Gain of Chromosome 6p Correlates with Severe Anaplasia, Cellular Hyperchromasia, and Extraocular Spread of Retinoblastoma.

Purpose: Gain of chromosome 6p has been associated with poor ocular survival in retinoblastoma and histopathologic grading of anaplasia with increased risk of metastatic spread and death. This study examined the correlation between these factors and other chromosomal abnormalities as well as results of whole genome sequencing, digital morphometry, and progression-free survival. Design: Retrospective cohort study from 2 United States tertiary referral centers. Participants: Forty-two children who had undergone enucleation for retinoblastoma from January 2000 through December 2017. Methods: Status of chromosomes 6p, 1q, 9q, and 16q was evaluated with fluorescence in situ hybridization, the degree of anaplasia and presence of histologic high-risk features were assessed by ocular pathologists, digital morphometry was performed on scanned tumor slides, and whole genome sequencing was performed on a subset of tumors. Progression-free survival was defined as absence of distant or local metastases or tumor growth beyond the cut end of the optic nerve. Main Outcome Measures: Correlation between each of chromosomal abnormalities, anaplasia, morphometry and sequencing results, and survival. Results: Forty-one of 42 included patients underwent primary enucleation and 1 was treated first with intra-arterial chemotherapy. Seven tumors showed mild anaplasia, 19 showed moderate anaplasia, and 16 showed severe anaplasia. All tumors had gain of 1q, 18 tumors had gain of 6p, 6 tumors had gain of 9q, and 36 tumors had loss of 16q. Tumors with severe anaplasia were significantly more likely to harbor 6p gains than tumors with nonsevere anaplasia (P < 0.001). Further, the hematoxylin staining intensity was significantly greater and that of eosin staining significantly lower in tumors with severe anaplasia (P < 0.05). Neither severe anaplasia (P = 0.10) nor gain of 6p (P = 0.21) correlated with histologic high-risk features, and severe anaplasia did not correlate to RB1, CREBBP, NSD1, or BCOR mutations in a subset of 14 tumors (P > 0.5). Patients with gain of 6p showed significantly shorter progression-free survival (P = 0.03, Wilcoxon test). Conclusions: Gain of chromosome 6p emerges as a strong prognostic biomarker in retinoblastoma because it correlates with severe anaplasia, quantifiable changes in tumor cell staining characteristics, and extraocular spread.

Clinical and Histopathological Features of Corneal Primary Acquired Melanosis and Melanoma.

AIM: The aim of this study is to describe the clinical and pathologic features of corneal primary acquired melanosis (PAM) and melanoma. METHODS: We describe 3 cases in total: two cases of corneal melanomas and 1 case of corneal PAM. The eyes were processed routinely for histopathological examination. Clinical histories, treatments, and outcomes were reviewed. RESULTS: Corneal melanomas arose from recurrence of conjunctival melanoma or conjunctival PAM at the limbus. One patient had a recurrence after excision of a limbal melanoma, another had a de novo corneal melanoma, and the last patient had corneal PAM in the setting of conjunctival PAM with atypia. All lesions were excised with adjuvant alcohol debridement and cryotherapy with no recurrence ranging from 1 week to 8 years. CONCLUSIONS: Corneal melanomas arise at the limbus from corneal PAM or conjunctival atypia. They can appear after excisional removal of a conjunctival melanoma. Surgical excision with alcohol debridement and adjuvant cryotherapy is successful.

Secondary ocular malignancies: A review.

Secondary ocular malignancies most commonly spread to the choroid. Previously, the prognosis was poor however, with newer treatments including immunotherapy, patient's life expectancy have increased. It is therefore, important that ophthalmologists diagnose this condition in a timely manner and offer treatment to maximize visual potential and refer them on to oncology colleagues in order to optimize their systemic treatment for their primary cancer.

Atypical Lipomatous Tumor/Well-Differentiated Liposarcoma of the Orbit: Three Cases and Review of the Literature.

The authors present 3 patients from this retrospective case series to review the clinical findings, imaging, pathology, and treatment of orbital atypical lipomatous tumor/well-differentiated liposarcoma. Pathology of biopsy specimens ranged from spindle cell proliferations mimicking neurofibroma to proliferations of well-differentiated adipocytes. Immunohistochemical stains were positive for murine double minute 2 in 1 case, and fluorescent in situ hybridization showed amplification of murine double minute 2 in 2 cases. Treatments ranged from serial debulking, proton beam irradiation, and exenteration. None of the patients developed metastases. A literature review supported the low-grade nature of this lesion. Orbital atypical lipomatous tumor/well-differentiated liposarcoma is a low-grade, indolent liposarcoma that may be locally invasive. The histologic diagnosis is enhanced with immunohistochemical staining for murine double minute 2 and fluorescent in situ hybridization analysis for amplification of murine double minute 2. Although treatment may vary according to the individual, conservative therapies may be attempted prior to radical surgery.

Natural course of adult-onset vitelliform lesions in eyes with and without comorbid subretinal drusenoid deposits.

PURPOSE: Adult vitelliform lesions (AVL) are associated with age related macular degeneration (AMD) and subretinal drusenoid deposits (SRDD). We evaluated the natural course of AVL, assessing the influence of SRDD on disease progression, visual function and incidence of macular atrophy (MA) and choroidal neovascular membranes (CNVM). METHODS: A retrospective cohort study was conducted between January 2011 and March 2016. Demographic, clinical and imaging data from 26 consecutive AVL patients were analysed following case note review. Optical coherence tomography images were graded for SRDD and patients divided into those with/without SRDD. Outcomes included presenting/changes in best corrected visual acuity (BCVA) and incidence of MA/CNVM. RESULTS: Mean age was 78.6 ± 7.6 years. Mean follow-up was 51.5 ± 25.6 months. Twelve patients (46.2%) had SRDD at presentation with 3 more (11.5%) developing them. Subjects with SRDD were older (mean 81.7 ± 6.1 years vs 74.3 ± 7.6 years, p = 0.010). Mean presenting BCVA was worse in SRDD eyes (0.39 ± 0.31 logMAR vs 0.19 ± 0.18 logMAR, p = 0.017). Eight of 15 patients with SRDD (53.3%) developed incident MA or CNVM; higher than those with no SRDD (1/11, 9.1%; p = 0.036). Two patients (7.7%) developed full thickness macular holes. CONCLUSIONS: Patients with AVL and SRDD likely represent an advanced pathological stage or phenotype with worse visual outcome and higher risk of MA/CNVM. Possible overlap with AMD exists. Follow-up, counselling and provisions for early detection/treatment of complications should be made. Better classification including improved understanding of phenotypic and genetic variations with reference to comorbid diseases including AMD is required. Presence of SRDD in AVL offers a dichotomous classification, indicating risk of future MA/CNVM formation.

Etiology and Clinical Features of Ocular Inflammatory Diseases in a Tertiary Referral Centre in Sydney, Australia.

PURPOSE: To report the pattern of uveitis in patients attending a tertiary uveitis service in Sydney, Australia. METHODS: The charts of patients seen between January 2009 and December 2015 were retrospectively reviewed. Data pertaining to patient demographics, eye examination on presentation, work-up and final diagnoses were collected. RESULTS: The total number of patients with uveitis seen over this period was 1165. There were 650 males (56%) and 515 females (44%). There were 838 patients aged 17-60 years (72%) and 327 patients aged >60 years (28%). Uveitis was anterior in 735 patients (63%), posterior in 234 patients (20%), pan in 109 patients (9%), and intermediate in 87 patients (8%). The most common associations were HLA-B27+ve (264 patients; 22.8%), sarcoidosis (78 patients; 6.7%) and Fuchs (33 patients; 2.8%), while VZV (51 patients; 4.4%), HSV (49 patients; 4.2%), tuberculosis (49 patients; 4.2%) and toxoplasmosis (48 patients; 4.1%) were the most common infectious causes of uveitis. No identifiable association was found in 389 patients (33.4%). HLA-B27 was more common in the younger age group compared with the older age group (p<0.001, χ2-test), but there was no difference between the age groups for no identifiable cause (p value 0.24) and sarcoidosis (p value 0.08). CONCLUSIONS: This retrospective case review reveals a broad spectrum of uveitis in a tertiary referral service in Sydney, Australia. It is comparable with other major studies around the world.

Efficacy of dexamethasone versus bevacizumab on regression of hard exudates in diabetic maculopathy: data from the BEVORDEX randomised clinical trial.

OBJECTIVE: To report the effect of bevacizumab versus dexamethasone on hard exudates (HEX) in diabetic macular oedema (DME). DESIGN: Post hoc analysis of 24-month data from the Randomised clinical trial of BEVacizumab OR DEXamethasone for diabetic macular oedema (BEVORDEX) phase 2 multicentre randomised clinical trial. Eyes with centre-involving DME resistant to or unlikely to benefit from macular laser therapy were included. Eyes were randomly assigned to bevacizumab every 4 weeks or Ozurdex dexamethasone implant (DEX) every 16 weeks, both as required. The 68 eyes from 48 patients that completed 24-month follow-up were analysed. Two masked graders assessed extent and location of HEX on baseline, 12-month and 24-month foveal-centred colour fundus photographs using validated grading software. RESULTS: Macular HEX was present in 60% of study eyes. Of these, 21 eyes were treated with DEX and 20 eyes with bevacizumab. Both treatments led to reduction in area of macular HEX at 12 months and 24 months. There was greater regression of HEX from the foveal centre in DEX-treated eyes (median change +890 µm, IQR=1040 µm) than bevacizumab-treated eyes (median change +7.0 µm, IQR=590 µm) at 12 months (p=0.04) but the difference was no longer statistically significant (p=0.10) by 24 months (DEX +1400 µm, IQR=1590 µm; bevacizumab +20 µm, IQR=2680 µm). Reassuringly, no study eye developed HEX at the foveal centre, a visually devastating consequence of diabetic maculopathy. CONCLUSIONS: Bevacizumab and DEX were effective in reducing area of HEX in eyes with DME. DEX provided more rapid regression of HEX from the foveal centre although bevacizumab-treated eyes started to catch up by 24 months. Distance from the foveal centre as well as total area of macular HEX should be assessed when evaluating treatments for foveal-threatening HEX. TRIAL REGISTRATION NUMBER: NCT01298076; Post-results.

Floppy eyelid syndrome: the coventry experience.

PURPOSE: Floppy eyelid syndrome is a condition that is difficult to identify and diagnose and with no clear guidelines on its management. We propose a method of reliably grading this syndrome and have proposed a management algorithm based on the grading. MATERIALS AND METHODS: Retrospective data collection of patients diagnosed with Floppy eyelid syndrome and treated under the care of a single oculoplastic surgeon over a 9 year period. RESULTS: First, 102 patients were included and were classified into 3 groups. Grade 1 (F1) 7.5%, Grade 2 (F2) 36.5% and Grade 3 (F3) 56%. Only 12% of our cohort required surgery, and 92% of these patients demonstrated improvement in their symptoms. DISCUSSION: Clinical grading of Floppy eyelid syndrome patients will help determine patient's management plan. In our experience, operating on both upper and lower eyelids at the same time where indicated helps to maintain the normal anatomical relationship and improve epiphora.

The collagen matrix of the human trabecular meshwork is an extension of the novel pre-Descemet's layer (Dua's layer).

BACKGROUND: The trabecular meshwork (TM) located at the angle of the anterior chamber of the eye contributes to aqueous drainage. A novel layer in the posterior part of the human cornea has recently been reported (the pre-Descemet's layer (Dua's layer (PDL)). We examined the peripheral part of this layer in relation to the origin of the TM. METHODS: The PDL and TM of 19 human donor eyes and one exenterated sample were studied. Samples were examined by light and electron microscopy (EM) for tissue architecture and by immunohistology for four matricellular proteins, five collagen types and CD34. RESULTS: EM revealed that beams of collagen emerged from the periphery of PDL on the anterior surface of the Descemet's membrane and divided and subdivided to continue as the beams of the TM. Long-spacing collagen was seen in the PDL and TM. Trabecular cells (CD34-ve) associated with basement membrane were seen in the peripheral part of the PDL and corresponded to the start of the separation of the collagen lamellae of PDL. Collagen VI was present continuously in PDL and extended into the TM. Matricellular proteins were seen predominantly in the TM with only laminin extending into the periphery of PDL. CONCLUSIONS: This study provides an insight into the origins of the collagen core of the TM as an extension of the PDL of the cornea. This finding adds to the knowledge base of the TM and cornea and has the potential to impact future research into the TM and glaucoma.

A Micro-RNA Connection in BRaf(V600E)-Mediated Premature Senescence of Human Melanocytes.

Recent high-throughput-sequencing of the cancer genome has identified oncogenic mutations in BRaf genetic locus as one of the critical events in melanomagenesis. In normal cells, the activity of BRaf is tightly regulated. Gain-of-function mutations like those identified in melanoma frequently lead to enhanced cell-survival and unrestrained growth. The activating mutation of BRaf will also induce the cells to senesce. However, the mechanism by which the oncogenic BRaf induces the senescent barrier remains poorly defined. microRNAs have regulatory functions toward the expression of genes that are important in carcinogenesis. Here we show that expression of several microRNAs is altered when the oncogenic version of BRaf is introduced in cultured primary melanocytes and these cells undergo premature cellular senescence. These include eight microRNAs whose expression rates are significantly stimulated and three that are repressed. While most of the induced microRNAs have documented negative effects on cell cycle progression, one of the repressed microRNAs has proven oncogenic functions. Ectopic expression of some of these induced microRNAs increased the expression of senescence markers and induced growth arrest and senescence in primary melanocytes. Taken together, our results suggest that the change in microRNA expression rates may play a vital role in senescence induced by the oncogenic BRaf.

Signalling pathways involved in ribonuclease-7 expression.

Antimicrobial peptides are host defence molecules that play a potential role in preventing infection at the epithelial surfaces. Ribonuclease (RNase)-7 has been shown to possess a broad spectrum of microbicidal activity against various pathogens. Here, we demonstrate that RNase-7 protein is localised to the superficial layers of ocular surface cells and increased in response to interleukin (IL)-1ß, suggesting an active role during inflammation related to ocular surface infection. Signal transduction pathways involved in RNase-7 expression are unknown. Involvement of transforming growth factor ß-activated kinase-1 (TAK-1) activated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway molecules [c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38] were studied because of their importance in infection and inflammation. Blocking the MAPKs resulted in inhibition of RNase-7 expression in response to IL-1ß. However, RNase-7 induction by IL-1ß was not affected by inhibiting the NF-κB signalling pathway. In conclusion, our results indicate that RNase-7 expression is specifically mediated via MAPKs but not NF-κB signalling pathways.

Comparative transcriptional profiling of the limbal epithelial crypt demonstrates its putative stem cell niche characteristics.

BACKGROUND: The Limbal epithelial crypt (LEC) is a solid cord of cells, approximately 120 microns long. It arises from the undersurface of interpalisade rete ridges of the limbal palisades of Vogt and extends deeper into the limbal stroma parallel or perpendicular to the palisade. There are up to 6 or 7 such LEC, variably distributed along the limbus in each human eye. Morphological and immunohistochemical studies on the limbal epithelial crypt (LEC) have demonstrated the presence of limbal stem cells in this region. The purpose of this microarray study was to characterise the transcriptional profile of the LEC and compare with other ocular surface epithelial regions to support our hypothesis that LEC preferentially harbours stem cells (SC). RESULTS: LEC was found to be enriched for SC related Gene Ontology (GO) terms including those identified in quiescent adult SC, however similar to cornea, limbus had significant GO terms related to proliferating SC, transient amplifying cells (TAC) and differentiated cells (DC). LEC and limbus were metabolically dormant with low protein synthesis and downregulated cell cycling. Cornea had upregulated genes for cell cycling and self renewal such as FZD7, BTG1, CCNG, and STAT3 which were identified from other SC populations. Upregulated gene expression for growth factors, cytokines, WNT, Notch, TGF-Beta pathways involved in cell proliferation and differentiation were noted in cornea. LEC had highest number of expressed sequence tags (ESTs), downregulated and unknown genes, compared to other regions. Genes expressed in LEC such as CDH1, SERPINF1, LEF1, FRZB1, KRT19, SOD2, EGR1 are known to be involved in SC maintenance. Genes of interest, in LEC belonging to the category of cell adhesion molecules, WNT and Notch signalling pathway were validated with real-time PCR and immunofluorescence. CONCLUSIONS: Our transcriptional profiling study identifies the LEC as a preferential site for limbal SC with some characteristics suggesting that it could function as a 'SC niche' supporting quiescent SC. It also strengthens the evidence for the presence of "transient cells" in the corneal epithelium. These cells are immediate progeny of SC with self-renewal capacity and could be responsible for maintaining epithelial turn over in normal healthy conditions of the ocular surface (OS). The limbus has mixed population of differentiated and undifferentiated cells.

Hormone therapy and intraocular pressure in nonglaucomatous eyes.

OBJECTIVE: The influence of sex hormones on intraocular pressure (IOP) has been the focus of recent debate. Previous studies investigating the effects of hormone therapy (HT) on IOP in postmenopausal women have produced conflicting results but have been limited by small numbers of participants. The aim of our study was to compare IOP in women without glaucoma taking HT with those not taking HT. METHODS: A prospective cross-sectional study of postmenopausal women visiting a single ophthalmic medical practitioner was conducted. All women with a history of intraocular disease, a family history of glaucoma, or refractive error exceeding +/-5 diopters were excluded. Applanation tonometry was used to measure IOP, and participants were then asked if they were current HT users. RESULTS: A total of 263 participants were recruited, of whom 91 reported current use of HT; 172 had never used HT. Within the HT group, 33 were taking an estrogen-therapy and 58 were taking a estrogen-progesterone therapy. Mean IOP in the HT group was significantly lower than that in the non-HT group; the mean difference was 1.41 mm Hg (P < 0.001). This difference remained statistically significant after statistical correction for age, use of systemic beta-blockers, and time of IOP measurement. There was no significant difference in mean IOP between women taking combined versus those taking estrogen-only preparations. CONCLUSIONS: Our study showed that IOP was significantly lower in women taking HT than in those who had never taken HT, even after removing other possible influences on IOP. The IOP-lowering effect of HT deserves further investigation to explore whether it may represent a possible new therapeutic modality for glaucoma.

Management of intraoperative floppy-iris syndrome-associated iris prolapse using a single iris retractor.

Tamsulosin is an alpha(1)-adrenergic antagonist known to be linked with intraoperative floppy-iris syndrome (IFIS), which is characterized by iris atonicity and a propensity toward progressive intraoperative pupil constriction and iris prolapse. We present 2 strategies for managing IFIS-associated iris prolapse. Placement of a single subincisional iris retractor following reposition of the prolapsed iris was the more successful approach. We recommend consideration of this approach in all cases of iris prolapse.

The role of limbal stem cells in corneal epithelial maintenance: testing the dogma.

OBJECTIVE: To study and characterize the epithelial cells in patients with a central "island" of normal epithelial cells surrounded with 360 degrees of clinically apparent limbal stem cell (SC) deficiency with conjunctivalization of the limbus and peripheral cornea. DESIGN: Observational, prospective, consecutive case series. PARTICIPANTS: Five human subjects (8 eyes) who presented with total limbal SC deficiency in 1 or both eyes with a central area of normal corneal epithelial cells. METHODS: Clinical slit-lamp examination, aided with fluorescein staining, for evidence of conjunctivalization and in vivo confocal microscopy (IVCM) of the conjunctivalized limbus and peripheral cornea and the normal central corneal epithelium. MAIN OUTCOME MEASURE: Long term survival of normal stratified corneal epithelial cell sheet in the presence of total limbal SC deficiency. RESULTS: In all 8 eyes the diagnosis of limbal SC deficiency was confirmed by clinical and IVCM examination. The conjunctivalized area extended circumferentially along the entire limbus, seen clinically by the presence of fluorescein staining cells, epithelial irregularity, and vascularization and by IVCM showing bright conjunctival epithelial cells, superficial and deep blood vessels, and goblet cells. The central corneal epithelial cells had a normal appearance with polygonal superficial cells, well-defined wing cells, and smaller basal cells. The central "islands" of normal epithelial cells remained unchanged over the mean follow-up period of 60 months (range, 8-12 years). CONCLUSIONS: The existence and survival of a healthy sheet of corneal epithelial cells over the follow-up period, in the presence of clinically apparent total limbal SC deficiency, suggests a limited role of limbal epithelial SC in physiologic homeostasis of the corneal epithelium. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed on this article.

Silicone oil-induced corneal perforation following complex retinal detachment.

PURPOSE: To report a case of silicone oil-induced corneal perforation following complex retinal detachment surgery. METHODS: Case report. RESULTS: Two months following a second retinal detachment repair, the patient presented to eye casualty with a corneal perforation secondary to silicone oil keratopathy. CONCLUSION: The pathophysiology of silicone oil-related perforation is not clearly understood. Poor corneal nutrition due to the presence of oil may be an important contributory factor. Close monitoring of patients for early signs of silicone oil keratopathy could preempt perforation.

Optimization of amniotic membrane (AM) denuding for tissue engineering.

INTRODUCTION: Amniotic membrane (AM) has gained increasing popularity as a useful carrier for ex vivo-expanded cells for tissue engineering, particularly in ocular surface reconstruction. However, current methods employed for denuding AM are highly variable, and the consequent effects on the structural and molecular composition of the AM basement membrane (BM) are ambiguous. We compare the effects of the main denuding procedures, and propose a highly effective standardized alternative. METHODS: AMs preserved for transplantation were denuded using published ethylenediaminetetraacetic acid (EDTA)- and dispase-based methodologies and our novel thermolysin-based procedure. Scanning and transmission electron microscopy and immunohistochemistry, for BM components (collagens IV and VII, laminin 5, and integrins alpha6 and beta4), were used to assess effectiveness of denuding epithelium, whilst maintaining the integrity of the BM. RESULTS: EDTA- and dispase-based denuding techniques resulted in the disaggregation and even destruction of the BM structure and molecular composition. Employing thermolysin effectively denuded epithelium whilst maintaining BM structural and molecular integrity. CONCLUSION: Current procedures for preparing AM are variable and often ineffective, resulting in nonstandard membranes. Our novel thermolysin-based technique effectively denudes the AM whilst preserving an essentially intact and consistent BM. Therefore, we propose that this novel thermolysin procedure may potentially improve overall generation of tissue-engineered constructs using AM.

Limbal epithelial crypt: a model for corneal epithelial maintenance and novel limbal regional variations.

OBJECTIVE: To determine the distribution of cell membrane proteins and extracellular matrix proteins around the limbal epithelial crypt (LEC) compared with adjacent limbus and corneal epithelium. METHODS: Serial histological sections of human corneoscleral limbus rims were stained with antibodies of interest by standard immunohistochemistry. RESULTS: Superficial cells of the limbus were desmoglein 3 positive, compared with the negative basal cells of the limbus that correspond to cells with more stemlike properties. The LEC had a much lower proportion of desmoglein 3 staining in comparison. Tenascin C staining demonstrated regional variations of the limbus depending on their association with the LEC. Limbus that was associated with or adjacent to the LEC had a greater tenascin C expression compared with normal limbus, whereas the LEC demonstrated the greatest tenascin C expression. CONCLUSIONS: Based on these and similar results previously reported for connexin 43, we propose a novel model on the mechanism of corneal surface epithelium maintenance involving 3 different limbal regions: zone 1, limbus including the LEC; zone 2, limbus associated with the LEC; and zone 3, limbus distant to the LEC. CLINICAL RELEVANCE: The noted limbal variations may influence the selection of the donor site for limbal grafts in the future.