Aqueous Humor Specimens Research Articles

Abstract 967: Clinical applicability of targeted panel sequencing in liquid biopsies for disease diagnosis and monitoring of patients with pediatric solid tumors

Abstract Liquid biopsy assays based on sequence analysis of cell-free DNA (cfDNA) have the potential to transform patient care. We recently validated a liquid biopsy platform, LBSeq4Kids, for patients with retinoblastoma, brain tumors and solid tumors using low pass whole genome sequencing (LP-WGS) of cfDNA from aqueous humor (AH), cerebrospinal fluid (CSF), and plasma (PL) to detect Copy Number Alterations (CNAs) in circulating tumor DNA (ctDNA). Herein, we present phase 2, a high-depth comprehensive Next-Generation Sequencing (NGS) panel using cfDNA that allows us to detect SNV’s, indels and gene fusions in clinically relevant genes for pediatric solid and brain tumors. Seventy-four cfDNA specimens have been analyzed to date, using plasma from 43 patients with solid tumors, CSF from 25 patients with brain tumors and AH from six patients with ocular tumors including retinoblastoma. cfDNA was extracted at diagnosis, during therapy, at remission and/or relapse. Sequencing libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of cfDNA. For the detection of recurrent mutations and fusions we designed a custom panel using a hybridization-based capture method (Twist Bioscience) that included full coverage of the exons of 136 genes harboring recurrent mutations and selected introns for five genes with recurrent rearrangement breakpoints including BRAF, EWSR1, SYT, FOXO1 and NTRK1. Hybridized libraries were paired-end sequenced (Illumina NextSeq 500) at an average 1000x collapsed coverage. Mutations identified in cfDNA were compared with those in the primary or metastatic tumors profiled with our OncoKids® NGS panel. A total of 35/43 (81%) plasma specimens were positive for either an SNV, deletion or fusion with the most common events being a somatic missense variant in TP53 (13/35, 37%) or translocation involving EWSR1 (9/35, 26%). A total of 16/25 (64%) CSF specimens were positive for SNVs and fusions, most often involving KIAA1549::BRAF (7/25, 28%) in low grade gliomas. A germline variant in RB1 was detectable in two AH specimens from the left and right eye of a patient with bilateral retinoblastoma while plasma from a patient with metastatic retinoblastoma demonstrated clinically relevant variants in RB1 and DICER1. Serial cfDNA profiling revealed a clinically reported TP53 c.746G>C missense variant in the plasma of a patient with osteosarcoma at multiple timepoints through the disease course, with evidence for relapse prior to standard imaging. These data suggest that clinical implementation of a second version of LBSeq4Kids that combines CNA and mutation analysis allows for diagnosis, residual disease detection and risk stratification for pediatric patients with diverse tumor types, regardless of histologic grade, with a variety of pediatric solid and brain tumors. Citation Format: Eirini Christodoulou, Venkata Yellapantula, Katrina O'Halloran, Liya Xu, Jesse L. Berry, Fariba Navid, Dejerianne Ostrow, Jaclyn A. Biegel. Clinical applicability of targeted panel sequencing in liquid biopsies for disease diagnosis and monitoring of patients with pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 967.

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma.

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Proteomic study of aqueous humour in diabetic patients with cataracts by TMT combined with HPLC-MS/MS

BackgroundThe purpose of this study is to identify the proteomic differences between the aqueous humour of diabetes patients with cataracts and that of non-diabetic sufferers of cataracts in a clinical setting.MethodsPatients were divided into the diabetic experimental group and the non-diabetic control group. Aqueous humour specimens were obtained via cataract surgery. Sample proteins were treated with a TMT reagent, separated using a cation chromatography column, and analysed using a C18 desalting column. Proteins were identified using HPLC-MS/MS. The differential proteins were identified using both a p value of < 0.05 and a fold change of > 1.2. GO classification enrichment analysis, KEGG pathway enrichment analysis, protein interaction network analysis, and ingenuity pathway analysis were all carried out. The expression level of four differential proteins were verified by Western blot, and GC and TTR expressions were further examined using an expanded sample pool.ResultsThe postprandial glucose levels between the experimental group (9.40 ± 1.35 mmol/L) and the control group (6.56 ± 0.81 mmol/L) were significantly different, with a p value of 1.16E-06. It is important to note, however, that the baseline levels of the parameters showed no statistical differences. In total, 397 aqueous humour proteins were identified; of these, 137 showed significant differences, with 63 upregulated ones and 74 down-regulated ones. The differential proteins play important roles in numerous biological processes and pathways, such as complement and coagulation cascades (p = 1.71E-09). Some of these differential proteins are associated with diabetic retinal degeneration and other diabetic complications. Differential proteins, such as HP, GC, and TTR, have high node degree in the protein interaction network. Western blot results further confirmed that GC were down-regulated while TTR was up-regulated in aqueous humour under diabetic condition.ConclusionA list of differential proteins in the human aqueous humour of diabetic patients was established. Proteins with high interaction scores as per protein interaction analysis, such as GC and TTR, were further verified and could potentially be used as early diagnostic markers for diabetic eye complications in clinical practice.

The Interplay Between Metabolites and MicroRNAs in Aqueous Humor to Coordinate Corneal Endothelium Integrity

The purpose of the study was to clarify the interplay between metabolites and microRNAs (miRs) in the aqueous humor (AqH) of bullous keratopathy (BK) patients to retain human corneal endothelium (HCE) integrity. Prospective, comparative, observational study. A total of 55 patients with BK and 31 patients with cataract (Cat) as control. A biostatic analysis of miRs and metabolites in the AqH, hierarchical clustering, and a least absolute shrinkage and selection operator (Lasso) analysis were employed. The miR levels in AqH of BK (n=18) and Cat (n=8) patients were determined using 3D-Gene human miR chips. Hierarchical clusters of metabolites detected by liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry in AqH specimens from 2 disease groups, BK (total n=55) and Cat (total n=31), were analyzed twice to confirm the reproducibility. The analytical procedure applied for investigating the association between metabolites and miRs in AqH was the exploratory data analysis of biostatistics to avoid any kind of prejudice. This research procedure includes a heat-map, cluster analysis, feature extraction techniques by principal component analysis, and a regression analysis method by Lasso. The cellular and released miR levels were validated using reverse transcription polymerase chain reaction and mitochondria membrane potential was assessed to determine the functional features of the released miRs. Identification of interacting metabolites and miRs in AqH attenuating HCE degeneration. The metabolites that decreased in the AqH of BK patients revealed that 3-hydroxyisobutyric acid (HIB), 2-aminobutyric acid (AB) and branched-chain amino acids, and serine were categorized into the same cluster by hierarchical clustering of metabolites. The positive association of HIB with miR-34a-5p was confirmed (P=0.018), and the Lasso analysis identified the interplay between miR-34a-5p and HIB, between miR-24-3p and AB, and between miR-34c-5p and serine (P=0.041, 0.027, and 0.009, respectively). 3-hydroxyisobutyric acid upregulated the cellular miR-34a expression, mitochondrial membrane potential, and release of miR-184 in dedifferentiated cultured HCE cells. Metabolites and miRs in AqH may synchronize in ensuring the integrity of the HCE to maintain efficient dehydration from the stroma. Proprietary or commercial disclosure may be found after the references.

Abstract 5553: Diagnostic aqueous humor proteome predicts metastatic potential in uveal melanoma

Abstract Introduction: Approximately half of patients with uveal melanoma (UM), a primary eye cancer, will develop metastatic disease. Gene expression profiling (GEP) is clinically validated to stratify risk of metastasis by assigning UM patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy which are limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding or tumor dissemination. Thus, liquid biopsy has emerged as a significantly less invasive alternative. Blood biopsy has largely been unsuccessful for clinical use in UM due to low detection rates in the setting disease limited to the eye, however eye-specific aqueous humor (AH) liquid biopsy may be a better alternative. In this study, we seek to determine the AH proteome related to the advanced GEP class 2 using the diagnostic AH specimens. Method: Twenty UM treatment naive AH were collected before plaque brachytherapy. Patients were sub-grouped by GEP classes into GEP 1 (n=12) and GEP 2 (n=5). Three patients were classified as GEP unknown (GEP NA, n=3) due to the unavailability of tumor biopsy. Ten microliters of AH samples were analyzed by proximity extension assay-derived multiplexed Olink platform. Protein expression levels of 1472 targets were analyzed, compared between GEP classes and correlated with clinical features. Significant differentially expressed proteins (DEPs, fold-change (FC) &amp;gt; 2 or FC &amp;lt; 0.5, P &amp;lt; 0.01) were subjected to Qiagen ingenuity pathway analysis for cellular pathway and upstream regulator identification. Results: GEP 2 class was correlated with AJCC stages (P = 0.012), advanced clinical tumor stages (P = 0.007) and mutated BAP1 (P = 0.018). 45 DEPs were identified when comparing GEP classes. Among them, 31 are up-regulated DEPs [fold-change (FC) &amp;gt;2, P &amp;lt; 0.01] and 14 are down-regulated DEPs (FC &amp;lt; 0.5, P &amp;lt; 0.01) in GEP 2 compared to GEP1. The unsupervised clustering analysis showed that the 45 DEPs well-differentiate AH samples by GEP classes, and the 3 GEP NA samples were clustered with GEP1 class. Pathway analysis showed that 45 DEPs contribute to metastatic-related pathways including cellular movement, cellular proliferation and epithelial to mesenchymal transition. Two upstream regulators, IL1 receptor and SPRY2, were predicted to regulate 8 out of the 45 DEPs. IL1R2 (FC = 3.4, P = 0.021) and SPRY2 (FC = 0.6, P = 0.052) protein expression levels were found matched as prediction in our Olink dataset. Conclusions: 45 AH DEPs could differentiate GEP class 1 and 2 at the diagnostic stage and could be detected even when the tumor was too small to biopsy. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy. Citation Format: Chen-Ching Peng, Liya Xu, Jesse Berry. Diagnostic aqueous humor proteome predicts metastatic potential in uveal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5553.

A pilot study of the use of dynamic analysis of cell-free DNA from aqueous humor and vitreous fluid for the diagnosis and treatment monitoring of vitreoretinal lymphomas.

The diagnosis of vitreoretinal lymphoma (VRL), a rare subtype of primary central nervous system lymphoma, is challenging. We aimed to investigate the mutational landscape of VRL by sequencing circulating tumor DNA (ctDNA) from aqueous humor (AH) and/or vitreous fluid (VF), as well as applying ctDNA sequencing to diagnosis and treatment monitoring. Baseline AH and/or VF specimens from 15 VRL patients underwent comprehensive genomic profiling using targeted next-generation sequencing. The molecular profiles of paired baseline AH and VF specimens were highly concordant, with comparable allele frequencies. However, the genetic alterations detected in cerebrospinal fluid ctDNA only partially overlapped with those from simultaneously collected AH/VF samples, with much lower allele frequencies. Serial post-treatment AH or VF samples were available for five patients and their changes in ctDNA allele frequency displayed a similar trend as the changes in interleukin-10 levels; an indicator of response to treatment. A cohort of 23 patients with primary central nervous system lymphoma was included as a comparison group for the genetic landscape and evaluations of the efficacy of ibrutinib. More MYD88 mutations, but fewer IRF4 mutations and CDKN2A/B copy number losses were observed in the baseline samples of primary central nervous system lymphoma than VRL patients. The objective response rate to ibrutinib treatment was much higher for patients with primary central nervous system lymphoma (64.7%, 11/17) than for those with VRL (14.3%, 1/7). In summary, we provide valuable clinical evidence that AH is a good source of tumor genomic information and can substitute VF. Moreover, molecular profiling of AH has clinical utility for the diagnosis of VRL and treatment monitoring.

Inflammatory Factors of Macular Atrophy in Eyes With Neovascular Age-Related Macular Degeneration Treated With Aflibercept.

BackgroundNeovascular age-related macular degeneration (nAMD) is a leading cause of blindness in older people. Low-grade inflammation is well-known as one of the pathogenic mechanisms in nAMD. Anti-vascular endothelial growth factor (VEGF) therapy is the first-line treatment for nAMD, although macula atrophy (MA) developed under anti-VEGF therapy causes irreversible visual function impairment and is recognized as a serious disorder. Here, we show specific expression patterns of aqueous humor (AH) cytokines in nAMD eyes developing MA under intravitreal injection of aflibercept (IVA) as an anti-VEGF antibody and present predictive cytokines as biomarkers for the incidence of MA in nAMD eyes under IVA treatment.MethodsTwenty-eight nAMD patients received three consecutive monthly IVA, followed by a pro re nata regimen for 2 years. AH specimens were collected before first IVA (pre-IVA) and before third IVA (post-IVA). AH cytokine levels, visual acuity (VA), and central retinal thickness (CRT) were measured.ResultsTwo-year incidence of MA was 21.4%. In nAMD eyes developing MA [MA (+) group], pre-IVA levels of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1β, VEGF and post-IVA level of MCP-1 were higher than those in nAMD eyes without MA [MA (−) group]. In hierarchical cluster analysis, pre-IVA MCP-1 and VEGF were grouped into the same subcluster, as were post-IVA MCP-1 and CRT. In principal component analysis, principal component loading (PCL) of pre-IVA interferon-γ-inducible protein 10 (IP-10) was 0.61, but PCL of post-IVA IP-10 decreased to −0.09. In receiver operating characteristic analysis and Kaplan–Meier curves, pre-IVA MCP-1, MIP-1β, and VEGF and post-IVA interleukin-6, MCP-1, and MIP-1β were detected as predictive factors for MA incidence. In 2-year clinical course, changes of VA in groups with high levels of pre-IVA MIP-1β (over 39.9 pg/ml) and VEGF (over 150.4 pg/ml) were comparable to those in MA (+) group.ConclusionSubstantial loss of IP-10 effects and persistent inflammation contribute to incidence of MA, and screening of AH cytokine levels could be a useful method to predict MA incidence in nAMD eyes under anti-VEGF therapy.

Microbial diagnosis of endophthalmitis using nanopore amplicon sequencing

ObjectivesWe investigated whether nanopore amplicon sequencing of aqueous humor was capable of rapid pathogen identification in infectious endophthalmitis. Methods5 cases of culture-positive bacterial endophthalmitis and 3 cases of fungal endophthalmitis (1 culture-positive and 2 presumed) were included. DNA was extracted from the aqueous humor and vitreous specimen, and PCR of bacterial rDNA (16S) and fungal rDNA (ITS1 and D1/2/3) was performed. Then, nanopore amplicon sequencing was performed for 2 h. The results of amplicon sequencing were compared to those of conventional culture studies. ResultsIn all cases, pathogens were identified by amplicon sequencing of aqueous humor specimens. In 3 cases of bacterial endophthalmitis, the identified microbes were confirmed by culture studies of both aqueous humor and vitreous specimens. In 2 cases of bacterial and 1 case of fungal endophthalmitis, the identified pathogens were confirmed only by culture studies of vitreous specimens. In all cases, amplicon sequencing identified pathogen in a shorter turnaround time than culture studies. In 2 cases with negative culture results, amplicon sequencing of aqueous humor identified fungal pathogens. ConclusionsOur data demonstrates the potential of amplicon nanopore sequencing using aqueous humor to enable rapid, sensitive and less invasive microbial diagnosis of endophthalmitis.

Polymorphisms of the cytomegalovirus glycoprotein B genotype in patients with Posner-Schlossman syndrome

AimsThe aim of this observational study was to report the distribution of glycoprotein B (gB) genotypes in the eyes of cytomegalovirus (CMV) positive patients with Posner-Schlossman syndrome (PSS), and to...

Sex-Based Analysis of Potential Inflammation-Related Protein Biomarkers in the Aqueous Humor of Patients With Diabetes Mellitus.

PurposeTo investigate whether men have higher inflammatory protein biomarker concentrations in their aqueous humor (AH) compared with women in groups of patients with varying levels of diabetic disease.MethodsThis cross-sectional study included AH specimens from 59 adult patients comprised of three groups: no diabetes mellitus (DM), DM without diabetic retinopathy (DR), and DM with proliferative diabetic retinopathy (PDR). Protein biomarker concentration values were quantified using a commercial proximity extension assay-based technique.ResultsIntersex comparisons of concentration values for each protein biomarker revealed no discoveries in patients with no DM or with PDR. In contrast, 24 discoveries were detected in patients with DM without DR. The mean concentration value for all 24 protein biomarkers was higher in men compared with women. Of these 24 proteins, 12 demonstrated a significant association with sex on multivariate linear regression analysis. The β coefficient results demonstrated a positive association between male sex and concentration value for all 12 of these proteins.ConclusionsHigher AH concentration levels of several potential biomarkers, including chemokines, proteases, proteins involved in programmed cell death, and a T-cell surface protein, were detected in men with DM with no DR. These findings suggest that men may have a more inflammatory disease phenotype compared with women in this group of patients.Translational RelevanceThe findings of this study help explain differences in epidemiologic patterns of diabetic retinopathy development between men and women.

Aqueous Humor Levels of Vascular Endothelial Growth Factor and Stromal Cell-Derived Factor-1α in Age-Related Macular Degeneration.

Objectives:This study investigated the contribution of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) angiogenic mediators in eyes with age-related macular degeneration (AMD).Methods:Aqueous humor specimens taken during cataract surgery in 7 cases of intermediate stage (nonexudative) AMD and 7 cases of late stage (exudative) AMD were evaluated using chemiluminescent immunoassay testing in this prospective case-control study. Mediator levels were compared with the normal reference values of 7 patients without any disease other than cataract.Results:The groups were similar in terms of age and gender (p>0.05). The aqueous humor levels of VEGF in both the intermediate AMD (median: 224.3 pg/mL, range: 44.8–380.4 pg/mL) and late-stage AMD (median: 108.7 pg/mL, range: 61.9-223.5 pg/mL) patients were similar to those of the control group (median: 121.1 pg/mL, range: 24.9–156.6 pg/mL) (p=0.256). Moreover, there was no significant difference in the SDF-1α concentrations between the intermediate AMD (median: 160.9 pg/mL, range 130–166.3 pg/mL), late AMD (median: 161 pg/mL, range: 154.1.9–171.6 pg/mL), and control group values (median: 161 pg/mL, range: 155.2–219 pg/mL) (p=0.763).Conclusion:The aqueous humor levels of VEGF and SDF-1α were within the normal range in patients with intermediate and late-stage AMD.

An Increase of Oxidized Phospholipids and the Role of Macrophages in Intraocular Inflammation

PurposeThe present study was conducted to examine the profile of oxidized phospholipids (OxPLs) in uveitis using rat model and clinical specimens, and to elucidate the role of macrophages in the metabolism of OxPLs.MethodsLewis rats were immunized with a bovine interphotoreceptor retinoid- binding protein (bIRBP) peptide with complete Freund's adjuvant (CFA) to induce experimental autoimmune uveitis (EAU). The aqueous humor (AH) was collected 2 weeks after immunization. Fifty-four human AH specimens, among which 21 eyes had a history of chronic uveitis, were collected during their cataract surgery. The profile of OxPLs in the AH specimens were analyzed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). In addition, the involvement of macrophages in the viability of cells treated by OxPLs was investigated through a WST-1 assay using ARPE-19 cells and C57BL/6 mouse alveolar macrophages (AMs). The influence of macrophages in the trend of OxPLs was traced by thin layer chromatography (TLC) using AMs.ResultsSix species of OxPLs were detected in the AHs of rats and humans. The content of each OxPL was higher in the uveitis group. Four kinds of OxPLs found in AHs showed cytotoxicity to ARPE-19 cells in a dose-dependent manner. The cytotoxicity was reduced by pretreatment of OxPLs with AMs. When the OxPLs were applied on AMs, a marked reduction of OxPLs in the medium was observed.ConclusionsThe OxPLs formed by intraocular inflammation could induce cytotoxicity. The present findings suggest that the phagocytic macrophages emerging in the inflammation site eliminate OxPLs, and prevent intraocular tissue damage following uveitis.

Aqueous humor and serum 25-Hydroxyvitamin D levels in patients with cataracts

BackgroundSerum 25-hydroxyvitamin D (25 (OH) D) levels are associated with various pathologic ocular conditions. Few studies have assessed 25 (OH) D concentrations in non-serum specimens, and none to date has assessed 25 (OH) D concentrations in human aqueous humor and their association with ocular diseases. This study investigated the possible correlations between 25 (OH) D concentrations in aqueous humor and serum and whether vitamin D concentrations in aqueous humor were associated with cataract.MethodsThis study prospectively enrolled 136 patients, including 87 with senile cataract and 49 with diabetic cataract, who underwent cataract surgery from January to November 2017. 25 (OH) D was measured in aqueous humor and serum specimens collected from all patients, and their correlation was analyzed statistically. Clinical and laboratory data, including the results of ophthalmologic examinations, were compared in the two groups of cataract patients.ResultsNo correlation was observed between 25 (OH) D concentrations in aqueous humor and serum (P = 0.381). 25 (OH) D concentrations in aqueous humor were significantly higher in patients with diabetic than senile cataract (P = 0.006). Multivariate logistic regression analysis showed that the adjusted odds ratio for diabetic cataract for the highest compared with the lowest quartile of 25 (OH) D concentration in aqueous humor was 4.36 ng/ml (95% confidence interval [CI]: 1.33–14.34 ng/ml; P = 0.015). Multivariate linear regression analysis showed that 25(OH) D concentration in aqueous humor was 2.68 ng/ml (95% CI: 0.34–5.01 ng/ml; P = 0.025) higher in patients with diabetic than senile cataract.Conclusions25(OH) D concentrations in aqueous humor and serum did not correlate with each other. Higher 25(OH) D level in aqueous humor was associated with diabetic cataract. These findings suggest that studies of vitamin D levels in patients with ocular conditions should include measurements of vitamin D levels in aqueous humor.

Bilateral mantle cell lymphoma of the ciliary body that responded to a combined local radiotherapy and chemotherapy regimen: a case report

BackgroundMantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) that often affects men over the age of 60. Systemic metastasis of MCL to eyes is rare and intraocular involvement is even rarer, which usually affects the choroid and iris. To the best of our knowledge, ciliary body metastasis of systemic MCL has not been reported.Case presentationA 59-year-old Han Chinese male with past-history of systemic MCL complained of redness, pain and blurred vision in the left eye. Ocular examination revealed a normal appearance in the right eye, and conjunctival injection, pseudohypopyon and anterior protrusion of peripheral iris in the left eye, all of which were unresponsive to corticosteroid treatments. Ultrasound biomicroscopy (UBM) and B-scan were then performed which detected ciliary body masses in both eyes with no vitreous and retino-choroidal anomalies. Combined liquid-based cytology tests and gene rearrangement assays of the aqueous humor specimen confirmed this to be a B-cell malignancy. Then both eyes were treated with external beam irradiation (40 Gy, delivered evenly in twenty fractions) over a course of one month. Additionally, the left eye received intravitreal methotrexate (MTX) (weekly for the first month, every two weeks for the second month, and monthly thereafter) over a course of twelve months. This therapy eventually led to complete remission of all symptoms in one month and disappearance of the ciliary body masses in twelve months.ConclusionHere we first reported a case of bilateral ciliary body MCL infiltration which was diagnosed by combined liguid-based cytology and gene rearrangement of aqueous humor cells. UBM may serve as a valuable tool in the diagnosis and serial assessments of anterior segment tumors.

Intraocular Oxygen and Antioxidant Status: New Insights on the Effect of Vitrectomy and Glaucoma Pathogenesis

The purpose of this study was to investigate correlations of partial pressure of oxygen (pO2) in the ocular anterior segment of human eyes and aqueous humor antioxidant levels of ascorbate (AsA) and total reactive antioxidant potential (TRAP) with glaucoma and vitreous status. This prospective, cross-sectional study stratified patients (n= 288 eyes) by lens and vitreous status and the presence of primary open-angle glaucoma for statistical analyses. Intraocular pO2 concentrations were measured using a fiberoptic probe in patients at the beginning of planned glaucoma and/or cataract surgery. Aqueous humor specimens were obtained for antioxidant analysis of AsA and TRAP. Following prior pars plana vitrectomy, pO2 levels were significantly higher than in the reference group of cataract surgery in the anterior chamber angle (16.2 ± 5.0 vs. 13.0 ± 3.9mm Hg; P= .0171) and in the posterior chamber (7.6 ± 3.1 vs. 3.9 ± 2.7mm Hg; P < .0001). AsA and TRAP levels were significantly lower (1.1 ± 0.4 vs. 1.4 ± 0.5mM, respectively; 403.3 ±116.5 vs. 479.0 ± 146.7 Trolox units, respectively; P= .004 and P= .024, respectively) in patients after vitrectomy. In patients with an intact vitreous, neither pO2 nor antioxidant status correlated with lens status or glaucoma. Increased pO2 and antioxidant depletion following vitrectomy suggests an alteration of the intraocular oxidant-antioxidant balance. Our study links physiologic factors such as increased pO2 in the anterior chamber angle and the posterior chamber to decreased antioxidant levels in aqueous humor following vitrectomy. Oxidative stress/damage to the trabecular meshwork in such post-vitrectomy cases may contribute to intraocular pressure elevation and increased risk of glaucoma. NOTE: Publication of this article is sponsored bythe American Ophthalmological Society.

Genomic cfDNA Analysis of Aqueous Humor in Retinoblastoma Predicts Eye Salvage: The Surrogate Tumor Biopsy for Retinoblastoma.

Tumor-derived cell-free DNA (cfDNA) has biomarker potential; therefore, this study aimed to identify cfDNA in the aqueous humor (AH) of retinoblastoma eyes and correlate somatic chromosomal copy-number alterations (SCNA) with clinical outcomes, specifically eye salvage. AH was extracted via paracentesis during intravitreal injection of chemotherapy or enucleation. Shallow whole-genome sequencing was performed using isolated cfDNA to assess for highly recurrent SCNAs in retinoblastoma including gain of 1q, 2p, 6p, loss of 13q, 16q, and focal MYCN amplification. Sixty-three clinical specimens of AH from 29 eyes of 26 patients were evaluated; 13 eyes were enucleated and 16 were salvaged (e.g., saved). The presence of detectable SCNAs was 92% in enucleated eyes versus 38% in salvaged eyes (P = 0.006). Gain of chromosome 6p was the most common SCNA found in 77% of enucleated eyes, compared with 25% of salvaged eyes (P = 0.0092), and associated with a 10-fold increased odds of enucleation (OR, 10; 95% CI, 1.8-55.6). The median amplitude of 6p gain was 1.47 in enucleated versus 1.07 in salvaged eyes (P = 0.001). The presence of AH SCNAs was correlated retrospectively with eye salvage. The probability of ocular salvage was higher in eyes without detectable SCNAs in the AH (P = 0.0028), specifically 6p gain. This is the first study to correlate clinical outcomes with SCNAs in the AH from retinoblastoma eyes, as such these findings indicate that 6p gain in the aqueous humor is a potential prognostic biomarker for poor clinical response to therapy.Implications: The correlation of clinical outcomes and SCNAs in the AH identified in the current study requires prospective studies to validate these finding before SCNAs, like 6p gain, can be used to predict clinical outcomes at diagnosis. Mol Cancer Res; 16(11); 1701-12. ©2018 AACR.

Herpesviridae prevalence in aqueous humor using PCR.

ObjectiveThe aim of this study was to determine the prevalence of Herpesviridae family in aqueous humor and the prevalence of antibodies against Herpesviridae family in serum.MethodsParticipants undergoing cataract surgery were included in the study. Serum for viral serology including herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein–Barr virus (EBV), and cytomegalovirus (CMV) was collected. Aqueous humor specimen was tapped for PCR analysis.ResultsNinety-two participants were included with a mean age of 67.67 years (SD ±12.51). The prevalence of positive serology tests was 83.3% for HSV, 94.0% for VZV, 98.8% for EBV, and 97.6% for CMV. A total of 66 aqueous humor specimens were negative for PCR of Herpesviridae family.ConclusionThis study showed previous HSV, VZV, EBV and CMV infections in >90% of the Thai population, while no viral presence was detected in aqueous humor. Thus, the serology test is unrelated to the presence of virus in the eye. We suggest that PCR is a valuable tool to diagnose intraocular viral infection and detect virus presenting active infection.

Comparative Study of Ocular Pharmacokinetics of Gatifloxacin Between Continuous Lavage and Hourly Topical Instillation in Rabbits

To investigate the ocular pharmacokinetics and distribution of gatifloxacin between continuous lavage through the Morgan lens and repeated topical drop instillation in rabbits. Thirty-six healthy rabbits were randomly divided into 2 groups, and 0.3% gatifloxacin ophthalmic solution was instilled by continuous lavage using the Morgan lens onto the left eyes at 15 mL/h (or 50 μL of the same solution repeatedly administered topically every hour). The animals were killed at 2, 4, 8, and 12 hours in the Morgan lens group or at 12 and 24 hours in the topical instillation group. Aqueous humor specimens were collected from the anterior chamber first, and other ocular tissues and plasma were collected subsequently. Pharmacokinetic and pharmacodynamic indices, other than the minimum inhibitory concentration (MIC), were used as therapeutic parameters in ocular tissues. The highest concentration was found in the corneas in both groups. With continuous lavage, gatifloxacin reached an efficacious high concentration in 4 hours in the cornea, which is equivalent to about 23 times the concentration reached in the same tissue by hourly topical instillation for 12 to 24 hours. Using historic data as references, C/MIC90 was 105 (>10) and AUC0-t/MIC90 was 194 h (>125) in the cornea in the continuous lavage group at 4 hours, whereas these values were 5 and 73 hours for the topical hourly instillation group. Compared with hourly topical instillation, continuous lavage through the Morgan lens could achieve significantly higher concentrations in ocular tissues within a short period, especially in the cornea. Continuous lavage may be a much more efficient way to treat recalcitrant corneal infections.

Expression of cytokines in aqueous humor from fungal keratitis patients

BackgroundAlthough a series of reports on corneal fungal infection have been published, studies on pathogenic mechanisms and inflammation-associated cytokines remain limited. In this study, aqueous humor samples from fungal keratitis patients were collected to examine cytokine patterns and cellular profile for the pathogenesis of fungal keratitis.MethodsThe aqueous humor samples were collected from ten patients with advanced stage fungal keratitis. Eight aqueous humor samples from patients with keratoconus or corneal dystrophy were taken as control. Approximately 100 μl to 300 μl of aqueous humor in each case were obtained for examination. The aqueous humor samples were centrifuged and the cells were stained and examined under optical microscope. Bacterial and fungal cultures were performed on the aqueous humor and corneal buttons of all patients. Cytokines related to inflammation including IL-1β, IL-6, IL-8, IL-10, TNF-α, and IFN-γ were examined using multiplex bead-based Luminex liquid protein array systems.ResultsFungus infection was confirmed in these ten patients by smear stains and/or fungal cultures. Bacterial and fungal cultures revealed negative results in all aqueous humor specimens. Polymorphonuclear leukocytes were the predominant infiltrating cells in the aqueous humor of fungal keratitis. At the advanced stages of fungal keratitis, the levels of IL-1β, IL-6, IL-8, and IFN-γ in the aqueous humor were significantly increased when compared with control (p<0.01). The levels of IL-10 and TNF-α also showed an ascending trend but with no statistical significance.ConclusionsHigh concentration of IL-1β, IL-6, IL-8, and IFN-γ in the aqueous humor was associated with fungal keratitis.

Quantitative Proteomic Analysis of Human Aqueous Humor Using iTRAQ 4plex Labeling.

iTRAQ 4plex method enables multiplexing of up to four samples for simultaneous quantitation to improve sensitivity and scope of proteomic analysis. Here, we describe iTRAQ 4plex labeling of human aqueous humor specimens followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and data analysis for peptide identification and quantitation using Proteome Discoverer software. The protocol can be applied for other animals as well; however, pooling of specimens may be required to obtain sufficient amount of protein for labeling.