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Conjunctival malignant melanoma in Denmark. Epidemiology, treatment and prognosis with special emphasis on tumorigenesis and genetic profile

Conjunctival malignant melanoma is a rare disease associated with considerable mortality.Most published data have been based on case reports or series of referred patients. In addition, very little is known about the genetic and epigenetic profile of conjunctival melanoma. The purpose of this thesis was: To relate clinicopathological features and choice of treatment of conjunctival melanoma patients to prognosis (paper I) and to determine the incidence rate of conjunctival melanoma in Denmark (paper II). A further aimwas to determine the prevalence of BRAF mutations in conjunctival melanoma, using droplet digital polymerase chain reaction (ddPCR) and immunohistochemistry (IHC), and to determine whether BRAFmutations are early events in pathogenesis and relate clinicopathological features and prognosis to BRAF mutation status (paper I and II). Finally, we wanted to identify tumour specific and prognostic microRNAs in conjunctival melanomas using a microarray-based analysis and to compare these with the microRNA expression of other melanoma subtypes (paper III). The study was based on: 139 conjunctival melanomas that had been surgically removed in Denmark over a period of 52 years (1960–2012). Conclusions: The overall incidence of conjunctival melanoma was 0.5/ 1 000 000/year and it increased in Denmark over 52 years. The increase was mainly caused by an increase in older patients (>65 years), bulbar lesions and lesions associated with a primary acquired melanosis with atypia (Larsen et al. 2016). Clinicopathological features significantly associated with a poor prognosis were extrabulbar location, involvement of adjacent tissue structures, tumour thickness exceeding 2 mm and local tumour recurrence. Patients undergoing incisional biopsy and/or treatment involving excision without adjuvant therapy fared worse than patients treated with excision and any type of adjuvant treatment (Larsen et al. 2015; Larsen 2016). We found that 35% (39/110) of conjunctival melanomas were BRAF mutated and the incidence of BRAF mutations was constant over time. BRAF mutation status corresponded in conjunctival melanoma and paired premalignant lesions. BRAF mutations were more frequent in males, in young patients and in tumours with a sunexposed tumour location (bulbar conjunctiva or caruncle), with a mixed or nonpigmented colour, with absence of primary acquired melanosis and with origin in a nevus. Immunohistochemistry was able to accurately detect BRAF V600E mutations (Larsen et al. 2016). In univariate analysis, distant metastatic disease was associated with BRAF mutations; however, no prognostic associations with BRAF mutations were identified in multivariate analyses (Larsen et al. 2015, 2016). MicroRNA expression analysis revealed 24 upregulated and one downregulated microRNAs in conjunctival melanoma, several of these microRNAs have key functions in the pathogenesis and progression of cutaneous melanoma. Additionally, we identified seven upregulated microRNAs (miR-30d-5p, miR-1385p, miR-146a-5p, miR-500a-5p, miR-5013p, miR-501-5p and miR-502-3p) specific for stage T1 and T2 conjunctival melanoma, whose expression was associated with increased tumour thickness. No stage T3-specific microRNAs were identified. Our analysis revealed that the microRNA expression did not differ significantly between conjunctival melanoma and sinonasal or laryngeal mucosal melanoma (Larsen 2016). From our findings of a distinct pattern of BRAF mutations and differentially expressed microRNAs, conjunctival melanoma seems closely related to cutaneous and other mucosal melanomas and bears less resemblance to uveal melanomas. This means that conjunctival melanoma patients may benefit from therapies that are effective for cutaneous and mucosal melanoma. Additionally, the identification of several upregulated microRNAs may prove to be useful as future prognostic or therapeutic targets in conjunctival melanoma.