Conjunctival Melanoma: Current Management.

To investigate the presence of KIT gene mutations and immunoreactivity in 85 conjunctival melanocytic tumours and to clarify the role of KIT as a potential therapeutic target in this group of patients. Eighty-five conjunctival pigmented tumours [27 melanomas, 12 primary acquired melanosis (PAMs) and 46 nevi] were immunostained for KIT. Intensity and pattern of expression were evaluated. Molecular analysis to identify KIT mutations was performed in 15 selected cases (tumour-rich areas >50%). KIT immunostaining score and pattern were statistically related to patients' age, sex, diagnostic category, presence of relapse, disease-free survival, presence of metastases, metastasis-free survival, limbal versus nonlimbal tumour location and thickness of melanomas. KIT stains were documented in 48% of melanomas, 50% of PAMs and 24% of nevi. The mean score of KIT staining in the melanomas/PAMs group was significantly different from nevi (p = 0.0076). No statistically significant differences were detected between either c-kit immunostaining score or pattern and each of the other clinico-pathologic parameters considered. No KIT gene mutations were detected in melanomas and nevi. A silent mutation/polymorphism in KIT exon 13 was found in one PAM. Despite the high level of KIT immunostains in PAMs and melanomas, this parameter seems not to be a good predictor of the presence of molecular mutations. KIT-activating mutations should be considered an uncommon event in this tumour.

Conjunctival melanocytic proliferations are diagnostically challenging, often complicated by small specimen size, and are separated into 3 broad categories. The first group includes benign nevi and primary acquired melanosis (PAM) without atypia. The second group includes junctional melanocytic proliferations with a risk of progression to invasive melanoma (PAM with atypia). The last category is conjunctival melanoma, of which 65% of tumors arise in the setting of PAM with atypia. Preferentially expressed antigen in melanoma (PRAME) immunohistochemistry has been widely adopted to differentiate cutaneous nevi and melanoma. However, there are limited studies on its utility in the evaluation of conjunctival melanocytic proliferations with little data regarding its potential utility in stratifying PAM. Twenty-eight clinically annotated cases (14 PAM without atypia and 14 PAM with atypia) were retrospectively evaluated with PRAME/MART-1 duplex immunohistochemistry and were assigned the commonly used PRAME immunoreactivity score: 0 for no staining, 1+ for 1%-25% of cells positive, 2+ for 26%-50%, 3+ for 51%-75%, and 4+ for >75%. PAM without atypia showed low (0-3+) PRAME expression in 14 of 14 cases (100%). PAM with atypia showed strong and diffuse (4+) PRAME expression in 12 of 14 cases (86.7%). Seven of eight (87.5%) PAM with severe atypia, 4 of 4 PAM (100%) with moderate atypia, and 1 of 2 PAM (50%) with mild atypia showed 4+ PRAME expression. In addition, all 5 cases that recurred or progressed (all classified as PAM with atypia) showed 4+ PRAME expression. Although additional larger studies are needed, PRAME seems to be a useful adjunct in evaluating junctional melanocytic proliferations of the conjunctiva.

Management of Pigmented Conjunctival Lesions

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 aim was to determine the prevalence of BRAF mutations in conjunctival melanoma, using droplet digital polymerase chain reaction (ddPCR) and immunohistochemistry (IHC), and to determine whether BRAF mutations 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 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 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-138-5p, miR-146a-5p, miR-500a-5p, miR-501-3p, 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.

To report 12-year follow-up experience with topical mitomycin chemotherapy for diffuse and multifocal primary acquired melanosis (PAM) with atypia and conjunctival melanoma. Interventional case series of 16 patients. Mitomycin was a primary treatment for residual epithelial disease in ten patients (eight with PAM with atypia and two with conjunctival melanoma) and as an adjuvant to excision and cryotherapy in six with conjunctival malignant melanoma. Primary treatments consisted of mitomycin 0.04% qid for 28 days (two 14-day cycles) and for 7 consecutive days as adjuvant therapy. Patients were followed for both local recurrence and metastatic disease. Sixteen patients were followed for a mean 81 months (range 13-144 months) after treatment. All tumors responded to chemotherapy. Recurrence was noted in eight (three adjuvant and five primary treatment patients). Three underwent orbital exenteration. The remaining five were treated conservatively. The mean time to recurrence was 36.9 months. The short-term mitomycin-related complications included transient keratoconjunctivitis (n=14), severe keratoconjunctivitis (n=1) and one corneal abrasion with scar formation. The long-term complications included pannus (n=2) and corneal haze (n=1). Visual acuity was maintained within two lines in 14 patients (including measurements just prior to exenteration). Three patients died, one of metastatic conjunctival melanoma. Conjunctival melanoma and PAM responded to mitomycin 0.04% topical chemotherapy; subepithelial nests appeared resistant to treatment. Treatment-related complications were acceptable. In this series, as primary and adjuvant therapy, topical mitomycin yielded an overall recurrence rate of 50%.

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 microRNAs in conjunctival melanoma, and to compare these with the microRNA expression of other melanoma subtypes. In order to investigate these rare tumours, we studied all the conjunctival melanomas that had been surgically removed in Denmark over a period of 52years (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 microRNA expression profiling, we investigated differentially expressed microRNAs. The overall incidence of conjunctival melanoma was 0.5/1000000/year, and it increased in Denmark over 52years. The increase was mainly caused by an increase in older patients (>65years) and bulbar lesions. Clinicopathological features significantly associated with a poor prognosis were extrabulbar location, involvement of adjacent tissue structures, tumour thickness exceeding 2mm 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 V600E mutations. In univariate analysis, distant metastatic disease was associated with BRAF mutations. No prognostic associations with BRAF mutations were identified in multivariate analyses. MicroRNA expression analysis revealed 25 tumour-specific microRNAs in conjunctival melanoma. Five possibly oncogenic miRNAs (miR-20b-5p, miR-146b-5p, miR-146a-5p, miR-506-3p and miR-509-3p) were up-regulated. Seven microRNAs (miR-30d-5p, miR-138-5p, miR-146a-5p, miR-500a-5p, miR-501-3p, miR-501-5p and miR-502-3p) were significantly and simultaneously up-regulated in both stage T1 and stage T2 tumours, and were associated with increased tumour thickness. The expression of the 25 tumour-specific microRNAs did not differ significantly between conjunctival melanoma and oral or nasal mucosal melanoma. In conclusion, the incidence of conjunctival melanoma increased in the Danish population from 1960 to 2012. From our findings of a distinct pattern of BRAF mutations and differentially expressed microRNAs, 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 microRNAs may prove to be useful as prognostic or therapeutic targets in conjunctival melanoma.

Hot-spot mutations in the promoter region of telomerase reverse transcriptase (TERT promoter mutations) occur frequently in cutaneous and conjunctival melanoma and are exceedingly rare in uveal melanoma. No information is available on the presence of these mutations in the conjunctival melanocytic precursor lesion primary acquired melanosis (PAM). We tested a cohort of uveal and conjunctival melanomas as well as conjunctival benign and premalignant melanocytic lesions for TERT promoter mutations in order to elucidate the role of these mutations in tumor progression. TERT promoter mutation analysis on fresh tumor DNA and DNA from formalin-fixed, paraffin-embedded specimens was performed by SNaPshot analysis in 102 uveal melanomas, 39 conjunctival melanomas, 26 PAM with atypia, 14 PAM without atypia, and 56 conjunctival nevi. Mutations of the TERT promoter were not identified in conjunctival nevi or PAM without atypia, but were detected in 2/25 (8%) of PAM with atypia and 16/39 (41%) of conjunctival melanomas. A single TERT promoter mutation was detected in 102 uveal melanomas (1%). We present the second documented case of TERT promoter mutation in uveal melanoma. In comparison with other types of melanoma, TERT promoter mutations occur at extremely low frequency in uveal melanoma. TERT promoter mutations are frequent in conjunctival melanoma and occur at lower frequency in PAM with atypia but were not detected in benign conjunctival melanocytic lesions. These findings favor a pathogenetic tumor progression role for TERT promoter mutations in conjunctival melanocytic lesions.

Purpose To summarise the epidemiology of conjunctival melanocytic neoplasms. Methods Review of population‐based data on 85 patients with primary conjunctival melanoma (CM) and recently published literature. Results CM accounts for 5‐7% of ocular melanoma in Europe. Its age‐adjusted incidence has increased 2‐fold in North Europe (Finland, from 0.40 to 0.80/million) and North America (USA, from 0.27 to 0.54) during the last 25 y. In both regions, age‐adjusted incidence is higher in men. Different rates between regions result from differences in registries, ethnicity and solar radiation. Age‐adjusted incidence of CM is 3‐fold in non‐Hispanic Caucasians and 2‐fold in Hispanics relative to Asians, African Americans and American Indians; among non‐Hispanic Caucasians it increases 2.5‐fold from 48 deg. (e.g. Paris) to 21 deg. (e.g. Mecca) of latitude. CM is rare below 30 y of age (age‐specific incidence, 0.06) but increases steadily thereafter (0.48, 1.05 and 1.57 for 30‐49, 50‐70 and >70 y, respectively). Median age at diagnosis is 58‐60 y. Most CM arise in limbal (57‐64%) followed by bulbar (12‐13%), palpebral (7‐9%) and caruncular (3%) conjunctiva. Tumor‐specific 5‐and 10‐y mortality estimates are 14‐20% and 29‐38%, respectively. Non‐limbal location, increasing tumor thickness and local recurrence are consistently associated with higher mortality. Clinically detectable primary acquired melanosis (PAM) and nevus precede or accompany CM in 57‐61% and 7‐23% of patients, respectively. Median age at diagnosis of PAM is 56 y. The risk of malignant change is not precisely known and depends heavily on subtype of PAM, ranging from 10 to 90%. Conclusion Recent studies provide epidemiological data on CM which are remarkably consistent. The epidemiology of conjunctival nevi and PAM is less precisely known.

Histological criteria for grading of atypia in melanocytic conjunctival lesions

Conjunctival nevi, conjunctival primary acquired melanosis (PAM), and conjunctival melanomas all arise from melanocytes that migrate from the neural crest to reside in the conjunctival epithelium. Conjunctival melanoma may arise de novo or from preexisting conjunctival nevus or PAM. In the medical literature, conjunctival melanoma is sometimes labeled together with uveal melanoma as “ocular melanoma.” The clinical behavior, molecular biology, and the histopathologic features of conjunctival and uveal melanoma are clearly different; therefore, conjunctival melanoma should be approached as an entity separate from uveal melanoma.

To report a case of conjunctival melanoma arising from diffuse primary acquired melanosis (PAM) with atypia in a young black woman in the context of previously published cases of this lesion in blacks. Retrospective case report with literature review. The number and percentage of conjunctival melanomas occurring in black patients were determined from case series in which race was specified, published from 1950 to the present. Nodular multifocal conjunctival melanoma in a 30-year-old black woman was treated using surgical excision and adjuvant cryotherapy. Extensive PAM with severe atypia, including areas of microinvasive melanoma, was treated using topical mitomycin C. Literature review revealed 35 cases of conjunctival melanoma occurring in black patients. No previous reports of mitomycin C use in black patients with melanoma or PAM were identified. Conjunctival melanoma is an exceedingly rare tumor in black patients. The current case brings the total of reported cases to 36. We successfully treated nodular melanoma and diffuse PAM in a young black woman using a combination of excision with cryotherapy and topical mitomycin C, suggesting that these lesions are amenable to the same types of therapy previously described for white patients.

To evaluate cell growth and the pattern of p53 suppressor gene expression in atypical primary acquired melanosis (PAM) and in recurrent conjunctival melanoma. Eighteen specimens of PAM with atypia and 24 specimens, comprising early and late lesions, from 12 patients with conjunctival melanoma were stained for the proliferating cell nuclear antigen using the PC10 antibody, and for the p53 gene product using the BP53-12-1, 1801 and DO7 clones. The immunoreactive cells were counted manually and the data evaluated statistically. Seven of nine PAM specimens progressing to melanoma expressed PC10. None of these lesions expressed the p53 gene product. The number of proliferating cells was higher in the late than in the early lesions of conjunctival melanoma. Four of the 12 recurrent melanomas displayed focal, but minimal, p53 expression. The proliferating cell count in the p53 positive tumours was very similar to that of the p53 negative conjunctival melanomas. Examination of the expression of proliferating cells in atypical PAM may be used as an adjunct to predict which lesions will progress to melanoma. The increase in the number of proliferating cells over time in recurrent conjunctival melanomas probably reflects more aggressive behaviour and may be used to monitor recurrence. The absence of p53 expression in PAM and minimal staining of conjunctival melanomas did not correlate with cell growth, suggesting that alterations in the p53 tumour suppressor gene are uncommon and late events in conjunctival melanoma, and that p53 expression is unlikely to be a useful prognostic indicator.

Reply

Malignant conjunctival melanoma is a rare disease with an incidence of 0.03 - 0.08. This tumour is potentially lethal, even after prompt and proper treatment, especially after delayed onset of therapy. Conjunctival melanoma arises from primary acquired melanosis (PAM), from a preexisting nevus or "de novo" without any precursor at all. In contrast to uveal melanomas, conjunctival melanoma metastasizes via the ipsilateral lymph nodes and in rare cases through the lacrimal duct into the nasal cavities. Removing the local tumour with preservation of visual functions and avoidance of metastases is the therapy of choice. Excision alone is followed by a high rate of recurrence. To minimize local recurrence rate surgical excision should be combined with an additional procedure such as cryotherapy, irradiation, or local chemotherapy with MMC. Surgical technique is characterized by a so-called "no-touch" method avoiding any direct manipulation of the tumour to prevent tumour cell seeding into a new area. The behaviour of conjunctival melanomas is individually unpredictable. Prognostic factors are tumour size and tumour location. Tumours growing extralimbal especially at the fornix, plica and caruncle have a significantly poorer prognosis than limbal tumours. In our own patients the 5-year, 10-year, and 15-year cumulative melanoma-specific survival rate was 84.4 %, 77.7 %, and 75.0 %, respectively. Up to now there is no effective treatment of the metastatic disease. In all cases with pigmented lesions of the conjunctiva exclusion of a malignant melanoma has to be the first aim. A patient suffering from a conjunctival melanoma should be referred to an ophthalmo-oncological center for proper treatment. An indefinite follow-up including photodocumentation is necessary since the rate of recurrence is high. An international prospective study would be worthwhile to answer open questions and to develop new kinds of treatment of this potentially fatal tumour.

Primary acquired melanosis (PAM) of the conjunctiva is a potentially serious melanocytic lesion that can lead to the development of a melanoma. A 60-year-old woman noticed pigmentation of the conjunctiva of her left eye for more than 10 years. She underwent excisional biopsy combined with cryotherapy and was diagnosed with PAM without atypia by intraoperative consultation. She was followed for 7 years, and no changes were observed. Fourteen years after the initial biopsy, she noted a growing conjunctival tumor, and a melanoma was suspected. She underwent orbital exenteration and skin grafting procedures. Histopathological examination of the specimen led to a diagnosis of conjunctival malignant melanoma. Re-examination of the initial biopsy specimen revealed that there was a proliferation of melanocytes that partially expanded over the basal layer of the conjunctiva which had been diagnosed as PAM with moderate atypia. We conclude that this case of conjunctival PAM had progressed to a conjunctival malignant melanoma after 14 years. Pathological evaluation of intraepithelial lesions has its limitations; thus, cases of PAM, even in the absence of obvious atypia, require careful follow-up.