Conjunctival Melanoma Research Articles

Photosubstitution in a trisheteroleptic ruthenium complex inhibits conjunctival melanoma growth in a zebrafish orthotopic xenograft model

In vivo data are rare but essential for establishing the clinical potential of ruthenium-based photoactivated chemotherapy (PACT) compounds, a new family of phototherapeutic drugs that are activated via ligand photosubstitution. Here a novel trisheteroleptic ruthenium complex [Ru(dpp)(bpy)(mtmp)](PF6)2 ([2](PF6)2, dpp = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2′-bipyridine, mtmp = 2-methylthiomethylpyridine) was synthesized and its light-activated anticancer properties were validated in cancer cell monolayers, 3D tumor spheroids, and in embryonic zebrafish cancer models. Upon green light irradiation, the non-toxic mtmp ligand is selectively cleaved off, thereby releasing a phototoxic ruthenium-based photoproduct capable notably of binding to nuclear DNA and triggering DNA damage and apoptosis within 24–48 h. In vitro, fifteen minutes of green light irradiation (21 mW cm−2, 19 J cm−2, 520 nm) were sufficient to generate high phototherapeutic indexes (PI) for this compound in a range of cancer cell lines including lung (A549), prostate (PC3Pro4), conjunctival melanoma (CRMM1, CRMM2, CM2005.1) and uveal melanoma (OMM1, OMM2.5, Mel270) cancer cell lines. The therapeutic potential of [2](PF6)2 was further evaluated in zebrafish embryo ectopic (PC3Pro4) or orthotopic (CRMM1, CRMM2) tumour models. The ectopic model consisted of red fluorescent PC3Pro4-mCherry cells injected intravenously (IV) into zebrafish, that formed perivascular metastatic lesions at the posterior ventral end of caudal hematopoietic tissue (CHT). By contrast, in the orthotopic model, CRMM1- and CRMM2-mCherry cells were injected behind the eye where they developed primary lesions. The maximally-tolerated dose (MTD) of [2](PF6)2 was first determined for three different modes of compound administration: (i) incubating the fish in prodrug-containing water (WA); (ii) injecting the prodrug intravenously (IV) into the fish; or (iii) injecting the prodrug retro-orbitally (RO) into the fish. To test the anticancer efficiency of [2](PF6)2, the embryos were treated 24 h after engraftment at the MTD. Optimally, four consecutive PACT treatments were performed on engrafted embryos using 60 min drug-to-light intervals and 90 min green light irradiation (21 mW cm−2, 114 J cm−2, 520 nm). Most importantly, this PACT protocol was not toxic to the zebrafish. In the ectopic prostate tumour models, where [2](PF6)2 showed the highest photoindex in vitro (PI > 31), the PACT treatment did not significantly diminish the growth of primary lesions, while in both conjunctival melanoma orthotopic tumour models, where [2](PF6)2 showed more modest photoindexes (PI ∼ 9), retro-orbitally administered PACT treatment significantly inhibited growth of the engrafted tumors. Overall, this study represents the first demonstration in zebrafish cancer models of the clinical potential of ruthenium-based PACT, here against conjunctival melanoma.

A Conjunctival Melanoma with Invasion of the Nasolacrimal Duct.

A Conjunctival Melanoma with Invasion of the Nasolacrimal Duct.

The effects of visible and blue light exposure on growth and melanogenesis of ocular melanoma cell lines

AbstractPurposeCumulative ultraviolet (UV) exposure is important in skin and conjunctival melanomas and melanogenesis. UV light does not reach the inner posterior eye, but visible light radiates the retina, underlying retinal pigmented epithelium (RPE) and the choroid. The effects of visible light on the choroidal microenvironment and potential roles in choroidal melanoma progression are unknown. We examined human ocular melanoma cells for responses to white or blue light exposure, specifically, cell viability and cytotoxicity, and melanin production.MethodsOcular melanoma cells were grown in RPMI/L‐glutamine/sodium pyruvate (Choroidal: MP38 and MP46 (RPMI + 20% FBS), 92.1 (RPMI + 10% FBS); conjunctival: CM2005.1 (RPMI + 10% FBS)). Cells were grown in 96 well black plates (8000/well; n = 5 per cell line; triplicate) for 18 hrs, then fresh medium (1% FBS) for 16 hr. Cells were exposed to: (1) white light (λ: 400–700 nm), 32,000 lux (direct sunlight) for 4 hr (short‐term), or 4 hr/day × 4 days (16 hr, cumulative); or (2) blue light (λ: 460 nm): 3100 lux for 4 hr (short‐term) or 16 hr/day X 2 days (32 hrs, cumulative). Unexposed control cells were grown in parallel. Viability and cytotoxicity of light‐exposed and control cells were measured with alamarBlue and LDH assays, respectively. Intracellular melanin was assessed after cumulative white or blue light exposure.ResultsShort‐term and cumulative white light reduced viability (alamarBlue) by 5–15% (92.1, MP46 and CM2005.1 cells, but not MP38) and 10–25% (92.1, MP38, MP46 and CM2005.1 cells), respectively. Short‐term blue light did not affect viability of any cell lines; cumulative blue light exposure reduced viability by 10% in MP38 cells only. No significant change in cell cytotoxicity (LDH) or intracellular melanins was noted after white or blue light exposure for any cell lines.ConclusionsMelanoma cells showed no light‐induced cytotoxicity (measured by LDH leak), although viability (measured by alamarBlue) was reduced after cumulative light exposure, suggesting reduced cell numbers rather than cell death. Although melanoma cell lines showed variable intracellular melanins, levels were unchanged with light exposure, despite cells expressing melanogenesis‐related proteins.

Histopathological analysis of juvenile patients with melanocytic lesions of the conjunctiva

Objective: This study aimed to examine the frequency of atypical features in conjunctival nevi in pediatric patients referred to the McGill University Health Center-McGill University Ocular Pathology and Translational Research Laboratory and to emphasize the importance of histopathological analysis to rule out malignant lesions, such as melanoma.Methods: Forty-four pediatric patients younger than 20 years of age previously diagnosed with melanocytic lesions of the conjunctiva were included in this study, and the database was analyzed for a 10-year period (2006–2015). Clinical information such as age, gender, location, type, size, and agreement between clinical and pathological diagnosis was also recorded and presented as means and percentages.Results: The mean age was 11.3 years, of which only 9.1% were older than 18 years. Gender predilection was found toward males (52.3%). An agreement was noted between clinical and pathological diagnosis in 77.2% of cases; only 22.72% showed atypia upon histopathological examination. The most common pathological diagnosis was compound nevus with different characteristics. The atypical lesions included conjunctival melanoma, junctional nevi with atypia, compound nevi with atypia, and cystic compound nevi. Moreover, 70% of the patients with atypical lesions were males and 60% of the specimens were from the right eye.Conclusion: An impressive number of 30% of all patients had some type of atypia after histopathological analysis. Therefore, based on these results, it is of extreme importance that all melanocytic lesions are sent for histopathological analysis, so an accurate diagnosis can be established.

Wide excision of eyelid tarsus with superficial lamellar lid margin tarsus excision for palpebral conjunctival melanoma

Conjunctival melanoma (CM) rarely occurs in the Chinese population. It is found more often in the non-bulbar area with worse prognosis compared with Caucasians. No consensus is reached regarding the standard surgical treatment of palpabral CM. We presented a 39-year-old Chinese female who developed CM at her right eye, involving superonasal palpebral conjunctiva, superonasal bulbar conjunctiva and medial caruncle. We excised the lesion including underlying Tenon capsule and the main part of underlying tarsus with a maximum clear margin of 4 mm, while sparing the tarsus of the eyelid margin. We applied a hemi-depth lamellar incision on the eyelid margin tarsus to preserve the contour of eyelid. Double freeze-thaw cryotherapy was applied to the excision margin, and the conjunctival deficit was closed by an amniotic membrane graft. We provided post-operation adjuvant chemotherapy with 0.02% mitomycin C three times daily for a schedule of three weeks on, one week off. Good cosmetic outcome was achieved with no recurrence in the following 36 months. Tarsus is important in lid function and for aesthetic appearance. To achieve wide-enough safety margin and intact eyelid contour, we presented a new simple approach with good cosmetic outcome and desirable disease control in palpebral CM surgery.

Second Primary Malignancies Following a Diagnosis of Conjunctival Melanoma

Background Although incidence of second primary malignancies (SPMs) has been investigated in patients with cutaneous melanoma and uveal melanoma, limited studies have investigated their occurrence subsequent to conjunctival melanoma (CM). We conducted a retrospective observational study to assess incidence of SPMs in patients with primary CM and to identify associated risk factors. Methods Cases of first primary CM diagnosed from 2000-2018 were extracted from the national cancer database Surveillance, Epidemiology, and End Results Program. Standardized incidence ratios (SIR) and excess absolute risk (EAR) of SPMs were calculated compared to a matched cohort from the general population with similar sex, race, age group, and calendar year. EAR was per 10,000 individuals, and a P-value of <0.05 was considered significant. Results 471 patients met inclusion criteria, 57 (12.1%) of whom developed second primary malignancies over an average (+/-SD) follow-up period of 6.8 (+/-5.0) years. Average age at diagnosis for the overall cohort was 60.2 (+/-18.6) years. Patients with CM demonstrated a significantly increased risk for overall SPMs relative to the general population, even after excluding eye and orbit melanomas (SIR 1.52; 95% confidence interval [CI], 1.15-1.97; EAR 67.58). Specific sites and malignancy types with increased risk were cutaneous melanoma (SIR 7.95; 95% CI, 4.45-13.12; EAR 45.34), ophthalmic non-melanoma malignancies (SIR 80.92; 95% CI, 2.05-450.84; EAR 3.41), and non-intrahepatic biliary malignancies (SIR 11.72; 95% CI, 1.42-42.32; EAR 6.32). Risk of overall SPMs was significantly increased 1-10 years from diagnosis date. Conclusions Patients with CM had an increased incidence of SPMs compared to the general population. Specifically, these patients developed more cutaneous, ophthalmic non-melanoma, and non-intrahepatic biliary malignancies. These second neoplasms could be due to shared pathophysiology or mutual risk factors. Patients with CM may benefit from surveillance for SPMs, such as annual age-appropriate screenings in the first ten years after diagnosis.

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.

IGF-1R is a molecular determinant for response to p53 reactivation therapy in conjunctival melanoma.

As the p53 tumor suppressor is rarely mutated in conjunctival melanoma (CM), we investigated its activation as a potential therapeutic strategy. Preventing p53/Mdm2 interaction by Nutlin-3, the prototypical Mdm2 antagonist, or via direct siRNA Mdm2 depletion, increased p53 and inhibited viability in CM cell lines. The sensitivity to Nutlin-3 p53 reactivation with concomitant Mdm2 stabilization was higher than that achieved by siRNA, indicative of effects on alternative Mdm2 targets, identified as the cancer-protective IGF-1R. Nutlin-3 treatment increased the association between IGF-1R and β-arrestin1, the adaptor protein that brings Mdm2 to the IGF-1R, initiating receptor degradation in a ligand-dependent manner. Controlled expression of β-arrestin1 augmented inhibitory Nutlin-3 effects on CM survival through enhanced IGF-1R degradation. Yet, the effect of IGF-1R downregulation on cell proliferation is balanced by β-arrestin1-induced p53 inhibition. As mitomycin (MMC) is a well-established adjuvant treatment for CM, and it triggers p53 activation through genotoxic stress, we evaluated how these alternative p53-targeting strategies alter the cancer-relevant bioactivities of CM. In 2D and 3D in vitro models, Nutlin-3 or MMC alone, or in combination, reduces the overall cell tumor growth ~30%, with double treatment inhibition rate only marginally higher than single-drug regimens. However, histopathological evaluation of the 3D models revealed that Nutlin-3 was the most effective, causing necrotic areas inside spheroids and complete loss of nuclear staining for the proliferative marker Ki67. These findings were further validated in vivo; zebrafish xenografts demonstrate that Nutlin-3 alone has higher efficacy in restraining CM tumor cell growth and preventing metastasis. Combined, these results reveal that β-arrestin1 directs Mdm2 toward different substrates, thus balancing IGF-1R pro-tumorigenic and p53-tumor suppressive signals. This study defines a potent dual-hit strategy: simultaneous control of a tumor-promoter (IGF-1R) and tumor-suppressor (p53), which ultimately mitigates recurrent and metastatic potential, thus opening up targeted therapy to CM.

Small but Challenging Conjunctival Melanoma: New Insights, Paradigms and Future Perspectives.

Simple SummaryConjunctival melanoma (CM) is a small but highly aggressive and infiltrative periocular malignancy. Despite wide surgical excision followed by adjuvant therapy, about one third and one quarter of patients will experience local recurrence and metastatic spread, respectively. The management of locally advanced (≥T2) tumours may require mutilating surgeries such as orbital exenteration to achieve local control. The last decade has been marked by the emergence of eye-sparing strategies based on wide surgical excision followed by adjuvant proton beam therapy. More recently, new genetic and immunological insights have incriminated several signalling pathways (MAPK, PI3K-AKT) and immune cells, making CM a “targetable” malignancy. Anti-BRAF and anti-MEK targeted therapies and immunotherapies have revolutionized the current management of CM through the use of new eye-sparing strategies and treatment of metastases.Although its incidence has increased over the last decades, conjunctival melanoma (CM) remains a rare but challenging periocular malignancy. While there is currently no recognized standard of care, “no-touch” surgical excision followed by adjuvant treatments is usually recommended. Despite its small size, managing CM is challenging for clinicians. The first challenge is the high risk of tumour local recurrence that occurs in about one third of the patients. The management of locally advanced CM (≥T2) or multiple recurrences may require mutilating surgeries such as orbital exenteration (OE). The second challenge is the metastatic spread of CM that occurs in about one quarter of patients, regardless of whether complete surgical excision is performed or not. This highlights the infiltrative and highly aggressive behaviour of CM. Recently, attention has been directed towards the use of eye-sparing strategies to avoid OE. Initially, wide conservative surgeries followed by customized brachytherapy or radiotherapy have appeared as viable strategies. Nowadays, new biological insights into CM have revealed similarities with cutaneous melanoma. These new findings have allowed clinicians to reconsider the management of locally advanced CM with “medical” eye-sparing treatment as well as the management of metastatic spread. The aim of this review was to summarize the current and future perspectives of treatment for CM based on recent biological findings.

Mucosal melanomas of different anatomic sites share a common global DNA methylation profile with cutaneous melanoma but show location-dependent patterns of genetic and epigenetic alterations.

Cutaneous, ocular, and mucosal melanomas are histologically indistinguishable tumors that are driven by a different spectrum of genetic alterations. With current methods, identification of the site of origin of a melanoma metastasis is challenging. DNA methylation profiling has shown promise for the identification of the site of tumor origin in various settings. Here we explore the DNA methylation landscape of melanomas from different sites and analyze if different melanoma origins can be distinguished by their epigenetic profile. We performed DNA methylation analysis, next generation DNA panel sequencing, and copy number analysis of 82 non-cutaneous and 25 cutaneous melanoma samples. We further analyzed eight normal melanocyte cell culture preparations. DNA methylation analysis separated uveal melanomas from melanomas of other primary sites. Mucosal, conjunctival, and cutaneous melanomas shared a common global DNA methylation profile. Still, we observed location-dependent DNA methylation differences in cancer-related genes, such as low frequencies of RARB (7/63) and CDKN2A promoter methylation (6/63) in mucosal melanomas, or a high frequency of APC promoter methylation in conjunctival melanomas (6/9). Furthermore, all investigated melanomas of the paranasal sinus showed loss of PTEN expression (9/9), mainly caused by promoter methylation. This was less frequently seen in melanomas of other sites (24/98). Copy number analysis revealed recurrent amplifications in mucosal melanomas, including chromosomes 4q, 5p, 11q and 12q. Most melanomas of the oral cavity showed gains of chromosome 5p with TERT amplification (8/10), while 11q amplifications were enriched in melanomas of the nasal cavity (7/16). In summary, mucosal, conjunctival, and cutaneous melanomas show a surprisingly similar global DNA methylation profile and identification of the site of origin by DNA methylation testing is likely not feasible. Still, our study demonstrates tumor location-dependent differences of promoter methylation frequencies in specific cancer-related genes together with tumor site-specific enrichment for specific chromosomal changes and genetic mutations. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Structural Protein Analysis of Driver Gene Mutations in Conjunctival Melanoma.

In recent years, there has been tremendous enthusiasm with respect to detailing the genetic basis of many neoplasms, including conjunctival melanoma (CM). We aim to analyze five proteins associated with CM, namely BRAF, NRAS, c-KIT, NF1, and PTEN. We evaluated each protein for its intrinsically disordered protein regions (IDPRs) and its protein-protein interactions (PPI) with the Predictor of Natural Disordered Protein Regions (PONDR®) and the Search Tool for the Retrieval of Interacting Genes (STRING®). Our PONDR® analysis found high levels of IDPRs in all five proteins with mutations linked to CM. The highest levels of IDPRs were in BRAF (45.95%), followed by PTEN (31.76%), NF1 (22.19%), c-KIT (21.82%), and NRAS (14.81%). Our STRING analysis found that each of these five proteins had more predicted interactions then expected (p-value < 1.0 × 10−16). Our analysis demonstrates that the mutations linked to CM likely affected IDPRs and possibly altered their highly complex PPIs. Quantifying IDPRs in BRAF, NRAS, c-KIT, NF1, and PTEN and understanding these protein regions are important processes as IDPRs can be possible drug targets for novel targeted therapies for treating CM.

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.

Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development.

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a peri-vascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development. Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a peri-vascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development. Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

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.

Definition of Biologically Distinct Groups of Conjunctival Melanomas According to Etiological Factors and Implications for Precision Medicine.

Simple SummaryConjunctival melanoma (ConjMel) is a rare but potentially deadly eye tumor developing on the ocular mucosal surface, which is partially exposed to sunlight. The relationships between potential etiological factors such as ultraviolet exposure and ConjMel mutational landscape have not been precisely described in large cohorts. Here, we report the sequencing of 400 cancer genes in 47 primary ConjMel and show several associations between mutations and etiological factors. BRAF- and CDKN2A-mutated ConjMel affect younger patients while NF1-mutated tumors tend to develop in older ones. CTNNB1 mutations are more frequent in nevi-derived ConjMel, suggesting that the Wnt pathway is pivotal in their tumorigenesis. We further identified concomitant KIT/SF3B1 mutations in BRAF/RAS-wild type, sun-protected tumors, suggesting a similar profile as previously observed in genital and anorectal melanomas, thus unveiling a distinct, mucosal-specific, tumorigenic pathway. Finally, we report for the first time new targetable oncogenic mutations, opening new therapeutic options for these patients.Conjunctival melanoma (ConjMel) is a potentially deadly ocular melanoma, originating from partially sunlight-exposed mucosa. We explored the mutational landscape of ConjMel and studied the correlation with etiological factors. We collected 47 primary ConjMel samples and performed next-generation sequencing of 400 genes. Hotspot mutations in BRAF, NRAS, HRAS, and KIT were observed in 16 (34%), 5 (11%), 2, and 2 cases, respectively. Patients with BRAF and CDKN2A-mutated ConjMel tended to be younger while the NF1-mutated one tended to be older. The eight tumors arising from nevi were enriched in CTNNB1 mutations (63% vs. 8%; Fisher’s exact p-test = 0.001) compared to non-nevi ConjMel and five were devoid of BRAF, RAS, NF1, or KIT mutations, suggesting a specific oncogenic process in these tumors. The two KIT-mutated cases carried SF3B1 mutations and were located on sun-protected mucosa, a genotype shared with genital and anorectal mucosal melanomas. Targetable mutations were observed in ERBB2, IDH1, MET, and MAP2K1 (one occurrence each). Mutational landscape of ConjMel characterizes distinct molecular subtypes with oncogenic drivers common with mucosal and skin melanomas. CTNNB1 mutations were associated with nevus-derived ConjMel. Concomitant KIT/SF3B1 mutations in sun-protected cases suggest a common tumorigenic process with genital and anorectal mucosal melanomas.

PRAME immunohistochemistry is useful in the evaluation of conjunctival melanomas, nevi, and primary acquired melanosis.

Many dermatopathologists find conjunctival melanocytic proliferations challenging because they are rare relative to their cutaneous counterparts and have nuanced morphology and nomenclature. PRAME immunohistochemistry has been widely adopted for distinguishing cutaneous nevi from melanoma, but limited data exist assessing its utility in evaluating conjunctival specimens. In particular, it is uncertain whether it can predict the risk of melanoma progression in primary acquired melanosis (PAM). Thirty clinically annotated cases (two melanomas, three PAM with atypia, seven PAM without atypia, 15 nevi, two combined nevi, and a diagnostically challenging nevus with atypical features) were retrospectively evaluated with PRAME. Strong, diffuse PRAME expression was present in melanomas and PAM with high-grade atypia, but not in PAM with low-grade atypia, PAM without atypia, or nevi. Scattered, faintly PRAME-positive intraepithelial melanocyte nuclei were identified in six nevi. A clonal nevus and nests of heavily pigmented type-A melanocytes in two additional nevi had cytoplasmic staining. PRAME was useful for distinguishing melanoma and its probable precursors from benign conjunctival melanocytic proliferations in our cohort. The data alert us to two diagnostic pitfalls in nevi: scattered, PRAME-positive intraepithelial melanocytes and cytoplasmic PRAME staining in type-A melanocytes and melanophages. Larger scale investigations are warranted to further substantiate these promising findings.

5-Hydroxymethylcytosine Loss in Conjunctival Melanoma.

Aims: Conjunctival and cutaneous melanoma partially share similar clinical and molecular backgrounds. As 5-hydroxymethylcytosine (5-hmC) loss has been demonstrated in cutaneous melanoma, we decided to assess if similar changes were occurring in conjunctival melanoma. Methods: 5-methylcytosine (5-mC), 5-hmC and TET2 were respectively identified by immunohistochemistry and RNA ISH in 40 conjunctival nevi and 37 conjunctival melanomas. Clinicopathological correlations were established. Results: 5-mC, TET2 and 5-hmC were respectively identified in 67.5%, 95% and 100% of conjunctival nevi and in 81.1%, 35.1% and 54% of conjunctival melanomas. A significant 5-hmC and TET2 loss was identified in conjunctival melanoma comparing to nevus, as well as a significant correlation between TET2 and 5-hmC expression. In the melanomas, 5-hmC expression was only significantly associated with local lymphatic invasion, but not with other clinicopathological parameters. There was a correlation between TET2 expression and the localization of the tumors. 5-mC expression was not associated with any clinicopathological parameters. Conclusions: We identified a significant 5-hmC loss in conjunctival melanoma similar to cutaneous melanoma. This loss may possibly be attributed to TET2 loss or IDH1 mutations. 5-hmC loss in conjunctival melanoma may help in the differential diagnosis between atypical conjunctival nevus and conjunctival melanoma.

Diagnostic Algorithm for Conjunctival Melanocytic Lesions.

This study aimed to develop an algorithm allowing the differentiation between conjunctival melanoma and other melanocytic infiltrations of the conjunctiva, on the basis of a dermatoscopic examination. A total of 160 conjunctival pigmented lesions were studied (40 melanomas and 120 non-melanoma conjunctival infiltrations). The clinical characteristics of the tumours were assessed with the use of dermatoscopic characteristics as described by Kittler, and with taking into consideration the typical characteristics of conjunctival lesions. On the basis of modified dermatoscopic criteria, an algorithm was generated consisting of an assessment of the presence of 9 suspicious characteristics, e.g. more than two colours, colour asymmetry, pattern asymmetry, vascular polymorphism, presence of short vessels, linear vascular pattern, the presence of a peripheral structureless area, the presence of a grey structureless area and black dots in any part of the lesion. The presence of any of these characteristics scores 1 point. If a melanocytic lesion scores ≥3 points, the probability of diagnosing melanoma is on the level of p>0.001. The use of the proposed algorithm, based on modified dermatoscopic characteristics, may be a valuable tool for the diagnosis of conjunctival melanoma.

Molecular Genetics of Conjunctival Melanoma and Prognostic Value of TERT Promoter Mutation Analysis.

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.