A multicenter analysis of nucleic acid quantification using aqueous humor liquid biopsy in retinoblastoma – Implications for clinical testing

Abstract

Purpose : Retinoblastoma (RB) is most often diagnosed with clinical features and not diagnosed with a tumor biopsy. This study describes tumor-derived analyte concentrations from aqueous humor (AH) liquid biopsy and its use in clinical assays.

Methods : 128 RB AH samples, taken from treatment naïve (TN) eyes, those undergoing treatment (TX), after completing treatment (END) and during bevacizumab injection for radiation therapy after completing RB treatment (BEV), and 14 control AH were analyzed for unprocessed analytes (dsDNA, ssDNA, miRNA, RNA, and protein) with Qubit fluorescence assays. dsDNA from two RB samples underwent shallow whole-genome sequencing (LP-WGS) to detect somatic copy number alterations (SCNAs). Logistic regression was used to predict disease burden given analyte 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 TN (3.08 ng/L) compared to TX (0.18 ng/L; P<0.0001) at an order of 17 times greater, and 20 times greater than END samples (0.15 ng/L; P=0.001). Using logistic regression, nucleic acid concentrations were useful in predicting higher versus lower RB disease burden. RB SCNAs were identified in a TX, but not in a BEV sample, indicating the correlation with retinoblastoma activity.

Conclusions : AH liquid biopsy in RB is a high yield source of dsDNA, ssDNA, miRNA and protein. TN samples are most useful for RB1 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.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.