Immunohistochemical Studies of Atypical Conjunctival Melanocytic Nevi
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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 per- formed 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 (REPRINTED) ARCH OPHTHALMOL / VOL 127 (NO. 8), AUG 2009 WWW.ARCHOPHTHALMOL.COM 970 ©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