Factors associated with diabetic macular edema in patients with proliferative diabetic retinopathy.

PURPOSE: To identify factors associated with diabetic macular edema (DME) and to characterize the types of DME present in eyes with proliferative diabetic retinopathy (PDR). METHODS: Observational, retrospective case series of PDR patients reviewed for demographic information, general medical history, ophthalmologic history, optical coherence tomography (OCT), and fluorescein angiogram image characteristics. DME and vitreomacular interface (VMI) status were determined using OCT images. DME was defined as center-involving DME (CI-DME) and noncenter-involving DME (NCI-DME). VMI was defined as vitreomacular adhesion (VMA), vitreomacular traction (VMT), or macular posterior vitreous detachment (PVD). RESULTS: A total of 293 eyes of 210 screened patients with PDR were included. Of the eyes, 194/293 (66.2%) had DME, and 99/293 (33.8%) had no DME; in univariable analysis, there were no significant differences in VMI status (p = 0.4) or epiretinal membrane (ERM, p = 0.1) between them. Of 194 eyes with DME, 90/194 (46.4%) had CI-DME, and 104/194 (53.6%) had NCI-DME. In univariable analysis, CI-DME eyes were significantly more likely than NCI-DME eyes to have a PVD (p = 0.029) and ERM (p < 0.001). In multivariable analysis, the presence of younger age (p = 0.028) and presence of ERM (p = 0.001) were significantly more likely to be observed in eyes with CI-DME. CONCLUSION: In this exploratory study focused on diabetic patients with PDR, we determined that VMI status did not have a significant association with DME in general, but VMI status, younger age, and presence of ERM may be associated with CI-DME.

ARID1A mutations in endometriosis-associated ovarian carcinomas.

BACKGROUND: Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described. METHODS: We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI­SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas. RESULTS: ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions. CONCLUSIONS: These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital­University of British Columbia Hospital Foundation.).

Factors Affecting Ophthalmology Trainees to Pursue Vitreoretinal Surgery Fellowship.

Objective The aim of this study was to understand the factors that ophthalmology trainees consider in pursuing vitreoretinal surgery (VRS) fellowship training. Methods This is a prospective observational survey study. Survey invitations were disseminated to postgraduate year 4 (PGY)-4 ophthalmology residents at Accreditation Council for Graduate Medical Education-accredited residency programs and surgical retina fellows at Association of University Professors of Ophthalmology Fellowship Compliance Committee-compliant fellowship programs in the United States. Survey questions on factors related to VRS were administered employing a 5-point Likert scale. Responses from ophthalmology residents pursuing surgical retina were combined with surgical retina fellows' responses and compared with responses from PGY-4 residents not pursuing vitreoretinal surgery. Results Eighty-one resident surveys were completed. Forty-three fellow surveys were completed. Fifty-seven out of eighty-one (70.4%) residents were not pursuing surgical retina, and a total of 67 trainees (24 residents, 43 fellows) were pursuing surgical retina. The following factors were associated with pursuing VRS training: male gender ( p = 0.031); having performed retina research during residency ( p ≤ 0.001); enjoying surgical retina procedures ( p ≤ 0.001), enjoying surgical retina patient outcomes ( p ≤ 0.001), and working with vitreoretinal surgeons ( p ≤ 0.001); finding surgical retina prestigious ( p ≤ 0.001); perceiving their residency having a strong record of matching surgical retina ( p = 0.039); liking the potential financial income from surgical retina ( p ≤ 0.001); and having vitreoretinal mentors during residency ( p = 0.014). A majority of trainees (31/57, 54.4%) not pursuing surgical retina disagreed or strongly disagreed with enjoying the patient outcomes in surgical retina. A third of female residents not pursuing surgical retina felt having a female surgical retina mentor would have made them more likely to pursue the field. Conclusion Longer retina rotations, encouraging resident participation in retina research, and increasing mentorship opportunities of female trainees from female retina specialists may increase resident interest in pursuing surgical retina fellowship.

Wedge Defects on Optical Coherence Tomography Angiography of the Peripapillary Retina in Glaucoma: Prevalence and Associated Clinical Factors.

PRCIS: Among subjects with glaucoma, wedge-shaped defects on optical coherence tomography angiography (OCTA) were associated with disc hemorrhages (DH), paracentral visual field (VF) defects, increased cup-to-disc ratio (CDR), and thinner retinal nerve fiber layer (RNFL). PURPOSE: To examine determinants of wedge defects on peripapillary OCTA in glaucoma. MATERIALS AND METHODS: A total of 278 eyes of 186 subjects with mild to severe primary open-angle glaucoma underwent 6×6 spectral-domain OCTA imaging of the superficial peripapillary retina from 2016 to 2020 at an academic practice. Wedge defects were defined as focal microvasculature loss that extends outward from the optic nerve in an arcuate, wedge shape. Logistic regression models controlling for intereye correlation identified variables significantly associated with wedge defects. Eyes with profound microvasculature loss in both hemispheres were excluded. Candidate variables included: age, sex, race or ethnicity, diabetes, hypertension, follow-up duration, baseline untreated intraocular pressure, intraocular pressure at time of imaging, DH history, paracentral VF defects, CDR, central corneal thickness, spherical equivalent, VF mean deviation, RNFL thickness, and glaucoma stage. RESULTS: Of 278 eyes, 126 (45.3%) had wedge defects in at least 1 hemisphere. In our multivariable logistic regression model, wedge defects were associated with DH history [odds ratio (OR): 3.19, 95% confidence interval (CI): 1.05-9.69, P=0.041], paracentral VF defects [OR: 4.38 (95% CI: 2.11-9.11), P<0.0001], larger CDR [OR: 1.27 (95% CI: 1.03-1.56), P=0.024, per 0.1 increase], and thinner RNFL [OR: 1.71 (95% CI: 1.25-2.34), P=0.0009, per 10 µm decrease]. CONCLUSION: DH history and paracentral VF defects were independently associated with wedge defects on OCTA, which was present in 45.3% of primary open-angle glaucoma patients. These findings may provide insight into glaucoma pathogenesis.

The testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice are not fully equivalent.

BACKGROUND: Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. RESULTS: We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot. CONCLUSIONS: Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.

S100B and neurofibromin immunostaining and X-inactivation patterns of laser-microdissected cells indicate a multicellular origin of some NF1-associated neurofibromas.

Neurofibromatosis 1 (NF1) is an autosomal dominant disease that predisposes individuals to developing benign neurofibromas. Some features and consequences of NF1 appear to result from partial deficiency of neurofibromin (Nfn), the NF1 gene protein product, as a result of haploinsufficiency for the NF1 gene. Other features and consequences of NF1 appear to involve total deficiency of Nfn, which arises as a result of either loss of function of the second NF1 allele or excess degradation of Nfn produced by the second allele in a particular clone of cells. We used immunofluorescence to assess the presence of Nfn in putative Schwann cells (S100B(+) ) and non-Schwann cells (S100B(-) ) in 36 NF1-derived benign neurofibromas classified histologically as diffuse or encapsulated. The S100B(+) /Nfn(-) cell population made up only 18% ± 10% (mean ± standard deviation) of the neurofibroma cells in both the diffuse and encapsulated neurofibromas. The proportion of S100B(+) /Nfn(+) cells was significantly higher and the proportion of S100B(-) /Nfn(-) cells was significantly lower in diffuse neurofibromas than in encapsulated neurofibromas. We isolated S100B(+) /Nfn(+) , S100B(+) /Nfn(-) , and S100B(-) /Nfn(+) cells by laser microdissection and, using X-chromosome inactivation profiles, assessed clonality for each cell type. We showed that, although some neurofibromas include a subpopulation of S100B(+) /Nfn(-) cells consistent with clonal expansion of a Schwann cell progenitor that has lost function of both NF1 alleles, other neurofibromas do not show evidence of monoclonal proliferation of Schwann cells. Our findings suggest that, although clonal loss of neurofibromin function is probably involved in the development of some NF1-associated neurofibromas, other pathogenic processes also occur.

Tumour size criteria for Group D and E eyes in the International Classification System for Retinoblastoma: effects on rates of globe salvage and high-risk histopathologic features.

PURPOSE: To determine the significance of large tumour size as a criteria for classifying advanced intraocular retinoblastoma, analysing rates of globe survival and high-risk (HE) histopathologic features. METHODS: Retrospective chart review of 212 eyes diagnosed with Group D (111 eyes) or Group E (101 eyes) retinoblastoma in at least one eye from January 1, 2006 to December 31, 2016 using the Los Angeles (LA) Classification System (no tumour size criteria for Group E). The 111 Group D tumours were then reclassified to Group E using 10, 12, 14, 16, 18 mm tumour size criteria, as determined by ultrasound or magnetic resonance imaging dimensions. RESULTS: For eyes in the original LA classification, 66.7% of Group D and 10.5% of Group E eyes undergoing globe preservation therapy avoided enucleation or radiotherapy (p < 0.0001; median follow-up of 33.0 months). In the LA classification, 8.5% of Group D and 26.3% of Group E enucleated globes had HE histopathologic features (p = 0.0065). When Group D eyes with tumours meeting the size criteria were reclassified to Group E, 65.7-74.4% of Group D and 16.1-36.7% of Group E eyes avoided enucleation or radiotherapy. Applying the tumour size criteria, 0-10.9% of Group D and 20.7-23.8% of Group E eyes had HE histopathologic features. CONCLUSION: Our retrospective analysis suggests that a large tumour size criteria for Group E retinoblastoma have no clinical basis, given that the LA classification system provided the greatest separation in globe salvage rates between Group D and E eyes. The LA classification system was also able to show a statistically significant difference in the rates of HE histopathologic features between Group D and E eyes. To avoid discrepancies in the literature, we recommend that centres use one uniform system for classifying advanced intraocular retinoblastoma.

Minimum altered regions in early prostate cancer progression identified by high resolution whole genome tiling path BAC array comparative hybridization.

BACKGROUND: Carcinoma of the prostate (CaP) is a serious health problem. The altered molecular mechanisms that lead to this disease are poorly understood. METHODS: Specimens from radical prostatectomies and blood were collected from 18 CaP surgery patients. For CGH studies, 20 CaP-related samples (16 Gleason grade 3, 3 higher grades, 1 BPH sample) and 18 samples of patient-matched normal epithelial cells were obtained by laser-assisted microdissection from frozen sections of the 18 prostatectomy specimens. High resolution SMRT aCGH was used to compare genomic profiles of prostatic samples to patient-matched blood and pooled female DNA. TMPRSS2-ERG fusion transcript analysis was performed by RT-PCR in relation to alterations detected at the TMPRSS2 locus. RESULTS: Our comprehensive aCGH approach allowed us to define 35 regions of recurrent alterations while excluding germline copy number polymorphisms. Novel regions identified include 2q14.2, containing INHBB, and 17q21.31. The TMPRSS2 locus at 21q22.3 may be a hotspot for rearrangements with 75% of the alterations resulting in the expression of a TMPRSS2-ERG fusion transcript. Differences in fusion expression in different areas in an individual tumor focus and expression in adjacent normal epithelium supported intrafocal heterogeneity and field cancerization, respectively. Both features challenge our efforts to develop more objective markers for diagnosis and prediction of the severity of CaP. CONCLUSION: The high-density array enabled precise mapping of genomic alterations and consequently definition of minimum altered regions smaller than previously reported thus facilitating identification of those genes that contribute to the cancer transformation process.

Posterior Vitreous Detachment and the Associated Risk of Retinal Toxicity with Intravitreal Melphalan Treatment for Retinoblastoma.

BACKGROUND/AIMS: The presence of a posterior vitreous detachment (PVD) may play a role in the development of severe retinal toxicity following intravitreal melphalan (IVM) injection for vitreous seeding. We aimed to evaluate the incidence of PVD in retinoblastoma eyes and its association with retinal toxicity after IVM. METHODS: We reviewed 112 eyes of 81 retinoblastoma patients with B-scan images available for review from 2010 to 2017. A cohort with vitreous seeding treated with IVM was compared to a cohort that did not undergo injection. The primary outcome measure was the presence of PVD at diagnosis and after treatment. Secondary measures included IVM-associated retinal toxicity and other ocular complications. RESULTS: The incidence of PVD was 20% at diagnosis, and in eyes with B-scans available both at diagnosis and after treatment 18% of eyes developed a PVD over the course of therapy, more frequently after IVM (p = 0.05). Of 34 eyes receiving IVM treatment, the incidences of posterior segment toxicity and globe salvage were similar between eyes with and without PVD (p = 0.4015 and 0.52, respectively). CONCLUSION: In this cohort of patients, there did not appear to be an association with the presence of PVD during IVM and the development of retinal toxicity.

Intensity calibration and automated cell cycle gating for high-throughput image-based siRNA screens of mammalian cells.

High-content microscopic screening systems are powerful tools for extracting quantitative multiparameter measures from large number of cells under numerous conditions. These systems perform well in applications that monitor the presence of objects, but lack in their ability to accurately estimate object intensities and summarize these findings due to variations in background, aberrations in illumination, and variability in staining over the image and/or sample wells. We present effective and automated methods that are applicable to analyzing intensity-based cell cycle assays under high-throughput screening conditions. We characterize the system aberration response from images of calibration beads and then enhance the detection and segmentation accuracy of traditional algorithms by preprocessing images for local background variations. We also provide a rapid, adaptive, cell-cycle partitioning algorithm to characterize each sample well based on the estimated locally and globally corrected cell intensity measures of BrdU and DAPI incorporation. We demonstrated the utility and range of our cell ploidy and probe density measurement methods in a pilot screen using a siRNA library against 779 human protein kinases. With our method, multiple image-based quantitative phenotypes can be realized from a single high-throughput image-based microtiter-plate screen.

Correlations of EGFR mutations and increases in EGFR and HER2 copy number to gefitinib response in a retrospective analysis of lung cancer patients.

BACKGROUND: Gefitinib, a small molecule tyrosine kinase inhibitor of the Epidermal Growth Factor Receptor (EGFR), has shown limited efficacy in the treatment of lung cancer. Recognized clinical predictors of response to this drug, specifically female, non-smoker, Asian descent, and adenocarcinoma, together suggest a genetic basis for drug response. Recent studies have addressed the relationship between response and either sequence mutations or increased copy number of specific receptor tyrosine kinases. We set out to examine the relationship between response and the molecular status of two such kinases, EGFR and HER2, in 39 patients treated with gefitinib at the BC Cancer Agency. METHODS: Archival patient material was reviewed by a pathologist and malignant cells were selectively isolated by laser microdissection or manual recovery of cells from microscope slides. Genomic DNA was extracted from 37 such patient samples and exons 18-24, coding for the tyrosine kinase domain of EGFR, were amplified by PCR and sequenced. EGFR and HER2 copy number status were also assessed using FISH in 26 samples. Correlations between molecular features and drug response were assessed using the two-sided Fisher's exact test. RESULTS: Mutations previously correlated with response were detected in five tumours, four with exon 19 deletions and one with an exon 21 missense L858R point mutation. Increased gene copy number was observed in thirteen tumours, seven with EGFR amplification, three with HER2 amplification, and three with amplification of both genes. In our study cohort, a correlation was not observed between response and EGFR mutations (exon 19 deletion p = 0.0889, we observed a single exon 21 mutation in a non-responder) or increases in EGFR or HER2 copy number (p = 0.552 and 0.437, respectively). CONCLUSION: Neither mutation of EGFR nor increased copy number of EGFR or HER2 was diagnostic of response to gefitinib in this cohort. However, validation of these features in a larger sample set is appropriate. Identification of additional predictive biomarkers beyond EGFR status may be necessary to accurately predict treatment outcome.

Mll5 is required for normal spermatogenesis.

BACKGROUND: Mll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Mll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects.

Retinoblastoma-binding proteins 4 and 9 are important for human pluripotent stem cell maintenance.

OBJECTIVE: The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation, expansion, and differentiation. MATERIALS AND METHODS: We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated from human PS cells and their differentiated derivatives), together with small interfering RNA (siRNA) screening to identify candidate pluripotency regulators. Validation of candidate regulators involved promoter analyses, Affymetrix profiling, real-time PCR, and immunoprecipitation. RESULTS: Promoter analysis of genes differentially expressed across multiple serial-analysis-of-gene-expression libraries identified E2F motifs in the promoters of many PS cell-specific genes (e.g., POU5F1, NANOG, SOX2, FOXD3). siRNA analyses identified two retinoblastoma binding proteins (RBBP4, RBBP9) as required for maintenance of multiple human PS cell types. Both RBBPs were bound to RB in human PS cells, and E2F motifs were present in the promoters of genes whose expression was altered by decreasing RBBP4 and RBBP9 expression. Affymetrix and real-time PCR studies of siRNA-treated human PS cells showed that reduced RBBP4 or RBBP9 expression concomitantly decreased expression of POU5F1, NANOG, SOX2, and/or FOXD3 plus certain cell cycle genes (e.g., CCNA2, CCNB1), while increasing expression of genes involved in organogenesis (particularly neurogenesis). CONCLUSIONS: These results reveal new candidate positive regulators of human PS cells, providing evidence of their ability to regulate expression of pluripotency, cell cycle, and differentiation genes in human PS cells. These data provide valuable new leads for further elucidating mechanisms of human pluripotency.