Clinical and immunohistopathological study of conjunctival melanocytic lesions in pediatric and adolescent patients. A case series

Abstract

Background: Conjunctival pigmented neoplasia can be benign, premalignant or malignant tumors. Our study aims to establish the epidemiological, gross morphological and immunohistopathological features of the conjunctival pigmented lesions in pediatric and adolescent patients (<18 years), to establish an accurate diagnosis.

Patients, material and methods: This is a retrospective case series study conducted within two Ophthalmology Clinics from Iaşi, Romania, on seven pediatric and adolescent patients. Using the Clinical Observation Chart and the Pathology Registers over a six-years period (January 2015-December 2021), we noted the patients' demographic data, clinical data, and ophthalmological investigations of the lesion, as well as the type of their treatment. All histological sections stained with Hematoxylin-Eosin (HE) and with five antibodies [pan-cytokeratin (pan-CK) AE1∕AE3, S100 protein, Melan A, human melanoma black 45 (HMB45), and Ki67] were re-examined by four pathologists for each case, to identify the type of the conjunctival lesion and its histological and immunohistochemical features.

Results: The mean age of all patients was 10.28 years, and the female∕male ratio was 1.3. Right eye was more often affected (71.42%). 71.42% of cases presented an elevated lesion, 57.14% of cases showed a lightly pigmented lesion, but 14.28% of cases exhibited a pink lesion and this feature described the inflamed juvenile conjunctival nevus. In all cases (100%) the conjunctival pigmented tumor was removed with safety margins. The microscopic examination revealed a compound melanocytic nevus in 57.14% cases, a junctional conjunctival nevus in 14.28% cases, an inflamed juvenile nevus in 14.28% cases, and a conjunctival melanoma arising from a pre-existing nevus in 14.28% cases. In all cases of nevi, the nevoid melanocytes showed strong immunopositivity for Melan A and S100 protein, variable and weak immunopositivity for HMB45, and a mean Ki67 labeling index of 1.71%. Conjunctival melanoma revealed strong immunopositivity of tumor cells for HMB45, Melan A and S100 protein, and a Ki67 labeling index of 20%. In all cases, the conjunctival epithelium showed strong immunopositivity for pan-CK AE1∕AE3. All our cases (100%) had a favorable outcome after the surgical removal of the tumor.

Conclusions: Any excision of a conjunctival pigmented lesion must be subject to a systematic immunohistopathological examination, and there is a set of antibodies (anti-HMB45 and anti-Ki67) that are useful for differential diagnosis between a conjunctival nevus and a conjunctival melanoma.

Figure 1

Diagram of the distribution of cases according to patients’ gender

Figure 2

Diagram of the distribution of cases according to immunohistopathological diagnosis of the lesion

Figure 3

Anterior segment photographs showing clinical morphology of conjunctival pigmented lesions correlated with their histological classification: (A) Case No. 3 – girl, 6 years old, presented a juxta-limbal, irregular, small, flat, lightly pigmented conjunctival lesion, which was diagnosed as a compound conjunctival nevus on her left eye; (B) Case No. 7 – boy, 7 years old, showed a solitary, sharply demarcated, prominent, heavy pigmented lesion, located on the temporal conjunctiva of her right eye that was diagnosed as a conjunctival melanoma arising from a preexisting nevus; (C) Case No. 5 – girl, 12 years old, exhibited on her right eye a juxta-limbal, irregular, slightly elevated, lightly tan conjunctival lesion, with few microcysts on its surface, which was diagnosed as a junctional conjunctival nevus

Figure 4

Microphotographs of compound conjunctival nevus. Case No. 3 – girl patient, 6 years old: (A) Compound nevus with nevoid melanocytes presence along the covering epithelium, with low pigmentation and significant inflammatory lymphoplasmacytic infiltrate at the base of the lesion. Nests of nevoid melanocytes around and among multiple cystic spaces lined by conjunctival epithelium. Nevoid cell nests on the base of the lesion and around cystic structures. Multiple large epithelial cysts could be seen in the histological section. Conjunctival epithelium shows squamous metaplasia and hyperplasia, with deep invagination and formation of cystic structures that are surrounded by tumor cells; (B) Cytoplasmic immunopositivity of the conjunctival epithelium for CK AE1/AE3 revealed its hyperplasia, but nevocytic cell nests do not show any immunostaining; (C) Immunopositivity for Melan A in the cytoplasm of the nevoid melanocytic cells demonstrates their organization into small nests around cystic spaces and at the base of the lesion, but non-staining cells represented the conjunctival epithelium; (D) Slight immunopositivity in the cytoplasm of the nevoid cell nests for HMB45; (E) The conjunctival epithelium and inflammatory cells did not stained with anti-HMB45 antibody; (F) Less than 1% of the nuclei were labeled for Ki67. HE staining: (A) ×20. Anti-CK AE1/AE3 antibody immunomarking: (B) ×100. Anti-Melan A antibody immunomarking: (C) ×400. Anti-HMB45 antibody immunomarking: (D and E) ×40. Anti-Ki67 antibody immunomarking: (F) ×200. CK: Cytokeratin; HE: Hematoxylin–Eosin; HMB45: Human melanoma black 45

Figure 5

Microphotographs of the inflamed juvenile conjunctival nevus. Case No. 6: boy patient, 13 years old: (A) The histological sections showed compound nevoid melanocytic proliferation made up of intraepithelial and subepithelial melanocytic nests organized around solid or cystic epithelial inclusions that also contain goblet cells. There was also a dense, diffuse, stromal inflammatory infiltrate with lymphocytes, plasma cells and eosinophils. At the base of the lesion, lymphocytes were organized into a lymphoid follicle with germinal center; (B) Cystic epithelial inclusions, with a predominance of goblet cells, surrounded by nests of nevoid melanocytes, along with dense stromal inflammatory infiltrate; (C) Nevoid melanocytic cells were located in conjunctival epithelium, but also in subepithelial connective tissue alongside with solid epithelial inclusions and eosinophils in a high number, which were diffusely dispersed among the nests of nevoid melanocytes; (D) Positive immunostaining for CK AE1/AE3 of cells organized into solid structures included into the tumor mass demonstrated their epithelial nature; (E) Positive immunostaining for Melan A in the cytoplasm of nevomelanocyte cells revealed their focal pagetoid spread in the conjunctival epithelium, as well the presence of tumor nests in the subepithelial conjunctive tissue; (F) Immunopositivity for S100 protein also showed the intraepithelial as well as the stromal nevomelanocytic nests; (G) Ki67 mean labeling index was 1% in nevocytic cells. HE staining: (A) ×100; (B and C) ×200. Anti-CK AE1/AE3 antibody immunomarking: (D) ×200. Anti-Melan A antibody immunomarking: (E) ×200. Anti-S100 antibody immunomarking: (F) ×200. Anti-Ki67 antibody immunomarking: (G) ×200. CK: Cytokeratin; HE: Hematoxylin–Eosin