Conjunctival malignant melanoma in D enmark: 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 and the resemblance to uveal, cutaneous and mucosal melanoma. The aim was to determine the incidence rate of conjunctival melanoma, and to relate clinicopathological features and treatment to prognosis. A further aim was to determine the prevalence of BRAF mutations in conjunctival melanoma, to determine whether BRAF mutations are early events in pathogenesis, and relate clinicopathological features and prognosis to BRAF ‐ mutation status. Finally, we wanted to identify tumour‐specific and prognostic micro RNA s in conjunctival melanoma, and to compare these with the micro RNA expression of other melanoma subtypes. In order to investigate these rare tumours, we studied all the conjunctival melanomas that had been surgically removed in D enmark over a period of 52 years (1960–2012). Tissue samples, clinical files, pathology reports and follow‐up data were collected and re‐evaluated. Using droplet digital polymerase chain reaction and immunohistochemistry, we investigated BRAF mutations; and using micro RNA expression profiling, we investigated differentially expressed micro RNA s. The overall incidence of conjunctival melanoma was 0.5/1 000 000/year, and it increased in D enmark over 52 years. The increase was mainly caused by an increase in older patients (>65 years) and bulbar lesions. 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. 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 sun‐exposed tumour location (bulbar conjunctiva or caruncle), with a mixed or non‐pigmented colour, with absence of primary acquired melanosis, and with origin in a nevus. Immunohistochemistry was able to accurately detect BRAF V 600 E mutations. In univariate analysis, distant metastatic disease was associated with BRAF mutations. No prognostic associations with BRAF mutations were identified in multivariate analyses. Micro RNA expression analysis revealed 25 tumour‐specific micro RNA s in conjunctival melanoma. Five possibly oncogenic mi RNA s (mi R ‐20b‐5p, mi R ‐146b‐5p, mi R ‐146a‐5p, mi R ‐506‐3p and mi R ‐509‐3p) were up‐regulated. Seven micro RNA s (mi R ‐30d‐5p, mi R ‐138‐5p, mi R ‐146a‐5p, mi R ‐500a‐5p, mi R ‐501‐3p, mi R ‐501‐5p and mi R ‐502‐3p) were significantly and simultaneously up‐regulated in both stage T 1 and stage T 2 tumours, and were associated with increased tumour thickness. The expression of the 25 tumour‐specific micro RNA s did not differ significantly between conjunctival melanoma and oral or nasal mucosal melanoma. In conclusion, the incidence of conjunctival melanoma increased in the D anish population from 1960 to 2012. From our findings of a distinct pattern of BRAF mutations and differentially expressed micro RNA s, it is evident that conjunctival melanoma is 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 up‐regulated micro RNA s may prove to be useful as prognostic or therapeutic targets in conjunctival melanoma.