Tumor biopsy can identify prognostic biomarkers for metastatic uveal melanoma (UM), however aqueous humor (AH) liquid biopsy may serve as an adjunct. This study investigated whether the AH of UM eyes has sufficient circulating tumor DNA (ctDNA) to perform genetic analysis. This is a case series of 37 AH samples, taken before or after radiation, and one tumor wash sample, from 12 choroidal and 8 ciliary body (CB) melanoma eyes. AH was analyzed for nucleic acid concentrations. AH DNA and one tumor wash sample underwent shallow whole-genome sequencing followed by Illumina sequencing to detect somatic copy number alterations (SCNAs). Four post-radiation AH underwent targeted sequencing of BAP1 and GNAQ genes. Post-radiation AH had significantly higher DNA and miRNA concentrations than paired pre-radiation samples. Highly recurrent UM SCNAs were identified in 0/11 post-radiation choroidal and 6/8 post-radiation CB AH. SCNAs were highly concordant in a CB post-radiation AH with its matched tumor (r = 0.978). BAP1 or GNAQ variants were detected in 3/4 post-radiation AH samples. AH is a source of ctDNA in UM eyes, particularly in post-radiation CB eyes. For the first time, UM SCNAs and mutations were identified in AH-derived ctDNA. Suggesting that AH can serve as a liquid biopsy for UM.

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. Case series study. Sixty-two RB eyes from 55 children and 14 control eyes from 12 children from 4 medical centers. 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. Unprocessed analyte (dsDNA, ssDNA, miRNA, RNA and protein) concentrations. 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. 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. Proprietary or commercial disclosure may be found after the references.

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 × 10 ⁹ –1.38 × 10 ¹⁰ 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.

Introduction: 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 cornea which enables use of AH as a liquid biopsy for intraocular disease. Retinoblastoma (Rb), an intraocular cancer in children, is unique in that direct tumor biopsy is prohibited, thus liquid biopsy has great clinical potential. cfDNA in the AH as a biomarker for Rb patients has been investigated and extracellular vesicles (EVs) are detectable in the AH from adults. However, AH EVs in Rb have previously never been explored. We know very little about the heterogeneity of AH EV populations in ocular cancers. Materials and Methods: 27 AH samples from 19 patients from 13 tumor-free eyes and 11 Rb eyes were collected. Rb eyes were further divided into treatment-naïve (Rb_Tn) and treatment-active (Rb_Tx) subgroups. Unprocessed AH samples were subjected to Nanoparticle Tracking Analysis (NTA) (Nanosight NS300) for size distribution and concentration, and to Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) for fluorescent-based immunophenotyping of EV marker expression (CD63, CD81, and CD9). Results: NTA demonstrated the concentration of AH EVs is 3.11 x 10⁹-1.38 x 10¹⁰ vesicles/mL; majority sized 76.8-103 nm. Study group comparisons showed that non-tumor containing eyes had a smaller nanoparticle mean size compared to Rb containing eyes (P = 0.002). More EVs were significantly detected in Rb_Tn containing eyes compared to Rb_Tx containing eyes (P = 0.022), suggesting the possible presence of tumor-derived EVs in Rb_Tn which were subsequently eliminated by treatment. SP-IRIS revealed distinct patterns of tetraspanin expression of AH small EVs (sEVs). Mono-CD63+ EVs were identified to be the most dominant sEV subpopulation from AH across non-tumor and Rb_Tx eyes. However, more diverse sEV subpopulation profile was detected in Rb_Tn AH samples. Significantly lower percentage of mono-CD63+ EVs could be determined in Rb_Tn eyes compared to Rb_Tx eyes (70.3% vs. 96.1%, P = 0.001). A significant accumulation of CD9/CD63 (4.3% vs. 1.1%, P = 0.035), CD63/CD81 (20.7% vs. 2.0%, P = 0.012) and CD9/CD63/CD81 (4.8% vs. 0.8%, P = 0.022) subpopulations in Rb_Tn was also observed. An enriched mono-CD63+ sEV subpopulation identified in AH indicates this is a potential AH-specific biomarker. In the setting of Rb there was a more heterogeneous population of sEVs which normalized with treatment. Conclusions: Small EVs are readily detectable in unprocessed AH with a dominant mono-CD63+ subpopulation in AH regardless of pediatric eye disease states. Tetraspanin colocalization analysis indicates the clearance of retinoblastoma-derived EV heterogeneity by chemotherapy. These novel finding suggests a potential clinical application of measurement of sEV subpopulations in AH samples to monitor response to therapy. Citation Format: Chen-Ching Peng, Deborah Im, Shreya Sirivolu, Bibiana Reiser, Aaron Nagiel, Paolo Neviani, Liya Xu, Jesse L. Berry. Clearance of tumor-derived extracellular vesicle heterogeneity in aqueous humor after chemotherapy in retinoblastoma eyes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3416.