Henrike Westekemper

Conjunctival Melanomas Harbor BRAF and NRAS Mutations and Copy Number Changes Similar to Cutaneous and Mucosal Melanomas

Purpose: Conjunctival melanoma is a rare but potentially deadly tumor of the eye. Despite effective local therapies, recurrence and metastasis remain frequent. Once the tumor has metastasized, treatment options are limited and the prognosis is poor. To date, little is known of the genetic alterations in conjunctival melanomas. Experimental Design: We conducted genetic analysis of 78 conjunctival melanomas, to our knowledge the largest cohort reported to date. An oncogene hotspot array was run on 38 samples, screening for a panel of known cancer-relevant mutations. Thirty tumors were analyzed for genome-wide copy number alterations (CNA) using array-based comparative genomic hybridization. Sanger sequencing of selected target mutations was conducted in all samples. Results: BRAF mutations were identified in 23 of 78 (29%) tumors. NRAS mutations, previously not recognized as relevant in conjunctival melanoma, were detected in 14 of 78 (18%) tumors. We found CNAs affecting various chromosomes distributed across the genome in a pattern reminiscent of cutaneous and mucosal melanoma but differing markedly from uveal melanoma. Conclusions: The presence of NRAS or BRAF mutations in a mutually exclusive pattern in roughly half (47%) of conjunctival melanomas and the pattern of CNAs argue for conjunctival melanoma being closely related to cutaneous and mucosal melanoma but entirely distinct from uveal melanoma. Patients with metastatic conjunctival melanoma should be considered for therapeutic modalities available for metastatic cutaneous and mucosal melanoma including clinical trials of novel agents. Clin Cancer Res; 19(12); 3143–52. ©2013 AACR.

Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency.

BACKGROUND Limbal stem cell deficiency (LSCD) leads to growth of abnormal fibro-vascular pannus tissue onto the corneal surface as well as chronic inflammation and impaired vision. Our aim was to investigate the clinical outcome of ocular surface reconstruction in LSCD using limbal epithelial cells expanded on amniotic membrane (AM). METHODS Forty-four eyes of 38 patients (27 male, 11 female) with total (n = 32) or partial (n = 12) LSCD were treated by transplantation of autologous (n = 30) or allogeneic (n = 14) limbal epithelial cells expanded on intact AM. LSCD was caused by chemical and thermal burns (n = 22), pterygium (n = 9), congenital aniridia (n = 6), tumor excision (n = 2), perforating eye injury, mitomycin C, epidermolysis bullosa, bilateral graft-versus-host disease and chlamydial conjunctivitis (each n = 1). RESULTS Mean follow-up time was 28.5 +/- 14.9 months. The corneal surface could be reconstructed to full stability in 30 (68%), and clear central cornea was achieved in 37 (84%) eyes. Grafting was significantly more successful in eyes treated by autologous than by allogeneic transplantation (76.7 vs. 50%, p < 0.05). The corneal surface could be successfully restored in 10 (83.3%) eyes with partial LSCD and in 20 (63.3%) eyes with total LSCD. Visual acuity (VA) increased significantly in 32 (73%) eyes, was stable in 10 (23%) eyes and decreased in 2 (4%) eyes. Mean VA increased significantly (p < 0.0001), from preoperative 1.7 +/- 0.9 log MAR (20/1,000) to 0.9 +/- 0.7 log-MAR (20/160). VA increased significantly after both autologous (p < 0.0001) and allogeneic transplantation (p < 0.005). CONCLUSIONS In most patients with LSCD, transplantation of limbal epithelium cultivated on intact AM restores the corneal surface and results in significantly increased VA.

TERT promoter mutations in ocular melanoma distinguish between conjunctival and uveal tumours

Background:Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma. We tested a cohort of ocular melanoma samples for similar mutations.Methods:The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas.Results:Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas. Mutations had a UV signature and were identical to those found in cutaneous melanoma.Conclusion:Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas. Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.

Expression of HSP 90, PTEN and Bcl-2 in conjunctival melanoma

Background In conjunctival melanoma, little is known about the tumour biology and protein-expression patterns. In this study, the authors analysed the expression of the antiapoptotic oncoprotein B cell leukaemia/lymphoma-2 protein (Bcl-2), the tumour-suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN), and the heat-shock-protein HSP-90 in conjunctival melanoma (CoM) and conjunctival nevi (CoN) by immunohistochemistry (IHC). Material and methods IHC was performed on 70 samples of CoM and 12 samples of CoN. Expression patterns between the diagnosis groups were compared. A receiver operating characteristic analysis was performed to determine the diagnostic value of the antigens. Results HSP-90 (p<0.0001) and PTEN (p=0.001) showed the potential to differentiate between CoM and CoN. Bcl-2 expression was higher in CoM than in CoN (p=0.04). The loss of nuclear PTEN expression was more pronounced in the malignant melanomas than in CoN (p=0.02). Tumours located at unfavourable sites (fornix, palpebral conjunctiva, caruncle) that had developed recurrences expressed almost twice as much HSP-90 than recurrence-free tumours. Conclusions Conjunctival melanocytes differentially express Bcl-2, HSP-90 and PTEN, depending on their entity. HSP-90- and PTEN expression may add relevant information for the differentiation between conjunctival melanoma and nevi.

Maligne epibulbäre Tumoren: Neue Strategien in Diagnostik und Therapie

In this article we discuss the complex diagnostic approaches and therapeutic options for the most important conjunctival malignancies. Conjunctival melanoma can be a diagnostic challenge as it can be difficult to distinguish from benign melanocytic conjunctival tumours. Complete surgical excision accompanied by a coherent adjuvant concept is the key for a curative therapy. Moderate and severe conjunctival intraepithelial neoplasias (CIN) are precancerous lesions and can progress to invasive squamous cell carcinoma. The involvement of large parts of the ocular surface can prevent an R 0-resection. Adjuvant therapeutic concepts are therefore especially important to gain tumour control and preserve the function of the affected eye. Lymphomas are the most common malignant primary tumours of the orbit and ocular adnexa. They can present as primary or secondary tumours of the conjunctiva, the lacrimal gland, the orbital fat, the eye lid or the lacrimal sac. The most common manifestation site of ocular MALT lymphoma is the conjunctiva with 20 - 33 % of all epibulbar lymphomas. More than 75 % of ocular lymphoma patients develop only one lymphomatous lesion. Immunophenotyping allows the exact differentiation between the lymphoma entities. Infectious agents (e.g., Chlamydia psittaci) seem to play a role in the pathogenesis. An overview over radiotherapeutic approaches that are conclusively applicable at the conjunctiva completes the article.

Clinical outcomes of amniotic membrane transplantation in the management of acute ocular chemical injury

Background Amniotic membrane transplantation (AMT) has been used in the management of acute ocular chemical burns to promote epithelialisation, reduce inflammation and restore ocular surface integrity. The aim of this study is to analyse the morphological and functional outcomes of patients receiving AMT after ocular chemical burn. Methods We performed a retrospective analysis of all patients treated for acute ocular chemical burn between 1998 and 2008 in two participating centres (University of Duisburg-Essen, Germany and Royal Victoria Infirmary, Department of Ophthalmology, Newcastle University, UK). Ocular chemical burns were classified by Roper-Hall and Dua classifications. Results 72 eyes of 54 consecutive patients aged 37.3 years (±SD 11.6 years) were included in this cohort study. 7 chemical burns were acid burns, 61 were alkaline and 4 were of unknown origin. In 37 eyes (51.4%), AMT was applied within the first 6 days after injury. Mean follow-up time was 36.4 months (median 18.5; 1.3–117.3  months). Overall, 29 eyes (40.3%) achieved a best-corrected visual acuity of LogMAR 0.2 (0.63 decimal) or better at final visit. Complete 360° limbal stem cell deficiency (LSCD) occurred in 33 eyes (45.8%), while partial LSCD occurred in 21 eyes (29.2%). Conclusion AMT is an effective adjunctive treatment in the management of acute ocular chemical burns to support epithelial healing and restore ocular surface integrity with potential to improve vision. However, long-term debilitated vision remained in those with severe burns complicated by LSCD.

Analysis of SDHD promoter mutations in various types of melanoma.

Objectives Recently, recurrent mutations in regulatory DNA regions, such as promoter mutations in the TERT gene were identified in melanoma. Subsequently, Weinhold et al. reported SDHD promoter mutations occurring in 10% of melanomas and being associated with a lower overall survival rate. Our study analyzes the mutation rate and clinico-pathologic associations of SDHD promoter mutations in a large cohort of different melanoma subtypes. Methods 451 melanoma samples (incl. 223 non-acral cutaneous, 38 acral, 33 mucosal, 43 occult, 43 conjunctival and 51 uveal melanoma) were analyzed for the presence of SDHD promoter mutations by Sanger-sequencing. Statistical analysis was performed to screen for potential correlations of SDHD promoter mutation status with various clinico-pathologic criteria. Results The SDHD promoter was successfully sequenced in 451 tumor samples. ETS binding site changing SDHD promoter mutations were identified in 16 (4%) samples, of which 5 mutations had not been described previously. Additionally, 5 point mutations not located in ETS binding elements were identified. Mutations in UV-exposed tumors were frequently C>T. One germline C>A SDHD promoter mutation was identified. No statistically significant associations between SDHD promoter mutation status and various clinico-pathologic variables or overall patient survival were observed. Conclusions Melanomas harbor recurrent SDHD promoter mutations, which occur primarily as C>T alterations in UV-exposed melanomas. In contrast to the initial report and promoter mutations in the TERT gene, our analysis suggests that SDHD promoter mutations are a relatively rare event in melanoma (4% of tumors) of unclear clinical and prognostic relevance.

Expression of MCSP and PRAME in conjunctival melanoma

Background To analyse the expression of melanoma chondroitin sulfate proteoglycan (MCSP) and the preferentially expressed antigen of melanoma (PRAME) in conjunctival melanoma (CoM), lymph node (LN) metastases of cutaneous melanoma (CM) and conjunctival nevi (CoN) by immunohistology. Methods Immunohistology was performed in 70 samples of CoM, 25 of LN metastases of CM and 12 of CoN, and assessed by an immunoreactive score (0–12 points). Statistical analysis was performed to disclose relevant differences in the expression pattern. The diagnostic value of the markers was tested by receiver operating characteristics (ROC) analysis. Results MCSP and PRAME were expressed at significantly higher levels in CoM and LN metastases of CM than in CoN (p<0.0001). Within CoM, an MCSP expression <9.0 points meant higher risk for recurrences (Cox HR=3.1) and a shorter recurrence-free survival (p=0.002) than an MCSP expression >9.0 points. ROC analysis showed an area under the curve of 91.3% for MCSP (p=0.0002) and 93.8% for PRAME (p<0.0001). Conclusions MCSP and PRAME are differentially expressed in conjunctival melanomas and nevi. MCSP might have an impact on the risk for recurrence in being inversely correlated to the event. Both markers have high potential to discriminate CoM from CoN. The results indicate that immunohistological characteristics gain relevance in the assessment of CoM.

Conjunctival Melanomas Harbor BRAF and NRAS Mutations— Response

In our recently published study ([1][1]), we describe genetic similarities between conjunctival and cutaneous melanoma and propose that the treatment rationale for patients with metastasized conjunctival melanoma should be adapted to that for metastasized cutaneous melanoma. Weber and colleagues ([2

Chemosensitivity of conjunctival melanoma cell lines to single chemotherapeutic agents and combinations.

Objective Two conjunctival cell lines (CRMM-1 and CRMM-2) have been established from recurrent conjunctival melanoma. The authors examined the chemosensitivity of these cell lines to cytotoxic substances and combinations to identify substances that inhibit cell growth efficiently in vitro. Material and methods CRMM-1 and CRMM-2 were exposed to cisplatin, mitomycin C (MMC), all-trans-retinoic-acid (ATRA), fotemustine or imatinib for 24 h. Sulforhodamine-B assays were used to assess the IC50. Isobolograms were performed to test possible synergism and antagonism with ATRA or imatinib. Results Cisplatin and MMC were efficient to inhibit the growth of CRMM-1 and CRMM-2. Combination of imatinib with MMC showed additive antitumoral effect on both cell lines. Combined treatment of imatinib with fotemustine or cisplatin resulted in antagonism. Strong antagonisms were also obtained with ATRA and fotemustine or cisplatin in both cell lines. A synergism was found for ATRA and mitomycin or imatinib in CRMM-2, in contrast to CRMM-1, where antagonism was obtained. Conclusions Cisplatin and MMC inhibit cell growth in conjunctival melanoma cell lines. The potential of ATRA was evident only in combination with MMC or imatinib in CRMM-2 cells. Imatinib and mitomycin increased their efficiency under combination therapy.