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 from intraepithelial lesions to invasive conjunctival melanoma. Methods: We exposed four 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 hybridisation (aCGH), RNA seq, fluorescence in-situ hybridisation (FISH) and immunohistochemistry. Results: Three out of four 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 of all grades (Low grade CMIL 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 compared to 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 three of these cases also showed 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 four of the invasive melanomas harboured classical driver mutations in NRAS and NF-1respectively. None of the cases showed mutations in BRAF, KIT and TERT mutations. RNAseq 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: Conjunctival melanocytic intraepithelial lesions 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 conjunctival melanocytic intraepithelial lesions. This study represents the first attempt to capture the mutational landscape of at all stages of conjunctival melanoma in a single tissue excision.

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

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.

Fluorescent in situ hybridization (FISH): A useful diagnostic tool for childhood conjunctival melanoma

Introduction: Conjunctival melanoma is extremely rare in children and has low rates of resolution. Definitive histopathological diagnosis based exclusively on microscopic findings is sometimes difficult. Thus, early diagnosis and adequate treatment are essential to improve clinical outcomes. Clinical case: We present the first case in which the fluorescent in situ hybridization (FISH) diagnostic technique was applied to a 10-year-old boy initially suspected of having amelanotic nevi in his right eye. Based on the 65% of tumor cells with 11q13 (CCND1) copy number gain and 33% with 6p25 (RREB1) gain as measured by the FISH analysis, and on supporting histopathological findings, the diagnosis of conjunctival melanoma could be made. Following a larger re-excision, adjuvant therapy with Mitomycin C (MMC), cryotherapy and an amniotic membrane graft, the patient has remained disease-free during 9 years of long-term follow-up. Case discussion: Every ophthalmologist should remember to consider and not forget the possibility of using FISH analyses during the differential diagnosis of any suspicious conjunctival lesions. Genetic techniques, such as FISH, have led to great advances in the classification of ambiguous lesions. Evidence-based guidelines for diagnosing conjunctival melanoma in the pediatric population are needed to determine the most appropriate strategy for this age group.

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.

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.

Fangchinoline suppresses conjunctival melanoma by directly binding FUBP2 and inhibiting the homologous recombination pathway

Conjunctival melanoma (CM) is a rare and fatal ocular tumour with poor prognosis. There is an urgent need of effective therapeutic drugs against CM. Here, we reported the discovery of a novel potential therapeutic target for CM. Through phenotypic screening of our in-house library, fangchinoline was discovered to significantly inhibit the growth of CM cells including CM-AS16, CRMM1, CRMM2 and CM2005.1. Further mechanistic experiments indicated that fangchinoline suppressed the homologous recombination (HR)-directed DNA repair by binding with far upstream element binding protein 2 (FUBP2) and downregulating the expression of HR factors BRCA1 and RAD51. In vitro and in vivo antitumour experiments revealed that fangchinoline increased the efficacy of cisplatin by blocking HR factors and reduced the drug dose and toxicity. In conclusion, our work provides a promising therapeutic strategy for the treatment of CM that is worthy of extensive preclinical investigation.