A Multicenter Analysis of Nucleic Acid Quantification Using Aqueous Humor Liquid Biopsy in Retinoblastoma: Implications for Clinical Testing.

Purpose: Retinoblastoma (RB) is most often diagnosed with clinical features and not diagnosed with tumor biopsy. This study describes tumor-derived analyte concentrations from aqueous humor (AH) liquid biopsy and its use in clinical assays. Design: Case series study. Participants: Sixty-two RB eyes from 55 children and 14 control eyes from 12 children from 4 medical centers. Methods: This study included 128 RB AH samples including: diagnostic (DX) samples, samples from eyes undergoing treatment (TX), samples after completing treatment (END), and during bevacizumab injection for radiation therapy after completing RB treatment (BEV). Fourteen-control AH were analyzed for unprocessed analytes (double-stranded DNA [dsDNA], single-stranded DNA [ssDNA], micro-RNA [miRNA], RNA, and protein) with Qubit fluorescence assays. Double-stranded DNA from 2 RB AH samples underwent low-pass whole-genome sequencing to detect somatic copy number alterations. Logistic regression was used to predict disease burden given analyte concentrations. Main Outcome Measures: Unprocessed analyte (dsDNA, ssDNA, miRNA, RNA and protein) concentrations. Results: Results revealed dsDNA, ssDNA, miRNA, and proteins, but not RNA, were quantifiable in most samples (up to 98%) with Qubit fluorescence assays. Median dsDNA concentration was significantly higher in DX (3.08 ng/µl) compared to TX (0.18 ng/µl; P < 0.0001) at an order of 17 times greater and 20 times greater than END samples (0.15 ng/µl; P = 0.001). Using logistic regression, nucleic acid concentrations were useful in predicting higher versus lower RB disease burden. Retinoblastoma somatic copy number alterations were identified in a TX, but not in a BEV sample, indicating the correlation with RB activity. Conclusions: Aqueous humor liquid biopsy in RB is a high-yield source of dsDNA, ssDNA, miRNA, and protein. Diagnostic samples are most useful for RB 1 gene mutational analyses. Genomic analysis may be more informative of tumor activity status than quantification alone and can be performed even with smaller analyte concentrations obtained from TX samples. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Evaluation of digital slit-lamp videos for paediatric anterior segment telemedicine consultations.

INTRODUCTION: This study aimed to assess the validity of telemedicine consultations using digital slit-lamp videos to detect anterior segment pathology in a paediatric population. METHODS: A paediatric anterior segment specialist simultaneously performed and recorded anterior segment examinations using the Topcon digital-ready slit lamp. Components of the examination included the eyelids/eyelashes, conjunctiva/sclera, cornea, anterior chamber, iris and lens. Masked to clinical findings, a paediatric ophthalmologist reviewed and graded the live video feed transmitted at 4 Mbps. At least three months later, both ophthalmologists graded the stored videos. We compared the sensitivity, specificity, percent agreement and weighted kappa (κ) of diagnosing anterior segment pathologies via live-streamed and store-and-forward video clips compared to the in-person standard examination. RESULTS: Examinations of 89 eyes from 45 children (5-17 years old) with known anterior segment pathology were included. Agreement between live-streamed and in-person standard examinations for conjunctiva/sclera, anterior chamber, iris and lens findings was almost perfect (sensitivity 89-96%, specificity 95-100%, κ = 0.87-0.97). Substantial agreement was found for cornea pathology (sensitivity 88%, specificity 90%, κ = 0.72), and moderate agreement was found for eyelids/eyelashes pathology (sensitivity 54%, specificity 92%, κ = 0.46). Store-and-forward results were similar, though slightly better for eyelids/eyelashes and slightly worse for conjunctiva/sclera. DISCUSSION: Digital slit-lamp videos hold promise for synchronous and asynchronous telemedicine in diagnosing paediatric anterior segment pathologies.

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.

Ophthalmic imaging in children: current practice patterns and perceived barriers.

Pediatric ophthalmologists were surveyed to determine current practice patterns regarding ophthalmic imaging for children and to identify perceived barriers to the adoption of imaging technologies in their practices. Some form of imaging was available in the majority of practices (94%), but its use varied widely among different clinical scenarios. The two most frequently perceived barriers to performing imaging in children were cooperation and lack of sufficient data supporting ophthalmic imaging in clinical practice.

Keratoconus diagnosis with optical coherence tomography pachymetry mapping.

OBJECTIVE: To detect abnormal corneal thinning in keratoconus using pachymetry maps measured by high-speed anterior segment optical coherence tomography (OCT). DESIGN: Cross-sectional observational study. PARTICIPANTS: Thirty-seven keratoconic eyes from 21 subjects and 36 eyes from 18 normal subjects. METHODS: The OCT system operated at a 1.3 microm wavelength with a scan rate of 2000 axial scans per second. A pachymetry scan pattern (8 radials, 128 axial scans each; 10 mm diameter) centered at the corneal vertex was used to map the corneal thickness. The pachymetry map was divided into zones by octants and annular rings. Five pachymetric parameters were calculated from the region inside the 5 mm diameter: minimum, minimum-median, inferior-superior (I-S), inferotemporal-superonasal (IT-SN), and the vertical location of the thinnest cornea. The 1-percentile value of the normal group was used to define the diagnostic cutoff. Placido-ring-based corneal topography was obtained for comparison. MAIN OUTCOME MEASURES: The OCT pachymetric parameters and a quantitative topographic keratoconus index (keratometry, I-S, astigmatism, and skew percentage [KISA%]) were used for keratoconus diagnosis. Diagnostic performance was assessed by the area under the receiver operating characteristic (AROC) curve. RESULTS: Keratoconic corneas were thinner. The pachymetric minimum averaged 452.6+/-60.9 microm in keratoconic eyes versus 546+/-23.7 microm in normal eyes. The 1-percentile cutoff was 491.6 microm. The thinnest location was inferiorly displaced in keratoconus (-805+/-749 microm vs -118+/-260 microm; cutoff, -716 microm). The thinning was focal (minimum-median: -95.2+/-41.1 microm vs -45+/-7.7 microm; cutoff, -62.6 microm). Keratoconic maps were more asymmetric (I-S, -44.8+/-28.7 microm vs -9.9+/-9.3 microm; cutoff, -31.3 microm; and IT-SN, -63+/-35.7 microm vs -22+/-11.4 microm; cutoff, -48.2 microm). Keratoconic eyes had a higher KISA% index (2641+/-5024 vs 21+/-19). All differences were statistically significant (t test, P<0.0001). Applying the diagnostic criteria of any 1 OCT pachymetric parameter below the keratoconus cutoff yielded an AROC of 0.99, which was marginally better (P = .09) than the KISA% topographic index (AROC, 0.91). CONCLUSIONS: Optical coherence tomography pachymetry maps accurately detected the characteristic abnormal corneal thinning in keratoconic eyes. This method was at least as sensitive and specific as the topographic KISA. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

In vitro measurement of rabbit corneal epithelial thickness using ultrahigh resolution optical coherence tomography.

The objective of this study was to reproducibly measure corneal epithelial thickness centrally and at the limbus in the rabbit cornea using ultrahigh resolution optical coherence tomography (OCT). Twelve freshly enucleated New Zealand white rabbit eyes were kept in a moist chamber at 4 degrees C. An ultrahigh resolution OCT system with a spatial resolution of 1.3 microm was used to image the cornea and its component layers. The central and peripheral (limbal) regions of all the samples were scanned within 6 h of harvest in order to minimize the post-mortem degradation of the corneal epithelium. The thickness of the corneal epithelium was determined by measuring the pixel equivalents of the obtained image. Unpaired Student's t-test was used to evaluate differences. The epithelial thickness centrally was found to be 45.8 +/- 2.2 microm, and 37.6 +/- 1.4 microm at the limbus (P < 0.001). Rabbit corneal epithelium is thicker centrally than at the limbus when measured by ultrahigh resolution OCT. This technique will aid in delineating the pathophysiology of diseases of the anterior cornea.

Association of herpesviruses in the aqueous humor of patients with serpiginous choroiditis: a polymerase chain reaction-based study.

PURPOSE: To determine the presence of herpesvirus DNA in the aqueous humor (AH) of patients with serpiginous choroiditis using polymerase chain reaction (PCR). METHODS: AH from nine patients previously diagnosed with serpiginous choroiditis were investigated for herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) by conventional virological methods and PCR. The PCR-positive DNA was gel-purified, extracted, and sequenced using a dye-based Applied Biosystems procedure. The sequences were processed through the National Cancer Institute's BLAST inquiry for species identification. RESULTS: Culture and cytological examination of AH from all nine patients were negative for HSV, VZV, and CMV. Five were positive for VZV, one was positive for HSV, and three were wholly negative using PCR. Subsequent DNA sequencing of the positive samples authenticated the presence of VZV and HSV DNA in the respective patients. CONCLUSION: VZV and HSV DNA were detected in a subset of patients with serpiginous choroiditis, suggesting that these viruses may function in the pathogenesis of this disease.