Conjunctival Melanocytic Lesions Research Articles

A Multicenter Study Validates the WHO 2022 Classification for Conjunctival Melanocytic Intraepithelial Lesions With Clinical and Prognostic Relevance

Several nomenclature and grading systems have been proposed for conjunctival melanocytic intraepithelial lesions (C-MIL). The fourth “WHO Classification of Eye Tumors” (WHO-EYE04) proposed a C-MIL classification, capturing the progression of noninvasive neoplastic melanocytes from low- to high-grade lesions, onto melanoma in situ (MIS), and then to invasive melanoma. This proposal was revised to the WHO-EYE05 C-MIL system, which simplified the high-grade C-MIL, whereby MIS was subsumed into high-grade C-MIL. Our aim was to validate the WHO-EYE05 C-MIL system using digitized images of C-MIL, stained with hematoxylin and eosin and immunohistochemistry. However, C-MIL cases were retrieved from 3 supraregional ocular pathology centers. Adequate conjunctival biopsies were stained with hematoxylin and eosin, Melan-A, SOX10, and PReferentially expressed Antigen in Melanoma. Digitized slides were uploaded on the SmartZoom platform and independently scored by 4 ocular pathologists to obtain a consensus score, before circulating to 14 expert eye pathologists for independent scoring. In total, 105 cases from 97 patients were evaluated. The initial consensus diagnoses using the WHO-EYE04 C-MIL system were as follows: 28 benign conjunctival melanoses, 13 low-grade C-MIL, 37 high-grade C-MIL, and 27 conjunctival MIS. Using this system resulted in 93% of the pathologists showing only fair-to-moderate agreement (kappa statistic) with the consensus score. The WHO-EYE05 C-MIL system (with high-grade C-MIL and MIS combined) improved consistency between pathologists, with the greatest level of agreement being seen with benign melanosis (74.5%) and high-grade C-MIL (85.4%). Lowest agreements remained between pathologists for low-grade C-MIL (38.7%). Regarding WHO-EYE05 C-MIL scoring and clinical outcomes, local recurrences of noninvasive lesions developed in 8% and 34% of the low- and high-grade cases. Invasive melanoma only occurred in 47% of the cases that were assessed as high-grade C-MIL. This extensive international collaborative study is the first to undertake a comprehensive review of the WHO-EYE05 C-MIL scoring system, which showed good interobserver agreement and reproducibility.

PRAME/MELAN-A double immunostaining as a diagnostic tool for conjunctival melanocytic lesions: A South American experience

IntroductionPRAME (PReferentially expressed Antigen in Melanoma) is an antigen that is predominantly expressed in human melanomas. In cutaneous melanocytic lesions, PRAME expression is associated with malignancy. The objective of this study was to evaluate the co-expression of PRAME and Melan A to evaluate their diagnostic value in different conjunctival melanocytic lesions (CML). Methods37 CML (23 nevi, 9 primary acquired melanosis (PAM), and 5 conjunctival melanomas) were evaluated by immunohistochemistry for PRAME and Melan-A. The percentage of melanocytic cells co-expressing PRAME and Melan-A was qualitatively evaluated as follows: negative, 0%; 1 + , 1–25%; 2 + , 26–50%; 3 + , 51–75% and 4 + , ≥ 76%. ResultsOf the invasive melanoma cases, 80% showed a 4 + pattern of marking, whereas 20% showed a 3 + pattern. 11% of the PAMs showed a 4 + pattern and 88.9% showed a 1 + pattern. All the nevi showed a 1 + pattern. The sensitivity and specificity of PRAME 4 + for differentiating high-grade CML from the benign and low-grade grouped CML are 93% and 100%, respectively. ConclusionPRAME/MELAN-A double immunostain is particularly useful to differentiate benign from malignant conjunctival melanocytic lesions.

ATRX Loss in the Development and Prognosis of Conjunctival Melanoma.

Metastatic disease is linked to TERT promoter mutations in conjunctival melanomas (CM). Both TERT promoter and ATRX mutations are associated with faulty telomere maintenance. This study aimed to determine the prognostic value of ATRX loss in conjunctival melanocytic lesions. Eighty-six conjunctival melanocytic lesions from the Rotterdam Ocular Melanoma Study group were collected. ATRX status and TERT promoter status were determined using immunohistochemical staining and molecular diagnostics, respectively. None of the nevi (n = 16) and primary acquired melanosis (PAM) without atypia (n = 6) showed ATRX loss. ATRX loss was found in 2/5 PAM with atypia without CM and in 8/59 CM. No cases with a TERT promoter mutation (n = 26) showed ATRX loss. Eight/eleven metastatic CM harbored a TERT promoter mutation, two other metastatic CM showed ATRX loss and one metastatic case showed no TERT promoter/ATRX alterations. In conclusion ATRX loss and TERT promoter mutations are only found in (pre)malignant conjunctival melanocytic lesions, with most metastatic cases harboring one of these alterations, suggesting that both alterations are associated with adverse behavior. Similar to TERT promoter mutations, ATRX loss may be used as a diagnostic tool in determining whether a conjunctival melanocytic lesion is prone to having an adverse course.

Consultations in Eye Pathology: Experience at the Ophthalmology Specialty Hospital.

Ophthalmic pathology is a discipline that relies heavily on a knowledge of clinical ophthalmology. The diagnosis of ocular and periocular lesions can be challenging because some lesions and diseases are unique to this region, whereas others may demonstrate site-specific differences from nonocular counterparts. Because of these challenges, ocular and periocular biopsies are frequently referred to specialized ophthalmic pathology centers for second opinion diagnoses. To analyze the referral patterns, diagnostic challenges, and diagnostic discrepancies for second opinion referrals at a dedicated ophthalmic pathology laboratory with an emphasis on lesions of special interest in ophthalmic pathology. Data sources included the pathology records of all slides and blocks received in consultation at the referral eye pathology center between December 1, 2015, and December 1, 2022, the personal experience of senior authors, and published peer-reviewed literature. Corneal, intraocular, and conjunctival biopsies are the most common types of cases received in consultation without the referring pathologist's diagnosis, likely reflecting diagnostic challenges. Degenerative intraocular processes occasionally raise concern for a neoplasm. Conjunctival melanocytic lesions are the most common conjunctival biopsies referred for second opinion diagnosis and require careful tissue sampling and clinical-pathologic correlation. Careful clinical-pathologic correlation, a high level of suspicion, and adequate sampling also are required for the accurate diagnosis of periocular sebaceous carcinoma. The diagnostic discrepancies involving uveal, retinal, conjunctival, eyelid, and temporal artery biopsies are most likely to adversely influence patient management and possible outcome. Such specimens may benefit from referral to specialized ophthalmic pathology laboratories.

PRAME expression by Immunohistochemistry and Reverse Transcription Quantitative PCR in conjunctival melanocytic lesions -A comprehensive clinicopathologic study of 202 cases and correlation of cytogenetics with PRAME expression in challenging conjunctival melanocytic lesions.

This study examined PRAME expression by immunohistochemistry and reverse transcription quantitative PCR (RT-qPCR) in 202 histologically unequivocal conjunctival melanocytic lesions: 76 nevi, 29 benign melanoses, 25 low-grade conjunctival intraepithelial melanocytic lesions (LGCMIL), 26 high-grade conjunctival melanocytic intraepithelial lesions/in-situ melanoma (HGCMIL) and 46 invasive melanomas. PRAME score 0 was seen in 96% of nevi (73/76), 96% of benign melanoses (28/29) and 88% of LGCMIL (22/25). PRAME score 4 was seen in 50% HGCMIL (13/26) and 76% invasive melanomas (35/46). PRAME score 4 had sensitivity of 50% and specificity of 100% in differentiating between HGCMIL and benign melanosis/LGCMIL. PRAME score 4 had sensitivity of 76% and specificity of 100% in differentiating between melanoma and nevi. Relative quantification of PRAME mRNA expression by RT-qPCR was performed on 49 (24%) cases: 17 nevi, 3 benign melanoses, 5 LGCMIL, 9 HGCMIL, and 15 invasive melanomas. The analysis generated two distinct groupings with 'high' relative PRAME expression for the HGCMIL and invasive melanoma and 'low/zero' expression for nevi, benign melanosis and LGCMIL. Thirty-three challenging conjunctival melanocytic lesions that had previous FISH analysis were studied: 18 nevi, 12 melanomas in a nevus, 2 nevoid melanomas, 1 in-situ melanoma. All nevi (100%) showed concordance between negative FISH and PRAME (scores 0-3). 4/13 (31%) melanomas (in-situ, invasive, isolated, and in association with nevus) showed concordance between positive FISH and PRAME score 4. In conclusion, PRAME score 4 has 100% specificity for diagnosis of HGCMIL and melanoma. PRAME is limited in its sensitivity in the evaluation of challenging melanocytic lesions.

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

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.

Single Time Frame Overview of the Genetic Changes in Conjunctival Melanoma from Intraepithelial Disease to Invasive Melanoma: A Study of 4 Exenteration Specimens Illustrating the Potential Role of Cyclin D1

Introduction: Despite advances in the understanding of the molecular pathogenesis of cutaneous melanoma, relatively little is known about the genetic changes that occur in the progression of conjunctival melanocytic intraepithelial lesions to invasive conjunctival melanoma. Methods: We exposed 4 exenteration specimens that each contained varying grades of intraepithelial conjunctival melanocytic neoplasia and invasive neoplasia to a combination of various techniques, including array comparative genomic hybridization (aCGH), ribonucleic acid sequencing (RNA-seq), fluorescence in situ hybridization (FISH), and immunohistochemistry. Results: Three out of 4 of the invasive melanomas showed gains in 11q13 (CCND1 locus) by aCGH. FISH demonstrated CCND1 gain in invasive melanoma and in conjunctival melanocytic intraepithelial lesions (CMILs) of all grades (low-grade CMILs and in situ melanoma), and this was paralleled by increased expression of Cyclin D1 protein within the atypical melanocytes by immunohistochemistry, using a double-staining method with a red end point for Melan A cytoplasmic staining and a brown end point for nuclear Cyclin D1 expression. Higher grades of melanocytic intraepithelial lesions showed more cells expressing Cyclin D1 than lower grade melanocytic intraepithelial lesions. The Cyclin D1 protein expression was in the same location as the amplified CCND1 signal by FISH. One out of 3 of these cases also showed the amplification of the 12q13-15 locus corresponding to MDM2 and FISH confirmed gains in the conjunctival melanocytic intraepithelial neoplasia and invasive melanoma. The remaining fourth case showed a homozygous deletion of 9p21 (CDKN2A) by aCGH only, with immunohistochemistry showing clonal loss of p16 protein expression in the invasive and conjunctival melanocytic intraepithelial lesion. Two out of 4 of the invasive melanomas harboured classical driver mutations in NRAS and NF-1, respectively. None of the cases showed mutations in BRAF, KIT, and TERT mutations. RNA-seq data showed secondary mutations in ARAF, PLCB4, MET, EZH2, MAP2K2, CTNNB1, CIITA, NF2, TP53, and MEN1, some of which are implicated in the MAPK pathway. Conclusion: CMILs harbour amplifications of CCND1 (3 cases), MDM2 (1 case), and loss of CDKN2A (1 case), which are also present when the lesion progresses to invasive melanoma, implicating these amplifications in the early pathogenesis of CMILs. This study represents the first attempt to capture the mutational landscape of all stages of conjunctival melanoma in a single tissue excision.

PRAME expression in melanocytic lesions of the conjunctiva.

PRAME (PReferentially expressed Antigen in MElanoma) is a tumour-associated antigen that is preferentially strongly expressed in most cutaneous melanomas but not or only focally in naevi. Our aim was to evaluate PRAME expression in melanocytic lesions of the conjunctiva. Surgical specimens of 114 conjunctival melanocytic naevi of different types (including 67 common, 25 combined deep penetrating and 21 inflamed juvenile naevi), 30 invasive melanomas, 10 in-situ melanomas, 23 primary acquired melanoses (PAM) without atypia and 11 PAM with atypia were analysed for PRAME expression by immunohistochemistry. Nuclear positivity for PRAME in melanocytes was assessed as the percentage of positive nuclei: negative (0%), 1+ (1-25%), 2+ (26-50%), 3+ (51-75%) and 4+ (> 75%). In 113 of 114 conjunctival melanocytic naevi, PRAME was either completely negative or focally 1+ positive. Diffuse 4+ PRAME expression was identified in 17 of 30 (57%) invasive melanomas, seven of 10 (70%) in-situ melanomas, four of five (80%) PAM with severe atypia, none of three PAM with moderate atypia, none of three PAM with mild atypia, one of 23 (4%) PAM without atypia and none of 114 naevi. Diffuse 4+ PRAME expression in invasive melanomas correlated with a higher mitotic count but was not related to age and gender of the patients, Breslow thickness, location or mutational status. Diffuse 4+ PRAME positivity is highly specific for malignant conjunctival melanocytic lesions. PRAME is therefore a useful ancillary marker to support the diagnosis of a suspected conjunctival melanoma.

Conjunctival Melanocytic Lesions.

Conjunctival melanocytic lesions consist of a variety of neoplastic and nonneoplastic conditions. These include benign processes such as primary intraepithelial hypermelanosis and melanocytic hyperplasia, secondary forms of intraepithelial hypermelanosis and melanocytic hyperplasia, melanocytic nevi, melanocytic proliferations with malignant potential, and melanoma. To provide a concise yet comprehensive resource regarding the histopathologic diagnosis of conjunctival melanocytic lesions. We aim to detail and clarify the numerous classification schemes that exist for junctional melanocytic proliferations of the conjunctiva (known as primary acquired melanosis or PAM; also termed conjunctival melanocytic intraepithelial neoplasia or C-MIN). Although not uniformly adopted, C-MIN is classified by using a numeric system based on a defined set of criteria. A less complex scheme (conjunctival melanocytic intraepithelial lesion or CMIL) has recently been proposed by the World Health Organization. Additionally, we aim to update the reader regarding molecular features and prognostic indicators. Peer-reviewed literature and archived cases for illustration. Accurate histologic classification is essential, as PAM/C-MIN/CMILs that have a significant potential to progress to invasive melanoma may be clinically indistinguishable from low-risk lesions. Conjunctival melanoma (CM) more closely resembles cutaneous melanoma in terms of its pathogenesis and molecular features, compared to melanoma arising at other mucosal sites or to uveal melanoma. Depth of invasion and ulceration status, among other factors, have emerged as important prognostic indicators in CM. Sentinel lymph node biopsy may provide further prognostic information. Lastly, integration of pathologic and clinical findings is essential at this anatomically sensitive location to determine appropriate clinical management.

Validation of the Newly Proposed World Health Organization Classification System for Conjunctival Melanocytic Intraepithelial Lesions: A Comparison with the C-MIN and PAM Classification Schemes

We sought to compare the sensitivity, specificity, accuracy, and interobserver agreement of the two most commonly used classification systems for conjunctival melanocytic intraepithelial lesions with the new World Health Organization (WHO) classification. Retrospective case series and evaluation of classification systems. We reviewed the pathology and medical records of all patients who underwent a primary biopsy procedure for conjunctival primary acquired melanosis (PAM) at Wills Eye Hospital between 1974 and 2002 who had ≥36months of follow-up. Data collected included age, sex, clinical findings, recurrence, and progression to melanoma. Twelve ophthalmic pathologists analyzed scanned hematoxylin and eosin-stained virtual microscopic slides using 3 classification systems: PAM, conjunctival melanocytic intraepithelial neoplasia, and the WHO 4th edition classification of conjunctival melanocytic intraepithelial lesions. Observer agreement, sensitivity, specificity, and diagnostic accuracy of each classification system were assessed. There were 64 patients who underwent 83 primary excisions with cryotherapy for conjunctival PAM who had adequate tissue for histopathologic evaluation. The interobserver agreement in distinction between the low- and high-grade lesions was 76% for PAM, 67% for conjunctival melanocytic intraepithelial neoplasia, and 81% for WHO classification system. Low-grade lesions provided the greatest interpretative challenge with all 3 classification systems. The 3 classification systems had comparable accuracy of 81%-83% in their ability to identify lesions with potential for recurrence. This study highlights the comparable strengths and limitations of the 3 classification systems for conjunctival melanocytic intraepithelial lesions and suggests that the simplified WHO classification scheme is appropriate for evaluation of these lesions.

Immunohistochemical Profiling of Conjunctival Melanocytic Intraepithelial Lesions, Including SOX10, HMB45, Ki67, and P16

To determine the usefulness of melan-A, SOX10, HMB45, and p16 immunohistochemical stains in the distinction between the low-grade and high-grade conjunctival melanocytic intraepithelial lesions, either independently or as components of an immunohistochemical panel. Retrospective observational case series. Institutional pathology records between 2014 and 2018 were searched for all patients with conjunctival melanocytic intraepithelial lesions. Biopsies without supporting clinical history or tissue available for review and immunohistochemical analysis were excluded. Clinical, histopathologic, and immunohistochemical (p16, SOX10, HMB45, and Ki-67) findings were recorded. Thirty-one patients underwent 47 biopsies for conjunctival melanocytic lesions between 2014 and 2018. Pathologic diagnoses were low-grade conjunctival melanocytic intraepithelial lesion (n= 18, 38%) and high-grade conjunctival melanocytic intraepithelial lesion/melanoma insitu (n= 29, 62%). The addition of melan-A and SOX10 immunohistochemical stains resulted in an upgrade of conjunctival melanocytic intraepithelial lesion from low-grade to high-grade in 2 (4%) of 47 cases. The addition of melan-A and SOX10 immunohistochemical stains did not downgrade any of the histomorphologically high-grade lesions. In a clinical-pathologic multivariable model, the parameters most predictive of high-grade melanocytic intraepithelial lesion/melanoma insitu were involvement of the caruncle (odds ratio [OR]= 19, confidence interval [CI] 1.6-212; P= .02] and p16 cytoplasmic H-score >30 (OR= 81, CI 2.7 to >999; P= .01) CONCLUSION: Although the stains for melanocytic markers melan-A and SOX10 facilitate assessment of melanocytic intraepithelial lesions, the current immunohistochemical panels have limited value in distinction between the low-grade and high-grade intraepithelial melanocytic proliferations and need to be used judiciously.

Conjunctival nevi and melanoma: multiparametric immunohistochemical analysis, including p16, SOX10, HMB45, and Ki-67

The role of p16 in the diagnosis and prognosis of conjunctival melanocytic lesions in the context of other clinical and immunohistochemical parameters has not been systematically explored. This study was conducted to determine whether p16 is a useful parameter in the diagnosis and prognosis of conjunctival melanocytic nevi and melanoma, either independently or as a component of immunohistochemical panels. Sixty-one patients underwent 61 biopsies for conjunctival melanocytic lesions between 2014 and 2018. Pathologic diagnoses were melanoma (n=25, 41%), nevus (n=21, 34%), and conjunctival melanocytic lesion of uncertain malignant potential (n=15, 25%). The biopsies were assessed for expression of p16, SOX10, HMB45, and Ki-67. In a multivariable model, the parameters most predictive of melanoma versus nevus were diffuse HMB45 staining (odds ratio [OR]=45, confidence interval [CI]=4.4-457, P=.02] and p16 nuclear H-score≤115 (OR=9.5, CI=1.2-77; P=.04). There was no association of p16 expression with melanoma thickness. Next-generation sequencing identified no CDKN2A mutations or copy number alterations in 12 conjunctival melanomas, including the tumors with absent p16 expression. This study demonstrates that p16 immunohistochemical stain is useful in distinguishing conjunctival melanocytic nevi from melanoma, particularly in combination with HMB45. P16 expression does not appear to correlate with CDKN2A status and melanoma thickness.

Differential diagnosis and management of melanocytic lesions of the ocular surface

Our purpose is to summarize the actual knowledge about melanocytic lesions of the ocular surface (conjunctival nevus, primary acquired melanosis and conjunctival melanoma),especially their clinical appearance, differential diagnosis and treatment. Conjunctival nevus is the most common benign, conjunctival melanocytic lesion. Primary acquired melanosis mainly presents in middle-aged or elderly individuals, characterized by proliferation of melanocytes of the conjunctival epithelial layer. Conjunctival melanoma is a rare tumor, it is the second most common malignant ocular surface tumor after ocular surface squamous neoplasia and the third most common ocular malignancy following choroideal malignant melanoma and ocular surface squamous neoplasia. Early recognition and proper management of conjunctival melanoma is indispensable due to its high malignant and metastatic potential. Due to frequent recurrences, the knowledge and use of intra- and postoperative adjuvant treatment modalities, and regular follow-up are necessary. Orv Hetil. 2020; 161(15): 563–574.

MicroRNA Profiling in Benign and Malignant Conjunctival Melanocytic Lesions

Abstract Increased levels of miR-196b-5p, miR-615-3p and miR-9-5p differentiate benign from malignant conjunctival melanocytic lesions, confirmed in an independent validation cohort. No microRNA could be identified to predict the metastatic potential of conjunctival melanoma.

Combined deep penetrating nevi of the conjunctiva are relatively common lesions characterised by BRAFV600E mutation and activation of the beta catenin pathway: a clinicopathological analysis of 34 lesions

BackgroundDeep penetrating nevus (DPN) is not a widely recognised lesion on the conjunctiva and only a few cases consistent with combined DPN have been reported.MethodsA review of all excised and...

Chemokine Receptor Expression Pattern Correlates to Progression of Conjunctival Melanocytic Lesions.

Chemokines play a role in the progression and metastatic spread of both cutaneous and uveal melanomas. The aim of this study was to examine the prognostic value of expression of chemokine receptors CCR7, CXCR4, and CCR10 in conjunctival melanocytic lesions. In total, 44 conjunctival nevi, 21 cases of primary acquired melanosis (PAM) with atypia and 35 conjunctival melanomas, were included. After immunohistochemical staining for CCR7, CXCR4, and CCR10 the immunoreactive score (IRS) was determined. The findings were correlated for association with melanoma and development of metastasis. For mechanistic evaluation, we used a mouse melanoma metastasis model using two human conjunctival melanoma cell lines, CM2005.1 and CRMM1. All tested chemokines showed a significantly higher expression in conjunctival melanoma than conjunctival nevi. There was a statistically significant difference between the IRS in nevi and PAM with atypia for nuclear IRS in CCR10 (P = 0.03) and both nuclear and cytoplasmic IRS in CXCR4 (P < 0.01 and P = 0.03, respectively); this was also true evaluating the groups PAM with atypia and melanoma all together (P < 0.01). Furthermore, a trend for lower IRS was seen in cases of melanoma without metastasis, with a suggestive pattern of a higher IRS in cases that did develop metastases, supported for CXCR4 using the mouse melanoma metastasis model. Expression of specific chemokines changes during the progression and metastatic spread of conjunctival melanocytic lesions. Differential chemokine profiles may hold prognostic value for patients with conjunctival melanomas and might be considered as a therapeutic target.

Immunohistochemical analysis of benign and malignant melanocytic lesions of the conjunctiva using double-staining

ObjectiveTo implement a double-staining technique to identify the most sensitive and specific combinations of melanoma antigen recognized by T cells (Melan-A), microphthalmia-associated transcription factor (MITF), human melanoma black 45 (HMB45), and Ki67 aiming to assist in the diagnosis of atypical melanocytic conjunctival lesions that are more prone to malignant progression. MethodsEight specimens of conjunctival melanoma and of primary acquired melanosis with moderate to severe atypia were double-immunostained with a combination of a cytoplasmic marker (anti-Melan-A or anti-HMB45), and a nuclear marker (anti-MITF or anti-Ki67). Eight specimens of normal conjunctiva and of conjunctival nevi served as controls. The specimens were processed using 3,3-diaminobenzidine substrate for nuclear stains and the fast-red substrate for cytoplasmic stains. Each slide was analyzed by light microscopy and provided a percent scale and a 0 to 4+ score for each nuclear and cytoplasmic component. ResultsMelan-A and MITF were strongly positive markers for all melanocytic cells, whereas Ki67 and HMB45 provided a variable response for identifying potentially proliferative or aggressive cells. HMB45 and MITF proved to be the best combination for differentiating between atypical and benign lesions on a percent scale and a 0 to 4+ scale (p = 0.0004), with the 3 other combinations providing mainly confirmatory diagnostic information (p < 0.05). ConclusionsOur study used an immunohistochemical double-staining approach to differentiate between atypical and benign melanocytic lesions of the conjunctiva. Our findings should aid in a more complete immunohistopathological diagnosis of conjunctival melanocytic lesions, particularly in diagnostically difficult cases.

Analysis of Melanin Structure and Biochemical Composition in Conjunctival Melanocytic Lesions Using Pump-Probe Microscopy.

PurposeWe analyze melanin structure and biochemical composition in conjunctival melanocytic lesions using pump-probe microscopy to assess the potential for this method to assist in melanoma diagnosis.MethodsPump–probe microscopy interrogates transient excited-state photodynamic properties of absorbing molecules, which yields highly specific molecular information with subcellular spatial resolution. This method is applied to analyze melanin in 39 unstained, thin biopsy specimens of melanocytic conjunctival lesions. Quantitative features of the biochemical composition and structure of melanin in histopathologic specimens are assessed using a geometric representation of principal component analysis (PCA) and principles of mathematical morphology. Diagnostic power is determined using a feature selection algorithm combined with cross validation.ResultsConjunctival melanomas show higher biochemical heterogeneity and different overall biochemical composition than primary acquired melanosis of the conjunctiva (PAM) without severe atypia. The molecular signatures of PAMs with severe atypia more closely resemble melanomas than other types of PAMs. Pigment organization in the tissue becomes more disorganized as diagnosis of the lesions worsen, but nevi are more inconsistent biochemically and structurally than other lesions. Relatively high sensitivity (SE) and specificity (SP) is achieved for differentiating between various melanocytic lesions, particularly PAMs without severe atypia and melanomas (SE = 89%; SP = 87%).ConclusionsPump–probe microscopy is a powerful tool that can identify quantitative, phenotypic differences between various types of conjunctival melanocytic lesions.Translational RelevanceThis study further validates the use of pump–probe microscopy as a potential diagnostic aid for histopathologic evaluation of conjunctival melanocytic lesions.

Melanocytoma of the Conjunctiva: Clinicopathologic Features of Three Cases

Background: Melanocytoma (magnocellular nevus) is a jet-black benign lesion histologically composed of polygonal tumor cells with small, inconspicuous nuclei and abundant cytoplasm. Melanocytomas in general are rare. Most cases occur in the optic nerve head. Conjunctival melanocytoma (magnocellular nevus) is extremely rare, and only 3 lesions of the ocular surface have been reported. Objectives: To describe the clinical and histological spectrum of conjunctival melanocytoma and discuss differential diagnoses of this rare lesion. Method: Four heavily pigmented conjunctival lesions were excised for slight tumor growth and histologically processed. The specimens were routinely stained with hematoxylin and eosin and periodic acid-Schiff. Sections were bleached and immunohistochemical stains were performed for CD68, HMB-45, S100, melanin, and Ki-67. Results: Histological examination revealed findings of a conjunctival melanocytoma in 3 cases. The fourth case was diagnosed histologically as a combined melanocytic lesion with a compound nevus and an inverted type A nevus. None of the lesions exhibited transition towards malignancy. The differential diagnoses included conjunctival melanoma, granular cell nevus, compound nevus with reactive changes, and blue nevus. Conclusions: Conjunctival melanocytic lesions suspicious for melanocytoma should be bleached to evaluate their cytologic features. CD68 can be helpful in identifying heavily pigmented melanomacrophages which may mimic a melanocytoma. As conjunctival melanocytomas are extremely rare, their pathogenesis may be different from that of other conjunctival nevi.

Identification of Feeder Vessels in Ocular Surface Neoplasia Using Indocyanine Green Angiography

ABSTRACTPurpose: To evaluate indocyanine green angiography (ICGA) for the identification and characterization of afferent (feeding) and efferent (draining) vessels in patients with ocular surface neoplasia.Materials and Methods: Consecutive patients with biopsy-proven benign, pre-invasive, or invasive ocular surface tumors of the bulbar conjunctiva were included. Patients underwent anterior segment optical coherence tomography, ICGA, and color photography for the evaluation of the thickness, location, number, and diameter of afferent and efferent vessels of the lesions.Results: Twenty-two eyes of 22 patients with papillomas (n = 4), intra-epithelial neoplasia lesion (n = 2) in situ or invasive carcinomas (n = 6), nevus (n = 5), conjunctival melanocytic intra-epithelial neoplasia lesion (n = 1), and in situ or invasive melanomas (n = 4) were investigated. Afferent (feeder) vessels were identified in all lesions. There were fewer afferent (3.1 ± 1.6) than efferent (7.5 ± 3.5) vessels per lesion (p < 0.001) and the mean diameter was smaller for afferent (101 ± 62 μm, 28–281) than efferent vessels (137 ± 51 μm, 31–652; p = 0.017). The number of afferent and efferent vessels was associated with the thickness of the lesion (p = 0.037, p < 0.01). Lesion filling times differed between benign and invasive or pre-invasive lesions (p = 0.018).Conclusions: ICGA is a useful adjunctive in vivo imaging method for the assessment of the vasculature in patients with suspected ocular surface neoplasia.