case to case. Furthermore, the discernment of the Medical Systems). cellular borders between melanocytes and keratinocytes was The staining was done on BenchMark XT automated tissue difficult in the immunolabeled and hematoxylin-eosin coun- staining systems (Ventana Medical Systems) using validated pro- terstained slides, thereby preventing a meaningful denomina- tocols. Endogenous peroxidase activity was blocked by hydro- tor from being determined. gen peroxide before antibody incubation. A combination of Clinical information, including location, ocular histories, and EDTA and boric acid in TRIS buffer (CC1 reagent; Ventana Medi- clinical photographs, when available, were reviewed and ana- cal Systems) was applied to the tissue sections for antigen re- lyzed after the histomorphologic and immunohistochemical find- trieval as needed, and the process was carried out before pri- ings were determined. The immunohistochemical data were com- mary antibody incubations. The tissue sections were washed pared with those previously published in the ophthalmic and and incubated with the primary antibodies, followed by incu- dermatologic literatures by conducting a PubMed search based bation with UltraView HRP-conjugated multimer antibody re- on the following keywords: nevus, melanoma, skin, conjunctiva, agent (Ventana Medical Systems). Antigen detection was per- immunohistochemistry, S-100, MART-1, HMB-45, and Ki-67. formed using UltraView and diaminobenzidine as the chromogen The bulk of the findings are summarized in Table 1 and (Ventana Medical Systems). Tissues were counterstained with Table 2. For comparative purposes, Ki-67 immunolabeling was hematoxylin. evaluated separately for the intraepithelial melanocytes in nevi, The immunohistochemical staining intensity was graded atypical PAM, and melanomas in situ, and the results are pre- as negative (–), weak/mild (ϩ), moderate (ϩϩ), or strong sented in Table 3. For the nevi, Ki-67–positive nevocytes in the (ϩϩϩ). If a large majority of cells stained, then the pattern most superficial stromal component of the junctional zone of the was regarded as diffuse; staining of scattered single cells or substantia propria (75 µm in depth) were also counted and are isolated small cell clusters was considered focal. In the com- reported in the “Nevi” subsection of the “Results” section. pound nevi (as distinct from purely junctional nevi or entirely Analyses using the Fischer exact test for HMB-45 positive stain- subepithelial nevi), nuclei exhibiting Ki-67 nuclear protein ing and the Mann-Whitney test for the Ki-67–positive staining positivity were located predominantly in the junctional were performed to determine the statistical significance of dif- region. This zone was subdivided into 2 parts: intraepithelial ferential immunostaining between the nevi with a subepithelial nevocytic nests and an immediately subepithelial population junctional component and the nodules of melanoma. With re- of nevocytes located in the most superficial conjunctival sub- spect to the HMB-45 analysis, any degree of positivity from weak stantia propria within a vertical depth of 75 µm from the epi- to strong, whether diffuse or focal, was considered positive; sta- thelial basement membrane. The latter region in nevi was tistical correlations of the varying degrees of intensity were not richest in Ki-67–positive cells. Fields were selected for the cell attempted. Statistical evaluation of S-100 and MART-1 immuno- counts that were as free as possible of lymphocytes. A 10ϫ10 staining was not performed because of the overwhelming preva- graticule at ϫ400 magnification was used to assist in accurate lence of positive results in the benign and malignant lesions. subepithelial cell counts in 4 different high-power fields, each encompassing a 100-µm horizontal span and a depth of 75 RESULTS µm. Two separate counts of Ki-67–positive cells are provided in the “Nevi” and “Intraepithelial Melanocytic Proliferations” subsections of the “Results” section for the 2 different compo- NEVI nents (intraepithelial and immediately subepithelial) of the junctional zone. For the invasive nodules of melanoma, Ki-67 nuclear protein staining was reported as a proliferation index Twenty discrete nevi were studied in 20 patients. Ten nevi (ie, the percentage of cells with positive nuclear staining out were located on the epibulbar surface away from the lim- of the total number of cells counted in four ϫ400 microscopic bus, 4 at the limbus, 2 in the caruncle, and 1 in the plica. fields), again using a 10ϫ10 graticule. The melanomas in situ Three were at the eyelid margin and involved the adja- were examples of the most severe intraepithelial atypical cent palpebral conjunctiva. Clinically, 16 nevi were pig- melanocytic proliferations diagnosed in 2 patients, that is, mented, and, in the remaining 4, no pigment could be markedly atypical primary acquired melanosis (PAM). visualized. There were 10 males and 10 females with a Because of the frequent and often prominent presence of a mean age of 40.1 years (median, 35 years; range, 6-76 lymphocytic infiltrate in the nevi and melanomas, care had to years) (Table 1). be taken to distinguish the deposition of the Ki-67 immuno- Twelve nevi possessed variably prominent intraepi- product in the nuclei of tumor cells from that in lymphocytes in S-phase, which was helped by CD-45 immunolabeling to re- thelial junctional activity (Table 1). Two were purely junc- veal the extent of the lymphocytic infiltrate. The immuno- tional and composed of conspicuous large intraepithe- stained nuclei were largest in the melanoma cells, smaller and lial nests of polygonal nevocytes (ie, theques) that were often ovoid in the junctional nevus cells, and smallest and clearly demarcated from the surrounding keratinocytes rounder in the lymphocytes. Large, germinal center cells of fol- (Figure 1A). A variable band of subepithelial lympho-

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Immunohistochemical Clinical Features Light Microscopy Analysis

Nevus Patient No./ Giant Sex/Age, y Location Lesion Diagnosis Cellsa Pigmenta Cystsa Lymphocytes S-100b MART-1b HMB-45b 1/M/10 Bulbar OD Pure junctional Neg 2ϩ Neg 1ϩ 2ϩ 3ϩ 1ϩ 2/M/6 Limbal OS Pure junctional Neg Neg 1ϩ 3ϩ 2ϩ 3ϩ 1ϩ Predominantly junctional, 2ϩ 3ϩ 1ϩ 3/F/9 Bulbar OD Subepithelial Neg 1ϩ 1ϩ 1ϩ 2ϩ 3ϩ Neg Compound-junctional 2ϩ 3ϩ 1ϩ (f ) 4/M/16 Bulbar OS Subepithelial Neg 1ϩ 3ϩ 3ϩ 2ϩ 3ϩ Neg Compound-junctional 3ϩ 3ϩ 3ϩ 5/M/15 Bulbar OS Subepithelial 1ϩ 3ϩ Neg Neg 3ϩ 3ϩ Neg Compound-junctional 3ϩ 3ϩ 1ϩ (f ) 6/F/32 Bulbar OS Subepithelial 1ϩ 3ϩ Neg 3ϩ 3ϩ 3ϩ 1ϩ (f ) Junctional 2ϩ 3ϩ 1ϩ (f ) 7/F/60 Plica OD Predominantly subepithelial 1ϩ 2ϩ 3ϩ 2ϩ 2ϩ 3ϩ Neg Junctional 3ϩ 3ϩ 1ϩ (f ) 8/M/36 Limbal OS Predominantly subepithelial Neg 1ϩ 2ϩ Neg 3ϩ 3ϩ Neg Junctional 2ϩ 3ϩ 0-1ϩ 9/M/17 Limbal OS Predominantly subepithelial Neg Neg Neg Neg 2ϩ 3ϩ Neg Junctional 3ϩ 3ϩ Neg 10/M/32 Bulbar OD Predominantly subepithelial 1ϩ 1ϩ 1ϩ 1ϩ 3ϩ 3ϩ Neg Junctional 2ϩ 3ϩ 1ϩ 11/F/34 Bulbar OD Predominantly subepithelial 1ϩ 1ϩ 2ϩ Neg 2ϩ 3ϩ 1ϩ (f ) Junctional 1ϩ 3ϩ Neg 12/M/21 Bulbar OS Predominantly subepithelial Neg 2ϩ Neg Neg 1ϩ 3ϩ Neg 13/M/52 Bulbar OD Pure subepithelial Neg 2ϩ Neg Neg 2ϩ 3ϩ Neg 14/F/76 Limbal OS Pure subepithelial Neg 2ϩ 3ϩ 1ϩ 2ϩ 3ϩ Neg 15/F/70 Caruncle OS Pure subepithelial 1ϩ 1ϩ 2ϩ Neg 2ϩ 3ϩ Neg 16/F/54 Bulbar OD Pure subepithelial Neg 1ϩ 2ϩ Neg 1ϩ 3ϩ Neg 17/M/54 Caruncle OD Pure subepithelial Neg 1ϩ 3ϩ Neg 3ϩ 3ϩ Neg 18/F/72 Mucocutaneous junctional OD Pure subepithelial 2ϩ 1ϩ Neg Neg 3ϩ 3ϩ Neg 19/F/68 Mucocutaneous junctional OD Pure subepithelial 1ϩ Neg Neg Neg 3ϩ 3ϩ Neg 20/F/68 Mucocutaneous junctional OD Pure subepithelial Neg Neg Neg Neg 3ϩ 3ϩ Neg

Abbreviation: Neg, not present or negligible. a For light microscopic analysis, 1ϩ indicates present; 2ϩ, mild to moderate; 3ϩ, conspicuous. b For immunohistochemical analysis, 1ϩ indicates light; 2ϩ moderate; 3ϩ, strong; generally diffuse unless focal (f ) is specified.

cytic inflammation was frequently present. One lesion dividual cell invasion of the epithelium (ie, pagetoid was compound with a predominantly junctional com- spread) nor a prominent lateral or radial intraepithelial ponent and only incipient colonization of the superfi- extension beyond the subepithelial component. Multi- cial substantia propria; 3 were balanced compound nevi nucleated nevus giant cells were present subepithelially with an active intraepithelial and a well-developed sub- in 8 lesions; there was no topographic predilection of the epithelial component (Figure 1B); and 6 were predomi- lesions with this feature. Other histomorphologic char- nantly subepithelial with scattered, widely separated over- acteristics are described in Table 1. lying junctional nests that did not extend laterally beyond The immunohistochemical staining of the lesions was the subepithelial body of the lesion. Eight lesions were monotonously repetitive and consistent (Table 1). We purely subepithelial with no evidence of persisting in- use the term junctional zone to include intraepithelial and traepithelial nevomelanocytic nesting and were com- superficial stromal nevocytic components, the latter con- posed of lymphocytoid cells with occasional intra- stituted by the 75 µm of stroma immediately beneath the nuclear vacuoles and often organized into large nests epithelium into which the intraepithelial nevocytes first (Figure 1C). The superficial cells were larger and more drop off (ie, abtropfung). All melanocytic cells were im- polygonal than deeper ones. munolabeled for S-100 and MART-1 (Figure 1D, bottom- Among the 16 microscopically pigmented nevi, 10 had left inset, and F); staining was diffuse and not focal or a junctional component and the remaining nevi were sectoral in the subepithelial cells of the nevi. The mul- purely subepithelial (Table 1). Pigmented cells were con- tinucleated nevus cells stained in the same fashion as the centrated in the superficial region of the subepithelial com- mononucleated cells. In 10 of 12 lesions with a junc- ponent; maturation into smaller cells usually occurred tional component, HMB-45 was typically lightly and spot- toward the base (Figure 1B). Among the 10 compound tily positive in some of the cells in the junctional nests nevi (predominantly junctional, balanced, and predomi- and immediately subepithelially (Figure 1F); in 1 case, nantly subepithelial types), there was no tendency for in- it was only faintly positive in the cells of the middle and

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Clinical Features Light Microscopy Immunohistochemical Analysis

Patient Ki-67 No./Sex/ Clinical Nodule/ Proliferation Age, y Location PAM a M-PAMb Cell Typec Pigment Cysts Lymphocytes S-100d MART-1d HMB-45d Index, %e 1/M/74 Bulbar OS Present Melanoma in situ Epithelioid 1ϩ Neg 2ϩ 2ϩ 3ϩ 3ϩ NA 2/F/69 Limbal OS Present Melanoma in situ Mixed 1ϩ (f ) Neg 1ϩ (f ) 1ϩ 3ϩ 3ϩ NA Nodule Epithelioid 2ϩ Neg 3ϩ 3ϩ 3ϩ 3ϩ 21.8 3/F/42 Limbal OS Present PAMϩ 3ϩ 3ϩ 3ϩ Nodule Epithelioid 1ϩ (f ) 3ϩ 3ϩ 3ϩ 3ϩ 3ϩ 7.1 4/M/59 Forniceal OD Present PAMϩ 3ϩ 3ϩ 3ϩ Nodule Epithelioid Neg 1ϩ 1ϩ (f ) Neg 1ϩ 3ϩ 14.8 5/F/64 Bulbar OS Absentf PAMϩf 1ϩ 1ϩ 3ϩ Nodule Mixed 1ϩ (f ) Neg 3ϩ 2ϩ 3ϩ 3ϩ 14.2 6/M/53 Bulbar OD Present PAMϩ 2ϩ 3ϩ 3ϩ Nodule Epithelioid 3ϩ/Neg Neg 3ϩ 2-3ϩ (f ) 3ϩ 2ϩ (f ) 15.1 7/M/40 Limbal OS Present PAMϩ 2-3ϩ (f ) 3ϩ 2ϩ (f ) Nodule Mixed 2ϩ Neg 3ϩ 3ϩ 3ϩ 3ϩ 17.0 8/M/85 Limbal OS Present PAMϩ 3ϩ 3ϩ 3ϩ Nodule Mixed Neg 3ϩ 3ϩ 3ϩ 3ϩ 2ϩ 21.8 9/M/24 Bulbar OD Present PAMϩ 3ϩ 3ϩ 2ϩ Nodule Spindle 1ϩ (f ) Neg 1ϩ 3ϩ 2ϩ 1ϩ 11.2 10/F/82 Forniceal OS Present PAMϩ 3ϩ 3ϩ 1ϩ Nodule Mixed 1ϩ (f ) Neg 3ϩ 2ϩ 3ϩ 3ϩ 16.6 Limbal OS Present PAMϩ 2ϩ 3ϩ 3ϩ 16.8 11/M/77 Nodule Mixed 1ϩ (f ) Neg 3ϩ 2ϩ 3ϩ 3ϩ Bulbar OS Present PAM– Nodule Epithelioid 1ϩ (f ) Neg 3ϩ 3ϩ 3ϩ 1ϩ (f ) 20.4 Bulbar OS Present PAM– 12/M/28 Nodule Epithelioid Neg Neg 3ϩ 3ϩ 3ϩ 1ϩ 20.3 Lateral Canthal OS Present PAM– Nodule Epithelioid 2ϩ Neg 1ϩ 2ϩ(f ) 3ϩ 3ϩ 22.5 13/F/96 Tarsal OD Absentg PAM– Nodule Epithelioid Neg Neg 3ϩ 2ϩ 3ϩ 3ϩ 21.0 14/M/59 Forniceal OD Present PAM– Nodule Spindle Neg Neg 3ϩ 1ϩ(f ) 3ϩ 2ϩ 18.7 15/F/73 Limbal OS Absent PAM–

Abbreviations: NA, not applicable in the 2 in situ melanoma lesions where mean number of Ki-67–positive cells was calculated and reported in Table 3; Neg, not present or negligible; PAM, primary acquired melanosis. a Clinical PAM was defined as apparent clinically flat golden brown patches of conjunctival pigmentation. b M-PAM was defined as microscopic PAM associated with nodules of melanomas. c Epithelioid indicates pure population; mixed, spindle and epithelioid; and spindle, pure spindle. d For immunohistochemical analysis, 1ϩ indicates light; 2ϩ, moderate; 3ϩ, strong; generally diffuse unless designated as focal (f ) with scattered single cells or isolated cell clusters. e Percentage of total cells that were immunolabeled. f PAM sine pigmento diagnosed only microscopically. g PAM was present for several years and was not apparent at the time of excision of last nodule due to earlier cryotherapy.

deeper layers of the substantia propria. In 2 nevi, nei- identified in the deeper stromal region beneath the su- ther the junctional nor subepithelial regions stained for perficial stromal junctional zone, as defined herein, ex- HMB-45. Intraepithelial dendritic melanocytes, when cept for reactivity detected in dispersed lymphocytes or present, were variably decorated with all 3 markers. Ki-67 small lymphoid aggregates. Widely separated epithelial positivity was restricted to nevus cells scattered within basal germinal cells immunoreacted with Ki-67, includ- the epithelial-stromal junctional zone (Figure 1F, in- ing those within epithelial inclusion cysts. set). In 9 of the 10 compound nevi, the total number of cells with Ki-67 nuclear immunostaining in the most superficial zone of the substantia propria (evaluated in each MELANOMAS case for a distance of 100 µm horizontally and a vertical depth of 75 µm beneath the epithelium) was 30 out of a total of 1583 cells counted in 4 high-power fields (mean, Thirteen patients had 15 nodules of conjunctival mela- 3.33 cells; median, 3 cells; range, 0-7 cells), or an over- noma (2 displayed 2 separate, nonoverlapping nod- all mean proliferation index of 1.89%. One lesion did not ules). There were 2 additional patients who were diag- allow an accurate subepithelial junctional cell count be- nosed with melanoma in situ with almost complete cause of the paucity of cells in the superficial stroma (ie, melanocytic replacement of conjunctival epithelium, the very early colonization). Ki-67 immunostaining was not most severe degree of preinvasive, atypical PAM, analo-

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 ing. Nine nodules of melanoma had associated micro- Table 3. Intraepithelial Ki-67–Positive Cells in Junctional scopic PAM; in the remaining 6 nodules, PAM was not and Compound Nevi, Melanomas In Situ, and Primary detected in the excised specimens but was noted in the Acquired Melanoses Associated With Melanoma clinical evaluation in all but 1 case (patient 15; Table 2). The results from immunolabeling of the cells in the Mean No. of No. of Ki-67–Positive nodules and of those responsible for the atypical intrae- Patients Diagnosis Cellsa pithelial melanoses were the same for each lesion, in- 2 Pure junctional nevib 7.8 cluding the 2 completely spindle-cell lesions. Fourteen 1 Predominantly junctional nevusc 9.0 of 15 invasive nodules and the 2 melanomas in situ stained 3 Balanced compound nevid 8.3 for S-100 diffusely or focally; results for 1 were nega- 6 Predominantly subepithelial nevie 6.0 tive. All 15 nodules and the 2 melanomas in situ stained 2 Melanomas in situf 34.5 for MART-1 with variable intensities in a diffuse pat- 9 PAM with melanoma nodules 19.7 tern. HMB-45 diffusely immunostained the cytoplasm of the cells of 13 of the 15 nodules and was marked in any Abbreviation: PAM, primary acquired melanosis. a Mean absolute counts of positively stained nuclei of intraepithelial cells in associated PAM (Figure 2C and D) and typically had a 4 high-power fields. granular character; it was focal in 2 nodules (Figure 2C, b Entirely intraepithelial nevocytic nests. inset). Staining was diffuse and strongly positive among c Small subepithelial component. d Equally prominent junctional and subepithelial components. the cells constituting the melanomas in situ (Figure 2D, e Small, persisting junctional component. inset bottom right). f Replacement of most of conjunctival epithelium by severely atypical Ki-67 staining was distributed throughout the nod- melanocytes without nodule formation. ules, sometimes in a somewhat uneven pattern (Figure 2E); no differences were noted among the le- gous to squamous cell carcinoma in situ. These 2 pa- sions composed of pure epithelioid cells, mixed epithe- tients had not had a previous biopsy nor a nodule of mela- lioid-spindle cells, or spindle cells (Figure 2F). The noma resected earlier. spindle-cell lesions had a prominent lymphocytic infil- In the invasive melanoma group, 8 patients were men trate as demonstrated by CD-45 immunostaining and 5 were women. The mean age at presentation in this (Figure 2F, inset). The Ki-67–positive cells counted in group was 60 years (median, 59 years; range, 24-96 years). 4 high-power (ϫ400) fields for each nodule (Table 2) Of the 15 melanomatous nodules, 5 were found at the ranged from 24.5 to 77.2 cells among all lesions (mean, limbus, 6 on the epibulbar surface, 2 in the fornices, 1 at 59.3 cells; median, 58.3 out of a mean total cell count of the lateral canthus, and 1 in the inferior tarsal conjunc- 343 cells). The overall mean Ki-67 proliferation index tiva. Ten nodules displayed pigmentation clinically and for the nodular melanomas was 17.3% (median, 17%; 5 did not; in 1 patient (patient 7; Table 2), the base of range, 7.1%-22.5%) (Table 2). the nodule was diffusely and intensely pigmented, but a Comparison of the HMB-45–positive immunostain- secondary outgrowth lacked pigment. The 2 patients with ing between the proportion of melanoma nodules (15 of melanoma in situ were a 74-year-old man and a 69-year- 15) vs the proportion of nevi (2 of 18) using Fisher ex- old woman; 1 lesion at the limbus was pigmented and act test revealed a P value of Ͻ.001. Comparison of the the other located elsewhere on the epibulbar surface was Ki-67–positive immunostaining between the percent- nonpigmented (patients 1 and 2; Table 2). age of cells in melanoma nodules vs the percentage of Twelve patients’ lesions were associated with clini- cells in the junctional zone of nevi using the Mann- cally apparent, flat PAM extending away from the main Whitney test revealed a P value of Ͻ.001. The differ- focus of disease. In 2 patients, there was no PAM seen ences in both categories of staining therefore proved to clinically or histopathologically (patients 13 and 15; be statistically significant. Table 2); a third patient with an amelanotic melanoma and no clinically detectable PAM was proven histopatho- INTRAEPITHELIAL MELANOCYTIC logically to have PAM sine pigmento (patient 5; Table 2). PROLIFERATIONS In patient 13, PAM had been clinically present for many years but had been successfully extirpated before the ap- Ki-67 immunoreactivity within the junctional nests pearance of the last nodule included in this study. (Figure 1F, inset) of 2 purely junctional, 1 predomi- Eight nodules were composed entirely of epithelioid nantly junctional, 3 balanced compound, and 6 predomi- cells (Figure 2A), 5 of a mixture of epithelioid and nantly subepithelial nevi (a total of 12 lesions) was com- spindle cells (Figure 2A, inset) and 2 completely of spindle pared with that of the intraepithelial melanocytic cells. Of the 2 melanomas in situ (Figure 2B), 1 was com- proliferations (Figure 2E, inset) diagnosed either as mela- posed of dyscohesive epithelioid cells and the other of nomas in situ without nodules (2 cases) or PAM with mixed epithelioid-spindle cells, both of which replaced atypia (a total of 11 lesions) accompanying 9 nodules of almost the total thickness of the epithelium (patients 1 melanoma (Table 3). The mean absolute Ki-67–positive and 2; Table 2). A lymphocytic infiltrate was univer- cell counts of the intraepithelial junctional nevus cells sally present within the nodules and was often conspicu- in the 12 nevi with such components ranged from 5.75 ous. The spindle-cell lesions displayed some desmoplas- to 9 (mean, 7.5) in 4 high-power fields. The mean posi- tic features, relative hypocellularity and a collagenous tive cell counts obtained in the 2 melanomas in situ were matrix, and an especially pronounced lymphocytic in- 25 and 44. In the atypical PAM components accompa- flammation, well demonstrated by CD-45 immunostain- nying the 9 nodules of melanoma, the mean positive cell

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B E

C F

Figure 1. Conjunctival nevi. A, Numerous, well-defined intraepithelial nevocytic nests (arrows) in a completely junctional nevus with a surface covering of keratinocytes. Note the subepithelial band of chronic inflammation (arrowheads) (hematoxylin-eosin, original magnification ϫ200). B, Compound nevus with junctional nests of cohesive nevocytes present in clear-cut intraepithelial cavities. There are subepithelial nests and sheets of stromal nevus cells, some of which are pigmented superficially toward the upper left (hematoxylin-eosin, original magnification ϫ200). C, Nests and diffuse collections of subepithelial nevus cells. Note the entrapped epithelial inclusion cysts and the total absence of intraepithelial nevocytic junctional nests (hematoxylin-eosin, original magnification ϫ100). D, Intraepithelial nests of nevocytes in a pure junctional nevus are uniformly and diffusely immunolabeled for S-100 protein. Bottom-left inset, MART-1 intensely stains nevocytes; bottom-right inset, HMB-45 also immunostains nevocytes in a lighter, more granular fashion (immunoperoxidase reaction, original magnification ϫ200 for panel D and inset). E, Compound nevus displays positive staining for S-100 in both junctional and subepithelial components. Nonstaining cells are inflammatory. Inset, positive S-100 immunostaining of a completely subepithelial nevus with uninvolved overlying epithelium (original magnification ϫ200). F, HMB-45–positive immunolabeling of a compound nevus is confined to the intraepithelial junctional nests and only the most superficial stromal nevus cells (arrows). Nonstaining nevus cells and inflammatory cells are present in the bottom half of the photomicrograph. The arrowhead identifies melanin-bearing cells, probably macrophages. The inset displays moderate Ki-67 positivity of dark-staining nuclei limited to the intraepithelial nevocytes within junctional nests; staining is absent from subepithelial nevocytic clusters (original magnification x200).

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 count was 19.7 (median, 20; range, 13-27). Compari- invasive melanomatous nodules were not included in that son of the positive cell counts of Ki-67 immunostaining study. The only other class of conjunctival lesions that between the nevi (n=12) and the combined group of atypi- has been productively assessed immunohistochemical PAM and melanomas in situ (n=11) was performed cally with the Ki-67 marker is the spectrum of squa- using the Mann-Whitney test, and the difference was sta- mous cell dysplasias and carcinomas.34 tistically significant at PϽ.001. Our study establishes that reproducible immunohistochemical staining differences exist between common conjunctival nevi and invasive melanomas with select COMMENT markers. With respect to the 18 of 20 nevi in this series with a subepithelial stromal component, the nevocytes Both benign and malignant melanocytic proliferations of of this region were positive for S-100 and MART-1, as the skin20-22 and conjunctiva23-26 are diagnostically chal- were the nevocytes in the junctional nests, when present. lenging because of their protean histomorphologic ap- There was, however, a characteristic immunopattern ex- pearances; immunohistochemical markers can offer ad- hibited by HMB-45. Patchy immunostaining was gener- ditional and supportive diagnostic information.16-20,27,28 ally restricted to the junctional zone, composed of in- With the application of monoclonal antibodies against traepithelial nevocytic nests and nevocytes in the the nuclear proliferation protein Ki-67, which is more immediately subepithelial substantia propria to a depth sensitive than counting mitotic figures for determining of 75 µm, in 10 of 12 lesions with this feature (2 lesions cellular proliferative activity (ie, identifying nuclei in late were entirely negative); the intraepithelial dendritic me- G1, S, M, and G2 phases of the cell cycle), the ability to lanocytes were also lightly stained. The nonjunctional ne- diagnose cutaneous benign and malignant disorders has vocytes in the deeper substantia propria were negative been further improved.5-13 This probe, however, has yet in all but 2 cases, in which only light staining was de- to be systematically used in studies of common conjunc- tected in small foci; these lesions were nevertheless cat- tival melanocytic lesions. In this article, our aim was to egorized as HMB-45 positive according to the design of resolve inconclusive results from earlier studies in an on- this study. Our results also included findings derived from going search for a diagnostically serviceable immuno- 2 purely junctional nevi. Both arose in boys aged 6 and histochemical approach to conjunctival melanocytic le- 10 years and had identical mild diffuse intraepithelial sions.29 One facet of our study that distinguishes it from HMB-45–positive staining. A gradient or zonal pattern preceding ones is that we did not try to correlate inten- for HMB-45 has been well described in common cuta- sities of immunohistochemical staining with different di- neous junctional and compound nevi1,2 and in Spitz nevi.3 agnostic categories, but rather combined all degrees of It has been commented upon by some ophthalmic in- positive staining together as a single indicium. vestigators, but less attention than it deserves has been Standard, commercially available antibodies for S-100 paid to this aspect of conjunctival nevi in differential immunolabeling stain benign and malignant melano- diagnosis.15,16,19 cytes, and our results are in keeping with this finding. On In our study, Ki-67 immunolabelling was consis- the other hand, there are at least 20 subtypes of S-100 pro- tently negative among the middle and lower subepithe- tein. S-100 A1 has shown some preliminary promise in dif- lial nevus cells, but was mildly positive within the in- ferentiating benign and malignant conjunctival melano- traepithelial junctional nests and spottily positive among cytes30 but was not used in this study. Other prior the most superficial stromal nevocytes (ie, to a depth of conjunctival immunohistochemical investigations with dis- 75 µm just beneath the epithelium). A mean prolifera- crepant results have explored the diagnostic utility of the tion index of 1.89% was discovered in this subepithelial HMB-45 and MART-1 genes coding for melanosome- zone, which is comparable to a 1.7% to 2.0% finding in related cytoplasmic antigens.14-21,30 A clear-cut set of re- cutaneous nevi.8,11 In the skin, dysplastic nevi (a class of sults that can be used in light microscopic diagnosis has lesion not included in this study because of their rarity not yet been successfully elucidated. To date, immunohis- in the conjunctiva) display an intermediate prolifera- tochemical techniques are not routinely used in the analy- tion index of 2.9%,11 which is still much closer to our sis and diagnosis of conjunctival melanocytic lesions.29 nevi than the melanomas. The mean Ki-67 proliferation Ki-67 immunolabeling has been attempted once be- indices of 1.89% for the nevi and 17.3% for the melano- fore with 69 conjunctival nevi and 2 melanomas, but only mas in the present study were statistically significant parsimonious descriptions of the results were pro- (PϽ.001). The mean Ki-67 index was 47% in an analo- vided.31 That particular study concluded that benign nevi gous study of cutaneous melanomas,11 which, at the time expressed nuclear proliferation marker Ki-67 at low lev- of clinical discovery, are typically larger and more ad- els and did not supply either quantitative or semiquan- vanced tumors than conjunctival melanomas. In a meta- titative information nor venture any comments regard- analysis reviewing many publications, a Ki-67 thresh- ing the 2 melanomas. A case report of a conjunctival Spitz old index as low as 10% was considered sufficient to nevus demonstrated low Ki-67 immunoreactivity con- diagnose a cutaneous melanoma.8 fined to the junctional zone but in none of the cells con- Of the 15 melanomatous nodules in 13 patients stituting the middle and lower levels of the lesion in the described herein, S-100 immunostaining was moderately substantia propria,32 which is comparable to what has been to strongly positive in 13 cases, mildly positive in 1, and found in cutaneous Spitz and dysplastic nevi.7,8,10,11 Ki-67 negative in 1. In the lesion negative for S-100, weak has been profitably applied in a single study33 to the evalu- MART-1 positivity and strong HMB-45 positivity were ation of cases of PAM, with and without atypia. Nevi and identified (patient 5; Table 2). MART-1 was vividly

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B E

C F

Figure 2. Conjunctival melanomas. A, Subepithelial nodule of melanoma composed of epithelioid cells and interspersed lymphocytes. Inset displays a mixed spindle-epithelioid cell melanoma (hematoxylin-eosin, original magnification ϫ200). B, One of the 2 melanomas in situ in this study manifests a nonnested continuous sheet of atypical medium-sized dyscohesive epithelioid melanocytes residing above the epithelial basement membrane. There is only a thin umbrella of covering squamous epithelial cells (hematoxylin-eosin, original magnification ϫ200). C, Uniform and intense HMB-45 immunoreactivity of large nests of mixed epithelioid and spindle melanoma cells. Note the prominent intraepithelial, nonnesting component of atypical melanocytes that is also strongly HMB-45 positive (Immunoperoxidase reaction, original magnification ϫ100). The inset (original magnification ϫ200) demonstrates an unequivocally positive but more erratic and modest cytoplasmic staining pattern in another case of pure epithelioid cell melanoma. D, The superficial epithelioid and deeper spindle-cell (arrows) populations of this nodule are strongly HMB-45 positive (original magnification ϫ100). The inset, bottom left, shows positive staining in a pure spindle-cell melanoma. The inset, bottom right, highlights HMB-45 cytoplasmic staining in a melanoma in situ that is far more intense than that observed in a junctional nevus (compare with Figure 1D, bottom-right inset) (both insets, original magnification ϫ200). A similar degree of staining is exhibited in primary acquired melanosis (PAM) with atypia. E, Ki-67 prominently stains the nuclei of the intraepithelial atypical melanocytes and those of many of the invasive melanoma cells. Inset shows dramatically dense Ki-67 nuclear positivity of PAM with atypia associated with a nodule of melanoma (original magnification ϫ200 for panel E and inset). F, Melanomatous spindle cells are admixed with a prominent inflammatory cell infiltrate. Ki-67 positively staining nuclei of the melanoma cells are larger than the smaller, round positive nuclei of the lymphocytes (original magnification ϫ200). The inset (original magnification ϫ400) reveals the CD-45–positive cell membrane staining of lymphocytes with their smaller nuclei among the tumor spindle cells.

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 positive in all but 1 lesion (also in patient 5). These data ings could also enhance the prediction of which cases of are in keeping with those reported for cutaneous and PAM with atypia have the potential to evolve into inva- nonconjunctival mucosal melanomas.1,2 Whether of sive melanoma. pure epithelioid or mixed epithelioid-spindle cell com- A cautionary word must be added regarding the grad- position, all of the melanomatous nodules in this series ing of Ki-67 nuclear positivity, which readily includes lym- were HMB-45 positive, most often diffusely but rarely phocytes. The presence of inflammatory cells is often focally. If not helpful in differential diagnosis, as a sec- prominent in childhood nevi owing to their mucosal mi- ondary practical consideration, MART-1 staining offers lieu and was detected frequently in the nevi and mela- the clearest and most reliable definition in conjunctival nomas in this series24,45,46 (Tables 2 and 3). Ki-67– specimens of normal dendritic melanocytes and of positive Langerhans cells, germinal basal cells in the intraepithelial melanocytic proliferations, as well as of surface epithelium and in epithelial inclusion cysts, con- the cells comprising subepithelial portions of benign and nective tissue histiocytes/macrophages, and vascular en- malignant lesions. It reveals the presence of melanocytes dothelial cells in the S-phase of the cell cycle must also to an extent that cannot be fully appreciated in be considered and excluded. Unfortunately, the immu- hematoxylin-eosin–stained sections. nodiagnostic laboratory at the Massachusetts General Hos- HMB-45 positivity was uniformly detected through- pital does not perform double immunohistochemical out all levels of the melanomatous lesions without a gra- staining. The outline of the nuclear immunoreactant in dient diminishing from superficial to deeper layers. Based lymphocytes is usually smaller and rounder than that of on our results, HMB-45 negativity in the subepithelial, melanoma cells but sometimes approximates that of ne- nonjunctional component of a conjunctival melano- vus cells. CD-45 staining of a sequential tissue section cytic lesion (observed in 18 of our 20 nevi) tends to in- in a high Ki-67–positive zone can sometimes assist in re- dicate a benign proliferation, whereas HMB-45 positiv- solving this issue by immunostaining lymphopoietic and ity throughout the entire extent of the subepithelial lesion hematopoietic cells (which are also S-100 and MART-1 should heighten suspicion of a malignant proliferation negative). (PϽ.001). Two extremely rare exceptions to this rule are In summary, although careful light microscopic analy- granular and clonal (ie, inverted) nevi.35-37 sis of conjunctival melanocytic lesions remains the main- There were 2 spindle-cell lesions in this series with a stay for diagnosis, not all ophthalmic pathologists and gen- suggestion of desmoplastic melanomatous features: 1 (pa- eral pathologists have had enough experience to diagnose tient 10) stained strongly for S-100, moderately for these lesions comfortably.29 Childhood lesions, in which MART-1, and lightly for HMB-45, whereas the second the junctional component and an inflammatory infiltrate (patient 15) stained lightly for S-100, strongly for MART-1, are often pronounced, can be disquieting. Therefore, our and moderately for HMB-45 (Table 2). Classic desmo- findings regarding HMB-45 and Ki-67 immunostaining plastic melanomas in the skin and other mucous mem- should provide an additional level of confidence in sepa- branes may be only positive for S-100.1,2,22,27,28 rating benign from malignant conditions. We believe that Although statistically significant, positive, Ki-67 our data further support the proposition that conjunctival nuclear-staining differential counts have been estab- melanomas are more akin to cutaneous than uveal mela- lished in PAM with and without atypia,33 the quantita- nomas; this topic continues to receive serious consider- tive Ki-67 results in our study should facilitate the dis- ation.20,30 There is no doubt that other markers will emerge tinction between active junctional nests in a nevus (low that will play an enhanced role in differential diagnosis. For counts) and atypical PAM (high counts); they were also example, Wilms tumor gene (WT1) has recently been dis- statistically significant at PϽ.001. Purely junctional con- covered to support the diagnosis of conjunctival mela- junctival nevi are encountered in the first decade of life noma along with HMB-45.47 We should point out that we when conjunctival melanomas and PAM occur only as did not attempt to assess the predictive value of our data curiosities.38-40 PAM with atypia as a precursor to the de- regarding ultimate behavior and the possible develop- velopment of nodules of melanoma is a disease of the sixth ment of metastases. or seventh decade or later.23,25,26 Although the patients’ ages establish a guideline, pathologists have relied chiefly Submitted for Publication: June 11, 2009; final revi- on histomorphologic criteria to separate atypical PAM sion received July 22, 2009; accepted August 7, 2009. from junctional nevi.23-25,38-44 This subject has been eclipsed Correspondence: Frederick A. Jakobiec, MD, DSc, David by the debate about whether PAM with atypia is identi- D. Cogan Laboratory of Ophthalmic Pathology, Massa- cal to melanoma in situ.42-44 chusetts Eye and Ear Infirmary, 243 Charles St, Ste 321, We propose that, in cases of PAM, immunohisto- Boston, MA 02114 ([email protected]). chemical staining for the presence of HMB-45 and the Financial Disclosure: None reported. detection of Ki-67 positivity should be routinely used Additional Contributions: Ann Marie Lane, MPH, De- when tissue is available (Table 3). HMB-45 cytoplasmic partment of Ophthalmology, Massachusetts Eye and Ear positive staining is much more intense in melanoma in Infirmary, performed the data analysis. situ and in PAM with atypia when compared with the staining of intraepithelial junctional nevocytes. Ki-67 posi- REFERENCES tivity is at least 2 to 3 times as great in the first 2 categories as in the last. Especially in childhood melanocytic 1. Blessing K, Sanders DS, Grant JJ. 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