Surgical Outcome After Treatment of Radiation-Induced Scleral Necrosis in Patients With Uveal Melanoma.

PURPOSE: Surgical repair might be required in patients with uveal melanoma (UM) that develop advanced forms of radiation-induced scleral necrosis (RISN). In this monocentric long-term observational study, we aimed at analyzing the treatment outcome after RISN surgery. METHODS: All consecutive cases with UM who underwent surgical intervention for RISN between 1999 and 2020 were included. Achievement of the tectonic stability and evaluation of incidence and the risk factors for a repetitive patch surgery (RPS) were the main endpoints. RESULTS: The final analysis included 57 patients (mean age: 58.7 years; 63.2% female patients), where 55 individuals underwent a patch grafting, and 2 cases were treated with conjunctival reconstructive surgery. The mean follow-up time after grafting was 38.5 months (0.03-221.1 months). Tectonic stability was achieved in 56 (98.3%) patients. Scleral graft (38/55, 69.1%) was the most frequent patching material, followed by Tutopatch (7/55, 12.7%), corneal graft (7/55, 12.7%), dura graft (2/55, 3.6%), and fascia lata (FL) graft (1/55, 1.8%). Eleven patients (20%) underwent RPS after the mean time of 12.9 months (0.3-50.3 months). In the final multivariate Cox regression analysis, the use of Tutopatch (5/7; 71.4%, adjusted hazard ratio = 4.66, P = 0.044) and RISN progression after patch grafting (9/11; 81.8%, adjusted hazard ratio = 9.67, P = 0.008) were independent risk factors for RPS. CONCLUSIONS: RISN surgery maintains long-term tectonic stability in most of the cases underwent surgical repair for RISN after brachytherapy for UM. Depending on graft material and, particularly, further RISN progression, an RPS might be necessary in certain cases.

P30-A119†Microbial contamination of amniotic membrane.

PURPOSE: This retrospective study aims to compare the rate of microbial contamination in fresh, non-preserved amniotic tissue as opposed to decontaminated cryopreserved tissue, thereby being able to determine the efficiency of the decontamination procedures applied during amniotic tissue preparation in the Cornea Bank Essen. METHODS: The amniotic tissue was retrieved from donor placentas acquired through elective c-section. Tissue preparation was performed according to standard operation procedures of the Cornea Bank Essen. Briefly, the tissue is rinsed with sterile balanced salt solution (BSS) and decontaminated with BSS containing anti-infectives. Preservation included the application of a cryopreservation solution containing anti-infectives and glycerin. The tissue is stored at a temperature of -80°C. Screening for microbial contamination of amniotic tissue in its pre- and post decontamination status is part of the process.In this study, data from 107 placentas prepared in the eye bank were retrospectively evaluated for the microbiological status to determine the effectivity of the procedure. RESULTS: Out of the fresh, non-preserved amniotic tissue, 53 were tested positive for microbial contamination. The most common species identified were C.acnes and Staphylococcus spp., which jointly comprised around 80% of the detected microorganisms. Others found in the remaining placentas were of the species: Acinetobacter, Bacillus spp., Faklamia, Lactobacillus, Rothia, Micrococcus, Penicillium, Ralstonia, Streptococcus and non-specific aerobic sporulating bacteria.In contrast, 8 samples of the decontaminated cryopreserved tissue were tested positive for microorganisms with 4 placentas inhabited by C.acnes, 2 by Bacillus spp. while the remaining consisting each of the species Staphyloccocus and Ralstonia. CONCLUSION: Overall, the decontamination measures applied during the preparation of the amniotic tissue can be regarded as effective. We found a significant reduction of the number of microorganisms detected in the amniotic tissue following antibiotic administration.However, some of the remaining species identified in the processed samples may be considered as contamination during the preparation and testing procedures.For instance, C.acnes can be considered a result of secondary contamination due to incorrect handling. Species such as Bacillus most likely managed to endure the decontamination process owing to its natural resilience against harsh circumstances.

Brachytherapy for Peripheral Retinal Capillary Haemangioblastoma in von Hippel-Lindau Disease. / Brachytherapie peripherer retinaler kapillärer Hämangioblastome beim Von-Hippel-Lindau-Syndrom.

AIM: To report our experience with 106ruthenium-brachytherapy of peripheral capillary haemangioblastomas in patients with von Hippel-Lindau disease. DESIGN: Retrospective case series. METHODS: A total of 53 haemangioblastomas, treated with 106ruthenium-brachytherapy, were included in our study. The applied radiation dose, visual outcome, angioma activity, need for vitreoretinal surgery and incidence of secondary complications such as macular oedema, secondary glaucoma, vitreous haemorrhage, and epiretinal gliosis were assessed. RESULTS: All treated eyes could be preserved. In 11 patients (20.8%), single brachytherapy did not achieve complete inactivation of the tumour. 31% developed macular oedema postoperatively. Tractional retinal detachment developed in 23.8%, and epiretinal gliosis was observed in 2.4% of patients. Vitreoretinal surgery was necessary in 50% of all treated eyes. At the end of the follow-up, 40.5% of all treated eyes achieved visual acuity (VA) of 0.6 or better, and one third reached a VA of less than 0.1. Mean irradiation dose to the tumour apex was 144 Gy. Higher apex doses correlated with better tumour control of irradiated haemanigoblastomas and lower complication rates. CONCLUSIONS: Brachytherapy of peripheral retinal capillary haemangioblastomas is an effective treatment modality. Higher irradiation doses seem to lead to more successful treatment.

SARS-CoV-2: Impact on, Risk Assessment and Countermeasures in German Eye Banks.

INTRODUCTION: Since the beginning of the COVID-19 pandemic there has been some debate regarding the risk of transmission through tissue transplantation and tissue banking processes. AIM OF THE STUDY: To analyze the changes that SARS-CoV-2 has caused regarding the harvesting of corneal donor tissue and eye bank activities in Germany. METHODS: A questionnaire was provided to 26 eye banks in Germany, consisting of questions about adaptations made in the screening of potential donors and the harvesting of corneal tissue following the pandemic spread of SARS-CoV-2. RESULTS: Eighteen eye banks actively reduced recruitment of donors and two banks ceased all activity. Additional diagnostic screening was performed in eight banks, using conjunctival swabs and/or nasopharyngeal swabs. In six eye banks, additional protective measures, such as FFP2 masks and/or facial shields, were implemented. Overall, a mean reduction in the number of obtained donor tissues of 17% was observed. DISCUSSION: Conjunctival and/or nasopharyngeal swabs of donors have been implemented by a minority. Reasons for not performing additional tests may be moderate sensitivity and lack of validation for postmortem use of RT-PCR testing. Also, the hazard of SARS-CoV-2 entering the corneal donor pool with subsequent transmission might be perceived as theoretical. Face shields provide a sufficient barrier against splash and splatter contamination but may be insufficient against aerosols. Additional face masks would provide support against aerosols, but it remains debatable if corneal harvesting can be considered an aerosol-producing procedure. In the future we expect to see changes in current guidelines because of a surge in scientific activities to improve our understanding of the risks involved with cornea donation in the COVID-19 pandemic, and because current practice may reduce the availability of donor corneas due to new exclusion criteria while the demand remains unchanged.

[Rehabilitation Following Limbal Stem Cell Deficiency: Current Treatment Options and Future Developments]. / Optische Rehabilitation nach Limbusstammzellinsuffizienz: aktuelle Behandlungsmöglichkeiten und Entwicklungen.

Limbal stem cell deficiency (LSCD) is a condition caused by the loss of corneal epithelial regenerative potential. The treatment of this condition is still a challenge. It results from various conditions both intrinsic as well as extrinsic. LSCD can be either uni- or bilateral and either partial or total. Today treatment options include a variety of techniques including transplantation of amniotic membrane and limbal tissue or tissue engineered cell sheets. This article summarizes the current techniques to treat LSCD and upcoming developments.

Frequent TERT Promoter Mutations in Ocular Surface Squamous Neoplasia.

PURPOSE: Ocular surface squamous neoplasia, including intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma (SCC), are one of the most common malignant tumors of the conjunctiva. Little is known of the genetic alterations involved in their pathogenesis. Promoter mutations in telomerase reverse transcriptase (TERT) have been identified in various cancers, including many associated with ultraviolet (UV) exposure. Our study analyzes the mutation rate and clinicopathological associations of TERT promoter mutations in ocular surface squamous neoplasia. METHODS: DNA was isolated and the region of the TERT promoter where hotspot mutations can occur analyzed by Sanger-sequencing in 48 ocular surface squamous neoplasia tumor samples (6 CIN and 42 SCC). An analysis of associations between TERT promoter mutation status and various clinicopathological parameters was performed. RESULTS: We identified TERT promoter mutations in 21 of 48 ocular surface squamous neoplasia samples (43.8%), including 4 in CIN and 17 in SCC. The mutations consisted of 8 Chr.5:1295228C>T, 1 Chr.5:1295228_1295229CC>TT, 5 Chr.5:1295242_1295243CC>TT, and 12 Chr.5:1295250C>T mutations. All mutations were C>T or CC>TT alterations, demonstrating a UV-signature. TERT promoter mutations showed no statistically significant associations with clinicopathological parameters. CONCLUSIONS: Telomerase reverse transcriptase promoter mutations are found in almost half of ocular surface squamous neoplasias and have a mutation profile supporting UV induction as the major source of mutagenesis. We conclude that UV induced TERT promoter mutations leading to aberrant overexpression of telomerase is a major pathogenetic factor in ocular surface squamous neoplasia.

Establishment of a Cell Line From Conjunctival Squamous Cell Carcinoma: PeCa-UkHb-01.

PURPOSE: Until now, no epithelial cell line from conjunctival squamous cell carcinoma (SCC), to our knowledge, has existed; therefore, the establishment of a model cell line would be a useful tool for further studies. In particular, the phenotypic and molecular characterization in comparison to other SCC cells is of high interest because this would enable the development of new treatment options for clinical application in ophthalmic oncology. METHODS: Epithelial cells were isolated from a bulbar conjunctiva SCC obtained from a 74-year-old male, harvested by stepwise trypsinization and named PeCa-UkHb-01. Cell doubling and the number of passages were determined. Short tandem repeats (STR) and karyotype analyses were performed. Semiquantitative real-time PCR and immunocytochemical fluorescence staining were carried out to detect tumor and epithelial cell markers. RESULTS: The cells had an epithelial and conjunctival phenotype. They grew above passage number 50 in a doubling time at approximately 34.5 hours. Short tandem repeat analyses confirmed the cell origin, although loss of heterozygosity occurred. Karyotype analyses revealed a heterogeneous composition of the cell culture and the karyogram itself showed aberrations and changes in the chromosome numbers. Real-time PCR and immunocytochemical fluorescence staining revealed the expression of the stem cell markers such as ABCG2, p63, OCT4, c-MYC, and SOX2 as well as the conjunctival cytokeratin K19. CONCLUSIONS: PeCa-UkHb-01 cells fulfill the criteria of a cell line. They display characteristics of ocular carcinoma cells and therefore the presented cell line might serve for further basic research in ophthalmic oncology.

Expression of pluripotency and multipotency factors in human ocular surface tissues.

AIM: Mechanisms that control ocular surface stem cells (SCs) are unclear. Recent studies have shown that several adult SCs express pluripotency markers. Our objective was to analyze the expression of key molecules of pluripotency in human ocular surface tissues as well as in cultivated limbal epithelium. METHODS: Four samples of human corneal, limbal and on amniotic membrane cultivated limbal epithelium (HLEC-AM), as well as bulbar and fornical conjunctiva were analyzed. Human embryonic stem (ES) cells and human umbilical vein endothelial cells served as controls. Expression of corneal epithelial differentiation markers (K3, K12, Cx43), putative limbal SC markers (ABCG2, p63, K15), and molecules associated with pluripotency/multipotency (NANOG, OCT4, SOX2, KLF4, KIT, NESTIN, PAX6, NOTCH1) was examined using real-time polymerase chain reaction (PCR) and immunohistochemical staining. RESULTS: Limbal epithelium showed a significantly (p < 0.05) higher expression of K15, ABCG2, OCT4, SOX2, NESTIN and NOTCH1, but a lower expression of K3 than corneal epithelium. Besides a higher expression of ABCG2 in fornix, the expression of pluripotency markers was similar in both conjunctival regions, although lower than in limbal epithelium. Expression of pluripotency factors in ES cells was significantly higher than in ocular surface SCs, whereas the expression in limbal epithelium was the closest to ES cells. HLEC-AM in comparison to limbal epithelium showed a lower expression of differentiation markers, a similar expression of ABCG2 but a significantly lower expression of pluripotency factors. CONCLUSION: Human ocular surface epithelial cells and especially limbal epithelial cell express genes are important for pluripotency and may have preserved some common mechanisms with pluripotent SCs.

Amniotic membrane transplantation in the human eye.

BACKGROUND: Amniotic membrane transplantation (AMT) has a long tradition in ophthalmic surgery and has become very popular recently because of newly developed methods of tissue preservation. METHODS: We selectively review the literature on recent developments, mechanisms of action, and established indications of AMT in the treatment of various diseases of the ocular surface. We searched the PubMed database for articles that appeared from 1994 to 2009 with the key words "amniotic membrane," "cornea," and/or "conjunctiva." RESULTS: Amniotic membrane (AM) can function in the eye as a basement membrane substitute or as a temporary graft. It has anti-inflammatory and anti-scarring effects and contains growth factors that promote epithelial wound healing on the surface of the eye. AMT has been found to be a good alternative for corneal and conjunctival reconstruction in many clinical situations, including acute burns, persistent epithelial defects of the cornea, and diseases that cause conjunctival scarring. Nonetheless, there have been no more than a few randomized and controlled trials of AMT to date. Other studies have shown that AM can serve as a culture substrate to expand epithelial progenitor cells for use in ocular surface reconstruction. CONCLUSION: AMT is an established technique in the treatment of various diseases of the external eye. In the last few years, AMT has brought about major advances in the reconstructive surgery of the ocular surface.

Comparison of cryopreserved and air-dried human amniotic membrane for ophthalmologic applications.

BACKGROUND: Cryopreserved amniotic membrane (Cryo-AM) is widely used in ocular surface surgery because of its positive effect on wound healing and its anti-inflammatory properties. A new peracetic acid/ethanol sterilized air-dried amniotic membrane (AD-AM) recently became available which might be an alternative to Cryo-AM. Our aim was to compare AM preserved with both methods with regard to the release of wound-healing modulating proteins, the preservation of basement membrane components, and the ability to serve as a substrate for the cultivation of human limbal epithelial cells (HLECs). METHODS: Pieces of Cryo-AM and AD-AM from three different donors were incubated in DMEM for five days. The culture supernatant was collected after an incubation period of 0.1, 24, 48, 72 and 120 h; in the case of AD-AM, this period was extended up to 14 days. TIMP-1, IL-1ra, CTGF and TGF-beta1 were detected in the culture supernatant using Western blotting. Twenty human limbal epithelial cultures were initiated on both AD- and Cryo-AM. The cultures were analyzed morphologically, and the outgrowth area was measured in 3-day intervals. Cryosections of Cryo- and AD-AM from three different donors were analyzed histochemically to detect the basement membrane components collagen IV, collagen VII, laminin, laminin 5 and fibronectin. RESULTS: The release of TIMP-1, IL-1ra and TGF-beta1 from Cryo-AM was constant for the studied period. CTGF showed a stronger signal after 120 h. None of the analyzed proteins, except for a small amount of IL-1ra, could be detected in the supernatant of AD-AM. An outgrowth of HLEC was observed in all cultures on Cryo-AM, but in only 30% of cultures on AD-AM. The outgrowth area on Cryo-AM was at all time points significantly higher than on AD-AM (p < 0.0001). Collagen IV, -VII, laminins and fibronectin were detectable in the basement membrane of Cryo-AM, but only collagen IV and fibronectin in AD-AM. CONCLUSIONS: Cryo-AM is a more suitable substrate for the cultivation of HLECs than AD-AM. The higher outgrowth rate of cultured limbal epithelium, release of intact soluble wound-healing modulating factors and a better preservation of basement membrane components suggest the superiority of Cryo-AM for use in ophthalmology in comparison to AD-AM.