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PurposeMolecular pathogenesis underlying persistent ocular surface inflammation in chronic Stevens-Johnson syndrome (SJS) still remains largely unexplored. The present study investigates the expression of matrix metalloproteinase 2 (MMP2), MMP3, MMP9, MMP11 and TIMP1 (tissue inhibittor of matrix metalloproteinase 1) in pannus tissues of chronic ocular SJS undergoing cultivated oral mucosal epithelial transplantation (COMET) and their prognostic relevance.MethodsIn this prospective study, 45 eyes with chronic SJS underwent COMET for visual and anatomical rehabilitation. Preoperative and postoperative clinical parameters were documented. MMP2, MMP3, MMP9, MMP11 and TIMP1 expression were assessed using immunohistochemistry and quantitative real time PCR. Inflammadry MMP9 assay was performed at 1-year follow-up. Kaplan-Meier curves and Cox proportional hazard models were used to correlate protein expression with clinicopathological parameters and COMET graft survival outcomes.ResultsMMP9 and MMP11 positivity was seen in both pannus epithelia (48% and 55%, respectively) and in stromal layer (57% and 33%, respectively) while MMP2 and MMP3 showed only pannus epithelial positivity in 35% and 51% cases, respectively. High MMP9 stromal expression was significantly associated with preoperative corneal keratinisation (p=0.011), conjunctival hyperaemia (p=0.014), symblepharon (p=0.028). High MMP9 and MMP3 epithelial expression were found to be independent risk factors for poor best-corrected visual acuity (BCVA) outcomes post-COMET (p=0.022 and p=0.048). Multivariate analysis revealed MMP9 to be the best prognostic marker (p=0.050).ConclusionOur findings suggest that differential expression of MMPs and TIMP1 is seen in SJS in chronic stage. Emergence of MMP9 as a poor prognostic predictor of BCVA post COMET and postoperative MMP9 immunoassay positivity could be a useful tool in further studies to understand the unresolved ocular surface inflammation seen in SJS.

Reactive oxygen species (ROS) are essential for cancer signalling pathways and tumour maintenance, making ROS targeting a promising anti-cancer strategy. Coenzyme Q 10 (CoQ10) has been shown to be effective against various cancers, but its impact on retinoblastoma, alone or with trolox, remains unreported. Cytotoxicity of CoQ 10 alone and with trolox was evaluated in normal human retinal pigment epithelium cells (ARPE-19) and Y79 retinoblastoma cells using CCK-8. Flow cytometry was used to assess apoptosis, cell cycle, ROS, and mitochondrial membrane potential (MMP). Anti-angiogenic potential was tested using human umbilical vein endothelial cells (HUVECs) and chick chorioallantoic membrane (CAM) assays. Mechanistic studies were conducted via RT-PCR and western blotting. CoQ 10 , alone and with trolox, reduced Y79 cell viability, induced apoptosis through excess ROS generation, and decreased MMP significantly. Both treatments caused G2/M phase cell arrest. The CAM assay showed a significant reduction in endothelial cell proliferation, evidenced by fewer number of co-cultured HUVECs when exposed to CoQ 10 or CoQ 10 with trolox. The combination of CoQ 10 and trolox significantly reduced VEGF-A, ERK, and Akt receptor levels, while CoQ 10 alone significantly inhibited ERK and Akt phosphorylation. Together, CoQ 10 and trolox reduced protein expression of VEGFA. CoQ 10 alone and with trolox, induces apoptosis in Y79 retinoblastoma cells by inhibiting the ERK/Akt pathway and downregulating VEGFA. This study is the first to report the in vitro and in-ovo anti-cancer potential of CoQ 10 alone or when combined with trolox, on human retinoblastoma Y79 cells.

Purpose: Eyelid sebaceous gland carcinoma (SGC) is an aggressive but rare malignancy of ocular region. Over-expression of PD-L1 and PD-1 has been demonstrated in a variety of solid tumors including conjunctival melanoma. PD-L1 is an immunoinhibitory molecule that suppresses the effective T cells response against tumor antigen leading to the progression of tumors. Inhibitors of the interaction of PD-L1 and PD-1 are associated with good clinical response various carcinomas. The prognostic value of the PD-1/PD-L1 axis in SGC remains unexplored. The purpose of this study was to evaluate expressions of PD-1 and its ligand PD-L1 in SGC and correlate its expression with clinicopathological features and patients survival. Methods: The immunohistochemical expression of PD-L1 and PD-1 was evaluated in 30 SGC cases. Results: PD-L1 immunopositivity was detected in 41.9% of the SGC cases. PD-1 expression in tumor infiltrative lymphocytes (TILs) was observed in 53.3% samples. Tumor PD-L1 positivity, PD-1 expression in TILs and tumor size (>10 mm) was associated with reduced disease-free survival. On multivariate analysis only tumor size (>10 mm) and a combined positivity of PD-L1 in tumor cells and PD-1 in TILs with an odds ratio of 5.212 (95% confidence interval 1.449-18.737) continued to be signifi cantly associated with SGC recurrence. Conclusion: PD-L1 is overexpressed in 50% of SGC cases. The combined tumor PD-L1 positivity and TILs showing PD-1 expression within the same SGC patient's samples predict high-risk SGC, suggesting that the up-regulation of PD-L1 in tumor cells and PD-1 positivity within the same SGC patient may aggravate tumor recurrence.

Background Bilateral ocular surface disease resulting from Stevens Johnson Syndrome (SJS) and chemical injuries are visually debilitating and difficult to treat. Ocular surface reconstruction by various means has been reported with variable results. This study addresses an unmet need for a prospective clinical trial comparing the outcomes of transplanting autologous oral and conjunctival epithelial cell constructs on human amniotic membrane by ex vivo tissue engineering. Methods A prospective, randomized controlled clinical trial was prospectively applied for registration, with the clinical trial registry of India (CTRI), with the approval of the Institute Ethics Committee number IEC/NP-99/11.04.2014 and CTRI No. REF/2018/10/021791, the study also registered with the WHO-recognized trial registry, International Standard Randomised Controlled Trial Number (ISRCTN) registration reference number 45780. The study was conducted to compare clinical outcomes of two different tissue-engineered cell grafts, Cultivated Oral Mucosal Epithelial Transplantation (COMET) and Conjunctival Cultivated Epithelial Transplantation (CCET) for ocular surface reconstruction in patients with bilateral ocular surface disease due to Stevens-Johnson Syndrome or chemical injuries. Fifty patients were enrolled and randomized to either the COMET or CCET group. A uniform pre-op and post-op protocol using standard medications was followed for all patients Parameters assessed at baseline, day 1, 1 week, 2 weeks, 1 month, 2 months, 3 months and 6 months postoperatively included patient comfort, best corrected visual acuity (BCVA), ocular surface status and corneal clarity. The efficacy was measured in terms of improvement of vision, reduction in vascularization, symblepharon and corneal clarity. Results In the study, 50 patients (50 eyes; mean ages of 29 ± 15.86 years and 26.36 ± 10.85 years, respectively; range, 12–65 years) were enrolled, with 25 patients each in the COMET and CCET groups. Out of them, 36% were female and 64% were male; the causes were Steven Johnson syndrome (48), and chemical injury (2). Mean pre-operative BCVA was log MAR 1.73 ± 0.57 for COMET and 1.99 ± 0.33 for the CCET group. Pre-operatively all 50 enrolled patients had opaque corneas pre-operatively, symblepharon that extended to the cornea categorised as grade 3 and corneal vascularization that went beyond the pupil’s boundary into the central zone encluaching on the visual axis. The minimal follow-up time was six months. Following surgery postoperatively, the BCVA considerably improved in the COMET group by 1.51 ± 0.58 compared to the CCET group by 1.91 ± 0.33 at 3 months. BCVA at 6 months was 1.73 ± 0.56 in the COMET group and 1.99 ± 0.31 in the CCET group, which is not statistically significant and comparable to the BCVA before surgery. The corneal clarity was significantly improved in COMET group 25 eye (100%) at 2 month, 3month and 19 eye (76%), 6eye (24%) at 6 months when compared to CCET group 15 eye improved (60%), 9 eyes (36%) not improved and one eye with opaque cornea (4%) at 2 months. 22 eye (88%) had not improved, 2 eye (8%) opaque cornea and 1 eye (4%) improved at 3 months. At 6 months 21 eye (84%) were not improved, 4 eye (16%) eye became opaqued at 6 months. Compared to preoperative conditions, both groups had improved corneal clarity significantly ( p  > 0.005). Of the 50 patients with grade 3 symblepharon extended to the cornea, were completely resolved 19 (76%) in COMET group when compared to CCET group 22 eye (88%) not improved. Similarly, 19 eye (76%) had a improvement in corneal vascularization when compared to the CCET group not improved 25 eye (100%) at 6months. No adverse event was observed in any of either group during the follow up periods. Conclusion Both cell types are effective to restore the ocular surface integrity in bilateral ocular surface disease. Whereas COMET is safe and efficacious in terms of improvement of clinical parameters including, BCVA, corneal clarity, reduction in vascularization and preventing the recurrence of symblepharon postoperatively 3months and 6 months. In addition, the CCET group maintained the stability of the ocular surface and had improvement in corneal clarity and a decrease in vascularization at 3 months compared to their pre-operative characteristics.

Existing clinical indicators for metastatic risk classification and patient treatment of uveal melanoma (UM) in the Asian population are limited. Preferentially expressed antigen in melanoma (PRAME) has gained attention in the prognosis of cancers and considered as a potential biomarker in many tumors including UM. Therefore, this study investigated the expression of PRAME and its association with loss of nuclear BAP1 (nBAP1) as well as its correlation with clinicopathological parameters and patient outcome. Immunohistochemical expression of PRAME and BAP1 proteins were assessed in 66 prospective cases of UM. mRNA expression level was measured by quantitative real-time PCR. Kaplan–Meier curves and Cox proportional hazard models were used to analyze the correlation of protein expression with clinicopathological parameters, metastasis-free survival and overall survival. Nuclear PRAME (nPRAME) expression and loss of nBAP1 were observed in 24 and 62% cases, respectively. PRAME mRNA expression level was found to be upregulated in 64% (7/11) of metastatic patients. mRNA and immunoexpression of nPRAME were statistically significant with many clinicopathological high-risk factors. On univariate and multivariate analyses, high mitotic activity, extraocular invasion and presence of nPRAME expression were statistically significant ( p  <  0.05 ). On Kaplan–Meier survival analysis, patients expressing PRAME had significantly reduced metastasis-free survival (MFS) and overall survival (OS). MFS and OS were also reduced in patients expressing PRAME along with loss of nBAP1. Our data show that nPRAME expression, in combination with loss of nBAP1, could be a useful predictive biomarker in the therapeutic management of UM patients at high risk.

Purpose: The evaluation of Herpes Simplex virus-1 (HSV-1) transcript by different investigative methods (qPCR, PCR and IHC) in corneal buttons from suspected viral keratitis patients and the comparison of results with histopathological findings and clinical diagnosis. Methods: Sixty corneal buttons, 30 suspected viral keratitis, and 30 controls (keratoconus and bullous keratopathy) obtained after primary penetrating keratoplasty, were included in the study. All the corneal buttons were subjected to reverse transcriptase quantitative PCR (qPCR) for the detection of latency-associated transcript (LAT) gene, conventional PCR for polymerase (pol) gene, and immunohistochemistry (IHC) for HSV-1 antigen respectively. After obtaining baseline preoperative clinical data, all the patients were followed up for three years. The results obtained were correlated with clinicopathological features and follow-up data. Results: Of the 30 suspected viral keratitis patients there were 6 females and 24 males with mean age 46.5 ± 24.62 years (3-80 yrs). There was a marked male preponderance (80%). HSV-1 LAT transcript was detected in 23% (7/30) corneal buttons by qPCR, HSV-1 DNA in 6.7% (2/30) and HSV-1 antigen in 30% (9/30) cases by conventional PCR and IHC respectively. A statistically significant association was found between qPCR and DNA PCR (P = 0.04). All the 30 control corneas were negative for HSV-1 LAT gene, DNA and antigen. Conclusion: Detection of HSV-1 LAT transcript by qPCR may be superior to HSV-1 DNA PCR (conventional) and IHC, which has low sensitivity. However, the utility of HSV-1 LAT mRNA analysis as a diagnostic modality by qPCR needs to be validated on a larger patient cohort.

Purpose: Mutations in human telomerase reverse transcriptase (TERT) are associated with increased telomerase activity in cutaneous melanomas. Conjunctival squamous cell carcinoma, also referred to as ocular surface squamous cell carcinoma, is cancer on the surface of the eye. Recent studies have identified UV signature mutations in TERT promoters in ocular melanoma and ocular surface squamous neoplasia. However, its immunohistochemical status has not been reported in ocular surface squamous cell carcinoma. This study aimed to explore the immunohistochemical and mutational status of TERT in ocular surface SCC. Methods: The immunohistochemical expression of TERT and mutational status of TERT promoter was evaluated in 19 ocular surface squamous cell carcinoma cases. Conjunctival melanoma tissue was used as a positive control. Results: The cytoplasmic overexpression of TERT was detected in 11/19 (57%), and TERT promoter mutations were identified in 6/19 (31%) of ocular surface squamous cell carcinoma. Out of these, 66% had a C228T mutation, and 33% had a C250T mutation. The TERT expression was found to be associated with a high (≥T3) AJCC category (P = 0.023), and TERT immunoexpression was significantly correlated with reduced disease-free survival (P = 0.024, log-rank analysis) in ocular surface squamous cell carcinoma patients. Conclusion: The present study demonstrates that TERT promoter mutations with UV signatures are frequent in ocular surface squamous cell carcinoma. The increased expression of TERT could be of biological significance in aggressive ocular surface squamous cell carcinoma.

PurposeUveal melanoma (UM) is an intraocular malignancy commonly arising from choroid which can cause visual loss or metastasis. Ataxia-telangiectasia mutated (ATM) protein is an activator of DNA damage response and its role in uveal melanoma (UM) is still unexplored. Therefore, the study aims to detect the expression and localization of ATM protein and its association with clinicopathological parametersMethodsExpression of nuclear ATM (nATM) was investigated on 69 formalin fixed paraffin embedded choroidal melanoma samples by immunohistochemistry and validated by western blotting. Results were then correlated with clinical and histopathological parameters. Prognostic significance was determined by the Kaplan–Meier analysis and the multivariate analysis by Cox’s hazard proportional method.ResultsLoss of nATM was observed in 65% of cases, which was statistically significant with the reduced disease-free survival (p = 0.042). This loss was more frequently found in cases with high-risk histopathological factors like epithelioid cell type, tumor infiltrating lymphocytes and high pigmentation which might help in the progression of melanoma. On multivariate analysis, extraocular spread and loss of nATM were found to be independent prognostic factors (p < 0.05).ConclusionOur data suggest that loss of nATM protein might serve as a poor prognostic marker in the pathogenesis of uveal melanoma which may lead to increased risk of metastasis.