Single-cell somatic copy number alteration profiling of vitreous humor seeds in retinoblastoma.

BACKGROUND: Heterogeneity can impact biomarker identification. Thus, we investigated the somatic copy number alterations (SCNAs) of individual tumor cells in the vitreous humor of a retinoblastoma patient using single-cell whole-genome profiling and explored the genomic concordance among vitreous and aqueous humor, vitreous seeds, and tumor. METHODS: Aqueous humor (AH), vitreous humor (VH), and tumor biopsy were obtained from an enucleated globe with retinoblastoma and vitreous seeding. Micromanipulation was used to manually isolate 39 live single tumor cells from vitreous seeds harvested from the VH. The SCNA profiles of these individual cells were generated via whole-genome sequencing and analyzed alongside profiles from the tumor mass and cell-free DNA (cfDNA) from AH and VH. RESULTS: Heatmap of VH single-cell SCNA profiles demonstrates heterogeneity among individual vitreous seeds with one clearly dominant subclone (23 of 37 cells). The SCNA profiles from the cells in this subclone demonstrate an average concordance of 98% with cfDNA profiles from acellular AH and VH and with the tumor profile. CONCLUSIONS: Our findings reveal some heterogeneity among single-cell SCNA profiles in individual VH seeds. Despite this heterogeneity, the dominant vitreous subclone exhibits extremely (>98%) high concordance with the SCNA profile from tumor and AH, suggesting AH cfDNA is representative of the dominant genomic subclone. This may facilitate tumoral biomarker identification via the AH. This preliminary work supports the potential of applying single-cell technology to VH seeds in retinoblastoma as a platform to study tumor subclones, which may provide insight into the genomic complexity of disease.

Comparative Single Vesicle Analysis of Aqueous Humor Extracellular Vesicles before and after Radiation in Uveal Melanoma Eyes.

Small extracellular vesicles (sEVs) have been shown to promote tumorigenesis, treatment resistance, and metastasis in multiple cancer types; however, sEVs in the aqueous humor (AH) of uveal melanoma (UM) patients have never previously been profiled. In this study, we used single particle analysis to characterize sEV subpopulations in the AH of UM patients by quantifying their size, concentration, and phenotypes based on cell surface markers, specifically the tetraspanin co-expression patterns of CD9, CD63, and CD81. sEVs were analyzed from paired pre- and post-treatment (brachytherapy, a form of radiation) AH samples collected from 19 UM patients. In post-brachytherapy samples, two subpopulations, CD63/81+ and CD9/63/81+ sEVs, were significantly increased. These trends existed even when stratified by tumor location and GEP class 1 and class 2 (albeit not significant for GEP class 2). In this initial report of single vesicle profiling of sEVs in the AH of UM patients, we demonstrated that sEVs can be detected in the AH. We further identified two subpopulations that were increased post-brachytherapy, which may suggest radiation-induced release of these particles, potentially from tumor cells. Further study of the cargo carried by these sEV subpopulations may uncover important biomarkers and insights into tumorigenesis for UM.

Diagnostic Aqueous Humor Proteome Predicts Metastatic Potential in Uveal Melanoma.

Gene expression profiling (GEP) is clinically validated to stratify the risk of metastasis by assigning uveal melanoma (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 is limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding, or tumor dissemination. Thus, ocular liquid biopsy has emerged as a less-invasive alternative. In this study, we seek to determine the aqueous humor (AH) proteome related to the advanced GEP class 2 using diagnostic AH liquid biopsy specimens. Twenty AH samples were collected from patients with UM, grouped by GEP classes. Protein expression levels of 1472 targets were analyzed, compared between GEP classes, and correlated with clinical features. Significant differentially expressed proteins (DEPs) were subjected to analysis for cellular pathway and upstream regulator identification. The results showed that 45 DEPs detected in the AH could differentiate GEP class 1 and 2 at diagnosis. 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.

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.

Single vesicle analysis of aqueous humor in pediatric ocular diseases reveals eye specific CD63-dominant subpopulations.

Aqueous humor (AH), the clear fluid in front of the eye, maintains the pressure and vitality of ocular tissues. This fluid is accessible via the clear cornea which enables use of AH as a liquid biopsy source of biomarkers for intraocular disease. Extracellular vesicles are detectable in the AH and small extracellular vesicles (sEVs) are present in the AH from adults. However, EVs in AH from pediatric eyes in vivo have never previously been explored. We know very little about the heterogeneity of AH EV populations in ocular disease. Twenty-seven processing-free AH samples from 19 patients across four different pediatric ocular diseases were subjected to Nanoparticle Tracking Analysis (NTA) and Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) analysis. NTA demonstrated the concentration of AH EV/EPs is 3.11 × 109-1.38 × 1010 particles/ml; the majority sized 76.8-103 nm. SP-IRIS revealed distinct patterns of tetraspanin expression of AH sEVs. An enriched mono-CD63+ sEV subpopulation identified in AH indicates this is a potential AH-specific biomarker. In the setting of retinoblastoma there was a more heterogeneous population of sEVs which normalized with treatment. This suggests a potential clinical application of direct measurement of sEV subpopulations in AH samples to monitor successful tumor response to therapy.

Chromosome 6p amplification detected in blood cell-free DNA in advanced intraocular retinoblastoma.

BACKGROUND: In patients with retinoblastoma, gains of chromosome 6p have been associated with less differentiated tumors. In cell-free DNA from the aqueous humor (AH), 6p gain has been associated with an increased risk of enucleation. While the identification of somatic copy number alterations (SCNAs) via the AH has been well established, these alterations are not routinely identified in the blood due to low tumor fraction. MATERIALS AND METHODS: SCNAs were considered positive at 20% deflection from the baseline. Somatic RB1 pathogenic variants were identified with targeted sequencing using a panel including all RB1 exons. RESULTS: A 24-month-old patient presented with unilateral retinoblastoma (Group D/AJCC Stage cT2B) and was treated with primary enucleation. In the peripheral blood, a heterozygous mutation (c.3920T>A) in the APC gene was reported. Genomic analysis of the tumor and AH revealed two novel somatic RB1 mutations (c.1589_1590del and c.2330dupC). Both also demonstrated highly recurrent RB-related SCNAs. Chromosome 6p gain was detected in the blood with an amplitude suggesting approximately 12% tumor fraction. At a follow-up of 24 months, there has been no evidence of metastatic disease. CONCLUSIONS: To our knowledge, this is the first time an SCNA has been detected in the blood of an RB patient, suggesting in some advanced eyes there may be a high enough tumor fraction to detect these alterations (>5% needed). It remains unclear whether 6p gain or increased tumor fraction in the blood is indicative of increased risk of metastatic disease or new primary cancer; studies to address this are ongoing.