Common variants in SOX-2 and congenital cataract genes contribute to age-related nuclear cataract.

Nuclear cataract is the most common type of age-related cataract and a leading cause of blindness worldwide. Age-related nuclear cataract is heritable (h2 = 0.48), but little is known about specific genetic factors underlying this condition. Here we report findings from the largest to date multi-ethnic meta-analysis of genome-wide association studies (discovery cohort N = 14,151 and replication N = 5299) of the International Cataract Genetics Consortium. We confirmed the known genetic association of CRYAA (rs7278468, P = 2.8 × 10-16) with nuclear cataract and identified five new loci associated with this disease: SOX2-OT (rs9842371, P = 1.7 × 10-19), TMPRSS5 (rs4936279, P = 2.5 × 10-10), LINC01412 (rs16823886, P = 1.3 × 10-9), GLTSCR1 (rs1005911, P = 9.8 × 10-9), and COMMD1 (rs62149908, P = 1.2 × 10-8). The results suggest a strong link of age-related nuclear cataract with congenital cataract and eye development genes, and the importance of common genetic variants in maintaining crystalline lens integrity in the aging eye.

An integrated automated multispectral imaging technique that simultaneously detects and quantitates viral RNA and immune cell protein markers in fixed sections from Epstein-Barr virus-related tumours.

BACKGROUND/AIM: Epstein-Barr virus (EBV) is an oncovirus that is commonly associated with the development of lymphomas and epithelial carcinomas. In the era of immunotherapy, histological evaluation of EBV-related cancers is currently a multi-sample, multi-technique process requiring separate time-consuming detection of EBV-encoded small RNAs by in situ hybridisation (ISH), and parallel labelling of sections for cancer-associated protein markers. METHODS: Using EBV-associated tumours as proof-of-concept for feasibility, here we developed an approach that allows simultaneous detection of EBV RNAs and multiple protein markers such as PD-L1, EBV-LMP, CD8, CD4, CD20, CD30 and CD15on a single tissue section based on our recently reported automated staining protocol. RESULTS: We successfully combined multiplex immunofluorescence (mIF) to detect 3 abovementioned protein markers involved in cancer, with ISH, and applied the protocol to f tissue samples from patients diagnosed with EBV-associated pulmonary lymphoepithelioma-like carcinoma (LELC), gastric carcinoma and Hodgkin's Lymphoma. Empowered by the Vectra 3 Automated Quantitative Pathology Imaging System, we demonstrate the utility and potential of this integrated approach to concurrently detect and quantitate viral RNA and protein biomarkers of immune and tumour cells. CONCLUSION: This study represents an important step forward in the research and diagnosis of EBV-associated cancers, and could be readily modified to include other proteins and RNA markers to apply to other malignancies. More importantly, the novel automated ISH-mIF protocol that we detailly described here could also be readily reproduced by most of the diagnostic and research lab to future projects that aim to look at both RNA and protein markers.

Primary angle closure glaucoma (PACG) susceptibility gene PLEKHA7 encodes a novel Rac1/Cdc42 GAP that modulates cell migration and blood-aqueous barrier function.

PLEKHA7, a gene recently associated with primary angle closure glaucoma (PACG), encodes an apical junctional protein expressed in components of the blood aqueous barrier (BAB). We found that PLEKHA7 is down-regulated in lens epithelial cells and in iris tissue of PACG patients. PLEKHA7 expression also correlated with the C risk allele of the sentinel SNP rs11024102 with the risk allele carrier groups having significantly reduced PLEKHA7 levels compared to non-risk allele carriers. Silencing of PLEKHA7 in human immortalized non-pigmented ciliary epithelium (h-iNPCE) and primary trabecular meshwork cells, which are intimately linked to BAB and aqueous humor outflow respectively, affected actin cytoskeleton organization. PLEKHA7 specifically interacts with GTP-bound Rac1 and Cdc42, but not RhoA, and the activation status of the two small GTPases is linked to PLEKHA7 expression levels. PLEKHA7 stimulates Rac1 and Cdc42 GTP hydrolysis, without affecting nucleotide exchange, identifying PLEKHA7 as a novel Rac1/Cdc42 GAP. Consistent with the regulatory role of Rac1 and Cdc42 in maintaining the tight junction permeability, silencing of PLEKHA7 compromises the paracellular barrier between h-iNPCE cells. Thus, downregulation of PLEKHA7 in PACG may affect BAB integrity and aqueous humor outflow via its Rac1/Cdc42 GAP activity, thereby contributing to disease etiology.

Serum IgG2 and tissue IgG2 plasma cell elevation in orbital IgG4-related disease (IgG4-RD): Potential use in IgG4-RD assessment.

AIMS: To determine the role of serum and tissue IgG2 in orbital biopsies with the histological features of IgG4-related disease (IgG4-RD) in comparison with non-IgG4-related orbital inflammatory disorders (OID), including autoimmune disorders. METHODS: This is an international (Sheffield, UK, and Singapore) collaborative, retrospective case review of 69 patients (38 from Singapore National Eye Centre and 31 from Royal Hallamshire Hospital, Sheffield) with orbital inflammatory biopsies between 2002 and 2016. Clinical information and histology were reviewed and cases were classified into three groups: Group 1: IgG4-RD orbital inflammation (n=43); Group 2: idiopathic OID (n=12) and Group 3: autoimmune OID (n=14). Serum IgG1, IgG2, IgG3 and IgG4 levels were collated where available and immunohistochemistry (IHC) for tissue IgG2 plasma cells was performed. RESULTS: Dual IHC showed IgG2 plasma cells as a distinct population from IgG4 plasma cells. Significant (twofold) serum IgG2 elevation was noted among IgG4-RD (group 1), idiopathic (group 2) and autoimmune OID (group 3). Similarly, significant elevation of tissue IgG2 plasma cells was also seen among IgG4-RD (group 1), idiopathic and autoimmune OID (groups 2 and 3). CONCLUSIONS: Significant elevations of serum IgG2 and tissue IgG2 plasma cells are present in orbital IgG4-RD in comparison with non-IgG4 orbital inflammation (idiopathic and autoimmune OID), suggesting that IgG2 may play a role in IgG4-RD. A serum IgG2 cut-off >5.3 g/L was found to be 80% sensitive and 91.7% specific for orbital IgG4-RD, with an accuracy of 0.90. Tissue IgG2 and IgG4 subclass reporting may provide additional insight regarding the 'IgG4-RD' pathogenesis.

Expression of the primary angle closure glaucoma (PACG) susceptibility gene PLEKHA7 in endothelial and epithelial cell junctions in the eye.

PURPOSE: The role of the recently identified primary angle closure glaucoma (PACG) susceptibility gene, pleckstrin homology domain containing, family A member 7 (PLEKHA7), in PACG is unknown. PLEKHA7 associates with apical junctional complexes (AJCs) and is thus implicated in paracellular fluid regulation. We aimed to determine PLEKHA7's localization in the eye and its association with AJCs to elucidate its potential role in PACG. METHODS: Total RNA from ocular tissues was isolated and analyzed by real-time PCR. Frozen and paraffin-embedded human globes were sectioned and used for immunohistochemistry and immunofluorescence analysis. RESULTS: Specific PLEKHA7 expression was found in the muscles, vascular endothelium, and epithelium of the iris, ciliary body and ciliary processes, trabecular meshwork (TM), and choroid. PLEKHA7 expression in musculature and vascular endothelium was confirmed with smooth muscle marker, SMA, and endothelium marker, PECAM-1, respectively. At the above sites, PLEKHA7 colocalization was seen with adherens junction markers (E-cadherin and ß-catenin) and tight junction markers (ZO-1). CONCLUSIONS: Specific localization of PLEKHA7 was found within PACG-related structures (iris, ciliary body, and choroid) and blood-aqueous barrier (BAB) structures (posterior iris epithelium, nonpigmented ciliary epithelium, iris and ciliary body microvasculature). The association of PLEKHA7 with AJCs in the eye suggests a potential role for PLEKHA7 in PACG via fluidic regulation. Novel expression of PLEKHA7 was also seen in the ocular smooth muscles and vascular endothelia.

p53 and c-kit (CD117) protein expression as prognostic indicators in breast phyllodes tumors: a tissue microarray study.

Breast phyllodes tumors are fibroepithelial neoplasms whose clinical behavior is difficult to predict on histology. There is relatively scant data on the role of biological markers. In this study, we determined if p53 and CD117 (c-kit) protein expression was predictive of behavior in a series of 335 phyllodes tumors diagnosed at the Singapore General Hospital, using immunohistochemistry on tissue microarrays. Representative areas from 250 (75%) benign, 54 (16%) borderline and 31 (9%) malignant phyllodes tumors were selected for construction of tissue microarrays using the 2 mm punch. Immunohistochemistry for p53 and CD117 was carried out using the streptavidin-biotin method. Staining proportion and intensity of both epithelial and stromal elements were analyzed. p53 immunostaining was observed in the epithelium of 28 (10%) of 278 microarrays; myoepithelium of 53 (21%) of 251 microarrays; and stromal cells in 105 (36%) of 289 microarrays. CD117 immunohistochemical reactivity was noted in epithelial and stromal components of 175 (of 267, 66%) and 17 (of 273, 6%) microarrays, respectively. Stromal p53 and CD117 protein expression was associated with tumor grade (P < 0.05). Of 43 (13%) women who suffered recurrences during the follow-up period, CD117 stromal staining predicted recurrent disease (P<0.05), but p53 was not correlative. We conclude that tissue microarrays are a convenient method for evaluating immunostaining results of large numbers of phyllodes tumors. Although positive p53 stromal immunohistochemical detection may corroborate histologic malignancy, it is CD117 protein expression in phyllodes tumor stromal cells that may be of potential utility in predicting recurrent disease.

Phyllodes tumors of the breast: the role of pathologic parameters.

We aimed to establish whether morphologic parameters were prognostically important in a large series of breast phyllodes tumors in Asian women. Of 335 phyllodes tumors diagnosed at the Department of Pathology, Singapore General Hospital, Singapore, between January 1992 and December 2002, 250 (74.6%) were benign, 54 (16.1%) borderline, and 31 (9.3%) malignant, based on histologic review of archival slides. Of the women, 43 (12.8%) experienced recurrences during the follow-up period. Recurrent disease was correlated with grade or classification (P = .028), stromal atypia (P = .016), stromal hypercellularity (P = .046), and permeative microscopic borders (P = .021). Multivariate analysis revealed that independent predictors of recurrence were pseudoangiomatous stromal hyperplasia (PASH) and margin status, whereby the presence of PASH and complete or negative margins reduced recurrence hazards by 51.3% and 51.7% respectively. The 7 women who died of disease during follow-up had malignant phyllodes tumor at the outset and experienced recurrences, and death was preceded by distant metastases.