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.

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.