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Purpose To investigate the cytotoxic effect of bleomycin, mitomycin C (MMC) and Fluorouracil (5‐FU) in combination with electroporation (EP) on human conjunctival melanoma (CM) and normal conjunctival cell lines using 2D and 3D cell culture systems in vitro. Methods Two CM (CRMM1, CRMM2) and one normal conjunctival epithelial cell line (HCjE‐Gi) were treated with various EP conditions and increasing concentrations of 5‐FU, MMC and bleomycin. Cell survival was assessed by MTT viability assay. All cell lines were seeded to create spheroids and were treated with bleomycin on day 3 and day 8 combined with EP. Spheroids were collected, fixed in buffered formalin and subsequently paraffin embedded for histological assessment of the effects of the treatment on cell viability. Results CM cell lines were resistant to electroporation alone and showed a reduction in cell number only when treated with 1000 Volts/cm and 8 pulses. HCjE‐Gi cells showed higher sensitivity to electric pulses over 750 Volts/cm. MMC and 5‐FU demonstrated a higher cytotoxicity for the HCjE‐Gi cell line. The CM cell lines were resistant to MMC and 5‐FU. Bleomycin (1 μg/ml) alone had no significant effect on the HCjE‐Gi even when combined with EP conditions ≥750 Volts/cm. In contrast, it significantly (p ‐, paired t‐test) reduced cell viability in the CM cell lines. Spheroids treated with bleomycin and EP showed a reduction in tumour mass and proliferation rates after treatment. Conclusion Our in vitro study using 2D and 3D models indicates that the application of EP may effectively enhance chemotherapy with bleomycin in CM. This may offer new viable perspectives for CM treatment.

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 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 microRNA expression profiling, we investigated differentially expressed microRNAs. 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) 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 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.

Purpose: We aimed to model and compare mortality rates for uveal melanoma (UM) and conjunctival melanoma (CM) by adjusting for differences in tumour size and local recurrence. Methods: Population-based mortality data for 240 and 85 patients with primary UM and CM and 91 and 23 patients with disseminated UM and CM, respectively, were compared with cumulative incidence analysis. Cox proportional hazards multivariate regression with time-dependent variables was used to adjust for differences in tumour diameter, thickness and recurrence rates. Results: The 10-year cumulative incidences of metastatic death from UM and CM were 39% (95% confidence interval [CI] 33–45) and 32% (95% CI 21–44), respectively. After adjusting for tumour size, risk of death from CM was higher than from UM (hazard ratio [HR] 1.9; p = 0.039). Additional adjustment for more frequent local recurrence of CM diminished the difference (HR 1.5; p = 0.25). Survival periods after systemic metastasis of UM and CM were comparable (median 8 months). Conclusions: Clinical observations show longer survival after primary CM than after primary UM. This reflects the smaller average size of CM. However, a primary CM of a given size is more deadly than a UM of equivalent size because primary CM tends to recur after treatment and, possibly, because additional lymphatic dissemination occurs with CM.

Purpose To investigate incidence, clinicopathological features and prognosis of BRAF-mutated conjunctival melanoma in Denmark. Furthermore, to determine BRAF mutations in paired premalignant lesions and evaluate immunohistochemical BRAF V600E oncoprotein detection. Methods Data from 139 patients with conjunctival melanoma (1960–2012) were collected. Archived conjunctival melanoma samples and premalignant lesions were analysed for BRAF mutations using droplet digital polymerase chain reaction (PCR). Results were associated with clinicopathological features and compared with BRAF V600E oncoprotein stainings. Results The overall incidence of conjunctival melanoma (0.5 cases/1 000 000/year) increased during the study period with 0.13 cases/1 000 000/10 years. The increase comprised a higher proportion of patients aged >65 years, epibulbar tumours and tumours developed from a primary acquired melanosis with atypia. BRAF mutations were identified in 39 of 111 (35%) cases. The rate ratio of BRAF-mutated versus BRAF-wild-type melanoma did not change over time. BRAF mutations were associated with T1 stage (p = 0.007), young age (p = 0.001), male gender (p = 0.02), sun-exposed location (p = 0.01), mixed/non-pigmented tumour colour (p = 0.02) and nevus origin (p = 0.005), but did not associate with prognosis. BRAF status in conjunctival melanoma and paired premalignant lesions corresponded in 19 of 20 cases. Immunohistochemistry detected BRAF V600E mutations with a sensitivity of 0.94 and a specificity of 1.00 in newer conjunctival melanoma samples (2000–2012, n = 47). Conclusion The incidence of conjunctival melanoma increased in Denmark over 50 years. The proportion of BRAF-mutated conjunctival melanoma was constant. BRAF mutations were identified as early events in conjunctival melanoma, associated with a distinct clinicopathological profile, similar to BRAF-mutated cutaneous melanoma. Immunohistochemical detection of BRAF can be used to assess BRAF V600E mutations.

Purpose As loss of cyclin dependent kinase inhibitors p27 and p21 have been identified in cutaneous melanoma and associated with a poorer prognosis, we decided to assess the expression of p27 and p21 in benign and malignant conjunctival melanocytic proliferations. Methods The expression of p27 and p21 was assessed by immunohistochemistry in 35 conjunctival naevi and 32 conjunctival melanomas. Statistical analysis was performed with JUMP 8.0 software. Immunohistochemistry was evaluated independently by two observers. Results There was a concordance in 94.02% and 92.54% of the cases in the evaluation of the expression of p27 and p21, respectively. Discrepant cases were simultaneously reviewed to achieve complete agreement. There were 13 subepithelial nevi and 22 compound nevi. There were 14 females and 21 males with a mean age was 36.9 ± 3.6 yo (SEM). Nuclear expression of p27 was found in all the nevi. p21 was identified in only 4 nevi (11.4%). The melanoma group was composed of 18 females and 14 males with a mean age of 65.9 ± 3.38 yo (SEM). p27 and p21 were respectively lost in 18 cases (56.25%) and in 24 cases (75%). There was a significant downregulation of p27 in conjunctival melanoma compared to benign conjunctival nevi (p > 0.0001). In the melanomas, there was a correlation between loss of p27 expression and depth of invasion (p = 0.0141) as well as non-bulbar tumor localization (p = 0.0279). Although the proliferation index was more elevated in melanomas with p27 loss, this was not significant. Conclusions The downregulation of p21 in benign melanocytic proliferations identified in the skin appears also to occur in the conjunctiva. Our results also demonstrate in vivo a significant downregulation of p27 in conjunctival melanoma. Restauration of p27 function in conjunctival melanoma might represent a potential therapeutical option for this tumor.

Purpose The prognosis for patients with disseminated ocular melanoma is poor. Responses to standard chemotherapy are low and serious toxicities are common. Here, we determine the feasibility of treating melanoma xenograft models in mice in order to guide the selection of treatments in metastatic patients. Methods A patient-derived xenograft model was established in SCID mice for metastatic choroidal and conjunctival melanoma. Mice bearing growing tumors were randomly assigned to the control or treatment groups. Mice were treated with dacarbazine and everolimus in the choroidal melanoma model. Dacarbazine and dabrafenib were used in the conjunctival melanoma model. Results Everolimus resulted in tumor stasis in the choroidal melanoma model while dacarbazine had no antitumor effect. In the conjunctival melanoma model the relative tumor volumes in mice treated with dacarbazine and dabrafenib were similar to the control group. Since this is a feasibility study we included only a limited number of cancer drugs. More drugs will have to be screened in future studies. The oncologists in our institution were aware of the results but it had no influence on the choice of treatments for the patients. To our knowledge this is the first report of a conjunctival melanoma xenograft model in SCID mice. Conclusion Our results suggest that ocular melanoma xenograft models can be used to determine which drugs achieve antitumor activity. These data could help oncologists in the choice of treatments for metastatic patients.

Purpose Since 2OOO, we have been using proton beam therapy after surgery for all invasive conjunctival melanomas. Patients with associated primary acquired melanosis also receive additional treatment with mitmycin 0, 04% drops at least two cycles. We have made a retrospective study to evaluate the results. Methods Patients were sent to Curie Institute for initial treatment, after the initial surgery or for recurrence. Surgery was performed under general anesthesia; proton beam was applied only on the area of invasive melanoma with a dose of 60 grays in 8 fractions. Results concerning the initial findings, the treatment and the follow up were prospectively registered. Results 62 patients were treated, 36 for initial treatment and 26 after surgery performed elsewhere. The median follow up is 24 months.(range 7 to 146 months) Median tumor diameter was 8 mm, with a thickness 2mm for 22 patients. TNM and histology will be described. Surgical excision was considered complete in 21 cases. All patients received proton beam irradiation and 25 received mitomycin drops. 10 patients (16%) presented local recurrence and 4 patients (6%) developed metastatic disease. Conclusion : Because of direct contact with the sclera, invasive conjunctival melanoma cannot be removed with sufficient margin if the eye is not removed. The use of proton beam therapy immediately after surgery allows good ocular preservation with very low rate of local recurrence .The prognostic of patients with primary acquired melanosis with atypia has been improved by the use of preventive treatment of the conjunctiva by mitomycin before invasive melanoma occurs.

Purpose As activating mutations of BRAF and NRAS have been identified in 47% of conjunctival melanoma, we evaluated the activation status of the MAP kinase and PI3K/mTOR pathways in benign and malignant conjunctival melanocytic proliferations. As loss of PTEN locus has been reported in conjunctival melanoma, we also assessed the expression of PTEN in conjunctival nevi and melanoma. Methods The phosphorylation status of MEK, ERK, AKT, S6 ribosomal protein and the expression of PTEN was evaluated by immunohistochemistry in 35 conjunctival naevi and 31 conjunctival melanoma. Statistical analysis was performed with JUMP 8,0 software. Immunohistochemistry was assessed independently by three observers. Results There were 13 subepithelial nevi and 22 compound nevi. There were 14 females and 21 males with a mean age was 36.9 yo. P-MEK, p-ERK, p-S6, p-AKT and PTEN were respectively found in 59%, 49 %, 49%, 83.9% and 100% of the nevi. The melanoma group was composed of 17 females and 14 males with a mean age of 67.1 yo. P-MEK, p-ERK, p-AKT, p-S6 and PTEN were respectively found in 87%, 87%, 94.3%, 94% and 96.7% of the melanoma. The phosphorylation of MEK and ERK, AKT and S6 was significantly elevated in the melanoma compared to the nevi (p=0,0050; p=0,0004; p=0,0031;p> 0,0001 respectively). There was also a significant correlation between the activation of MEK and ERK (p=0,0045). Conclusion Our results demonstrate ex vivo a significant increase in the activation of the MAP kinase pathway in malignant conjunctival melanocytic proliferations as well as a significant increase in the phosphorylation of pAKT and S6 ribosomal protein, suggesting a contribution of the PI3K/mTOR pathway to conjunctival melanoma development.

Purpose To investigate the expression of HSP90 in human conjunctival melanoma (CM) cell lines and primary human melanocytes, and to assess the effects of HSP90 inhibitors on cell growth and proliferation. Methods CM cell lines (CRMM1 and 2; CM2005.1) and primary human melanocytes (n=3, P2 to P4) were immunolabelled for HSP90, and visualised using immunofluorescence and confocal microscopy. HSP90 expression was also examined in cell lysates from CM cell lines (CRMM1 and 2; CM2005.1) and primary melanocytes (n=3). The effect of several HSP90 inhibitors (17DMAG, SM252 and SM122) on cell growth and viability was assessed at 24hrs. Results CRMM1 and 2, and CM2005.1 cells showed strong cytoplasmic expression of HSP90, with low levels of cytoplasmic expression in primary melanocytes. This was confirmed using western blot. The HSP90 inhibitors induced dose-dependent cell death in CM cells, 17DMAG (water soluble) being most effective (LD50 at 24 hrs ~30uM compared with ~50um for SM122 and SM122).Primary human melanocytes viability was not significantly reduced by the HSP90 inhibitors. Conclusion HSP90 is a widely expressed chaperone protein that can regulate cell growth, survival and angiogenesis pathways. CM cell lines expressed high levels of HSP90 protein compared to melanocytes, consistent with a recent study of primary conjunctival melanoma specimens, that found over-expressed HSP90 in CM, particularly recurrent tumours. HSP90 inhibitors may be useful in inhibiting CM growth alone or in combination therapies. Funding: National Foundation for Medical Research & Innovation (MCM & RMC); Lions NSW Eyebank

Purpose : In cutaneous melanoma, downregulation of miR-211 has been implicated in migration and invasion. As we have previously demonstrated the downregulation of miR-211 in conjunctival melanoma in vivo, we evaluated in vitro the role of miR-211 in the migration of 4 conjunctival melanoma cell lines compared to a cutaneous melanoma cell line. Methods : Migration assays were performed using a cutaneous melanoma cell line and 4 conjunctival melanoma cell lines. Taqman RT-PCR was performed to evaluate the level of miR-211 in each cell line. Results : Compared to the cutaneous melanoma cell line with a high migration potential, 2 conjunctival melanoma cell lines demonstrated moderate migration abilities while 2 others no migration. The migration potential of the cell lines was significantly inversely correlated with Taqman evaluation of miR-211 expression (p=0,0239). Conclusions : The migration ability of conjunctival melanoma cell lines is lower than a cutaneous melanoma cell line. The level of miR-211 expression in conjunctival melanoma appears in vitro to inversely correlate with the migration potential of the cell lines, confirming the downregulation of miR-211 observed in vivo in conjunctival melanoma.

Purpose To present a case report of a patient with atypical presentation of a conjunctival melanoma as an amelanotic tumor. Methods A 82-year-old female presenting a large amelanotic tumor in inferior tarsal conjunctiva of the right eye was referred to our hospital. Previous histological analysis diagnosed intraepithelial carcinoma. Imaging digital analysis techniques showed infiltration of the extrinsic ocular muscles but not the scleral tissue. Excisional biopsy was obtained by removing the tumor with free margins and preserving as many unaffected tissue as possible. Amniotic membrane graft was used to cover the surgical injury. Results Histopathological diagnosis confirmed amelanotic conjunctival melanoma. Systemic evaluation with imaging techniques (MRI, TC scan, Ecography…) revealed liver and kidney masses suggestive of metastasis and chemotherapy was prescribed to control the disease. Ophthalmological controls showed remission of tumor for 4 months, but new conjunctival masses appeared. At the present the patient is treated with palliative chemotherapy. Age and systemic status do not allow aggressive therapies in this patient. Conclusion Amelanotic melanoma is an atypical subtype of melanoma lacking of pigmentation. Its macroscopic aspect makes it almost impossible to clinically distinguish this tumor from less aggressive tumors, such as intraepithelial carcinomas. The diagnosis delay makes early treatment difficult in this malignant tumors.

Purpose Amplification of RUNX2 (6p21.2), a transcription factor involved in bone formation and in tumor progression of prostate and breast cancer, has previously been identified in 76% (16/21) of primary conjunctival melanoma and in 100% (4/4) of metastatic conjunctival melanoma. The aim of this study was to investigate RUNX2 expression at the protein level in a panel of conjunctival melanocytic proliferations Methods Expression of RUNX2 was assessed by immunohistochemistry in 26 conjunctival naevi (14 compound naevi, 11 subepithelial naevi and 1 cellular blue naevus), 13 PAM (9 PAM with atypia) and 25 conjunctival melanoma. Statistical analysis was performed with JUMP 8,0 software. Expression was regarded as negative if less than 10% of the tumor cells expressed RUNX2. Examination was independently assessed by two observers. Results RUNX2 was found in 3 naevi (11,5 %), in one PAM with atypia (7,6%) and in 5 melanoma (20 %) without significant variation between the 3 groups. In the melanoma group, there was no correlation between RUNX2 expression and TNM category, depth of invasion, proliferation index (Ki67), local lymphatic invasion and the presence of metastasis. There was however a significant correlation between RUNX2 expression and death (p=0,0191). Conclusion RUNX2 is expressed in a limited number of conjunctival melanocytic proliferations without significant differential expression between benign and malignant lesions. The biological significance of RUNX2 in conjunctival melanoma tumor progression requires further functional investigations.

Purpose To describe the experience, the indications, the outcome, complications and prognosis of strontium brachytherapy in bulbar conjunctival melanoma. Methods 46 with conjunctival melanoma, treated with strontium brachytherapy in Leuven, Leiden or Amsterdam, were revised. Clinical indications, visual outcome, prognosis and complications were registered. Results In 38 patients 6x10 Gy was used, with 2 recurrences and 1 scleral melting. In 7 cases 6x5,9 Gy was used with 4 recurrences. In 3 cases a second strontium brachytherapy application was needed. Although there were in almost all cases transient dry eye symptoms, no major anterior segment problems were seen. With the exception of 1 scleral melting,in a patient where also a previous extended surgery was done with excision of sclera. Conclusion Strontium brachytherapy is a safe treatment for conjunctival melanomas, when a dosis of 60 Gy is used. It gives rarely complications.

Purpose Downregulation of TRPM1, a transient receptor potential cation channel, has been correlated with a higher metastatic risk in skin melanoma. The promoter of TRPM1 contains MITF binding sites and MITF regulates in vitro the transcription of TRPM1. We have showed a partial loss of TRPM1 mRNA expression in a limited number of conjunctival melanoma. The aim of this study was to further investigate TRPM1 and MITF expression in a broader panel of conjunctival melanocytic proliferations Methods Expression of MITF and TRPM1 was assessed by immunohistochemistry in 17 conjunctival naevi, 8 PAM (6 PAM with atypia) and 16 conjunctival melanoma. Statistical analysis was performed with JUMP 8,0 software. Results A complete preservation of both MITF and TRPM1 expression was identified in all the naevi and the PAM. A partial loss of TRPM1 expression was found in 44% of the conjunctival melanoma (2 cases with a scattered loss of expression and 5 cases with a regional loss of expression). There was a significant partial loss of TRPM1 expression in the melanoma group compared with the naevi group (p=0,0027) or with the PAM group (p=0,0331). A partial loss of MITF was identified in 50% of the melanoma with significant reduction compared with the naevi group (p=0,0005) or with the PAM group (p=0,0262). There was a significant correlation between partial loss of TRPM1 expression and partial loss of MITF expression (p

Purpose To show the use of anterior segment optical coherence tomography (AS-OCT) in conjunctival, limbal, subconjunctival tumors and to compare the data with histopathologic specimens. Methods 15 patients (15 eyes) with conjunctival, limbal and subconjunctival lesions were examined using AS-OCT (Visante OCT 2.0, Carl Zeiss Meditec, Dublin, CA). Eyes were scanned using Anterior Segment Single, High Resolution Corneal and Raw Image modes. Gray-scale, OCT Color and Rainbow Color images were analyzed using built-in software. All tumors were excised and histopathologicaly examined. Results In all small tumor cases AS-OCT allowed to see the deeper lesion structure with complete penetration. In conjunctival melanoma, papilloma, subepithelial naevus and pterygia cases AS-OCT showed tumor structure, layers and extension clearly. Underlying cornea was also visible. In larger and solid tumors (squamous cell carcinoma, fibroma and lipoma) anterior surfaces were hyper-reflective, but the deeper structures of the lesions were incompletely penetrated. However, in all cases it was possible to differentiate cystic lesions from solid lesions. Histopathologic examination revealed the diagnosis and in small tumors confirmed the AS-OCT data showing tumor epithelium and underling layers. Conclusion AS-OCT may be a useful non-invasive diagnostic tool for the evaluation of ocular surface tumors in selective cases. AS-OCT may give useful information before planning the surgery.

Purpose Understanding molecular and biochemical events of pterygium may allow to use less invasive treatments. Several antigens have been investigated to study the physiopathology of this disease. TROP1 protein, a cell adhesion regulatory molecule, and TROP2, a cell surface calcium signal trasducer, are expressed by a large variety of human cancers. Methods The aim of this work is to evaluate TROP1 and 2 protein expression in primitive and recurrent of pterygia. The expression of these antigens was evaluated in limbal crypts (4 cases), corneal epithelium (2 cases), conjunctival melanoma (1 case), normal conjuntiva (4 cases) and pterygia(8 primitive and 4 recurrent). All samples were fixed in formalin, embedded in paraffin and stained by immunohistochemistry. The different expression of TROP1 and 2 was evaluated and correlated with markers normally expressed in pterygia,such as vimentin, ki67, survivin, MMP7, p63, cyclin D1 and p53. Results No expression of TROP1 was observed in corneal epithelium and in limbal crypt, few positive cells were observed in healthy conjuntival epithelium. TROP2 was observed in some cells of limbal crypt, but was not expressed in corneal epithelium, while was positive in conjuntival epithelium. A strong TROP1 and 2 expression was detected in primary pterygia. TROP 1 and 2 positivity correlated with expression of vimentin, ki67, survivin, MMP7, p63, cyclin D1 and p53. Interestingly TROP1 positivity decreased in recurrent pterygia. Conclusion TROP1 and TROP2 antigens can be use as marker also for ocular surface.

Purpose To characterize malignant conjunctival melanomas with extension and recurrence in the nasolacrimal system Methods Localization of the primary tumor and recurrences of 210 conjunctival melanomas treated in the Netherlands were reviewed for orbital and nasal tumors (1978-2008). On the basis of these cases and literature data, characteristics for nasolacrimal system extension and metastasis were reviewed. Results Six patients (3%) showed a recurrence of the primary conjunctival melanoma in the nasolacrimal system. Two of the six primary tumors were limbal tumors, the other four were diffuse tumors involving the fornix. In all six patients, the primary conjunctival melanomas were associated with primary acquired melanosis (PAM). During follow-up period (mean 11.6 ± 3 years; median 8.7 years) two patients developed metastases, and died during follow-up. Conclusion Patients should be advised to contact the treating ophthalmologist in the case of symptoms of epiphora, nose obstructions and epistaxis, especially non-bulbar and diffuse cases associated with PAM.

Purpose To describe the classification, grading and staging of conjunctival melanocytic proliferation. Methods We have audited our experience with conjunctival melanomas, using a novel mapping system and have found shortcomings in the current Tumour Node Metastasis (TNM) staging system. We have also reviewed our cases of intra-epithelial melanocytic neoplasia and confirmed other authors’ impressions that conjunctival ‘primary acquired melanosis with atypia’ is histologically similar to cutaneous in situ melanoma. To improve objectivity in the reporting of conjunctival intra-epithelial melanocytic neoplasia, we propose a scoring system based on pattern of melanocytic infiltration, density of melanocytes & degree of cellular atypia. Results The term ‘conjunctival melanosis’ should be used only to describe the slit-lamp appearance of hyperpigmentation. Histologically, this abnormality should be categorized as ‘hypermelanosis’ or ‘melanocytosis’. Hypermelanosis can either be primary or secondary to ocular or systemic disease. Benign melanocytosis comprises conjunctival melanocytic hyperplasia and naevi. Malignant melanocytosis is essentially melanoma, which is primary (in situ or invasive) or secondary (i.e., spreading to conjunctiva from adjacent tissues) or rarely metastatic. We suggest that the TNM staging system for conjunctival melanoma should be revised to: (1) include a Tis stage; (2) take account of superficial extent, invasion of adjacent tissues and caruncular involvement, in stages TI to TIII; and (3) to sub-categorize TIV disease so that there is better correlation with likely mortality. Conclusion We have revised the classification of conjunctival melanocytic proliferations & improved the grading and staging of melanoma. These developments should be useful in treatment & research.

Purpose Analysis of patients with conjunctival melanoma. Methods Retrospective analysis of 61 consecutive patients with histopathologic diagnosis of conjunctival melanoma treated between 1991-2007 at the Department of Ophthalmology and Ocular Oncology of Jagiellonian University in Krakow. There were 30 (49.2%) women and 31 (50.8%) men in mean age of 57.6 years (25-89). Results The tumors involved in 29 cases right eye and in 32 cases left eye. 26 (42.6%) melanomas were located in temporal quadrant; 37 (60.6%) tumors involved limbus and 22 (36.1%) bulbar conjunctiva; in 2 (3.3%) patients tumors infiltrated tarsal conjunctiva. 53 tumors (86.9%) were pigmented, 5 (8.2%) - amelanotic and 3 tumors (4.9%) - mixed. In 59 (96.7%) patients the tumors were nodular and in 2 (3.3%) diffuse, superficial. In all cases surgical excision was performed and in 44 (73.13%) cases adjunctive Ru-106 brachytherapy. In 20 (32.7%) cases recurrence of melanoma necessitating secondary treatment was observed. The mean time to recurrence was 24.65 months (2-91). In 5 (8%) cases documented metastasis occurred. Among all patients 5 (8%) died because of metastatic disease, 3 because of other reasons. 6 cases were lost to follow-up because they moved to other countries. Conclusion Conjunctival melanoma most commonly occurs in form of a melanocytic, nodular tumor localized in corneal limbus and bulbar conjunctiva. Local recurrence of the tumor may be expected despite the surgical excision combined with adjunctive therapies.

Purpose To evaluate the treatment of conjunctival melanoma at the Institut Curie in term of local recurrences, ocular preservation, distant metastasis and survival Methods Retrospective study of patients with conjunctival melanoma treated for their first tumoral localisation between January 2000 and December 2005. The clinical records, histologic features, ocular treatments were rewiewed. The rate of local recurrences, ocular preservation, regional and distant metastasis, and survival were recorded. Results During the study period, 91 patients were referred for conjunctival melanoma. 67 of them were treated for a first occurrence of malignant melanoma (38 women and 29 men). The mean age was 62 years (25 – 90). 32/67 patients (47 %) presented a primary acquired melanosis (PAM). Most cases (55 patients) were treated by surgical excision and adjunctive radiotherapy (Iodine plaque, protonbeam or external beam irradiation), 5 cases were treated by surgery alone, and the last 6 by surgery and adjunctive cryotherapy or adjunctive topical chemotherapy. 3 patients were lost of follow-up. With a mean follow-up of 52 months (15-101months) 20/64 patients developped at least one local recurrence (between 1 and 7 recurrences). 16 of them presented a PAM. An enucleation was finally necessary for 1 patient and an exenteration for 2 patients. 7 patients developped metastatic disease (all but one presented a PAM) and 6 of them died. Conclusion Local recurrences, metastatic disease and tumor related death appear to occur at higher frequency when melanoma is associated with PAM. This risk must be taken into account in the therapeutic approach of conjunctival melanoma.

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.

Purpose Since 1985, a new therapeutic strategy for the conservative treatment of conjunctival melanoma has been developed at the Jules Gonin Eye Hospital (Lausanne). In order to evaluate its long-term results, we had to identify and classify our patients. Methods We looked retrospectively in our clinical and histopathological databases for all cases of conjunctival melanoma treated in Lausanne since 1985. Results 189 patients were identified. We studied patients’ parameters, clinical presentation and histopathological characteristics of all consecutive conjunctival melanoma cases. Conclusion A database of 189 conjunctival melanoma patients treated with the same therapeutic strategy since 1985 was established. This collective will allow for further studies with regard to the long-term results of the Lausanne conservative therapeutic strategy of this rare ocular tumour.

A case of primary acquired melanosis of the conjunctiva is reported. During the 18 years of observation the patient developed a malignant melanoma in the iris, and seven years later a malignant melanoma appeared in the lower lid. The iris melanoma may be an extension from the precancerous melanosis, but the most likely explanation is that the condition represents two independent diseases.