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 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.

Keywords: Aqueous humor; Circulating analytes; Liquid biopsy; Multicenter; Retinoblastoma.

Figure 1

Quantification of aqueous humor analytes in all retinoblastoma (RB) samples and controls: (A) double-stranded DNA (dsDNA), (B) single-stranded DNA (ssDNA), (C) RNA (D) micro-RNA (miRNA), and (E) protein concentration of RB eyes and control eyes were analyzed. SEM = standard error of the mean.

Figure 2

Quantification of retinoblastoma (RB) aqueous humor analytes from: diagnostic (DX), during RB treatment (TX), during bevacizumab injection at the end of RB treatment (BEV), and end of RB treatment (END) samples. A, double-stranded DNA (dsDNA),(B) single-stranded DNA (ssDNA), (C) RNA, (D) micro-RNA (miRNA), and (E) protein concentration. NA = not available; SEM = standard error of the mean.

Figure 3

Quantification of retinoblastoma (RB) aqueous humor (AH) analytes from diagnostic AH samples grouped by International Intraocular Retinoblastoma Classification. A, double-stranded DNA (dsDNA), (B) single-stranded DNA (ssDNA), (C) RNA, (D) micro-RNA (miRNA), and (E) protein concentration. NA = not available; SEM = standard error of the mean.

Figure 4

Receiver operating curves (ROC) curve analysis of the utility of aqueous humor analyte concentrations to determine retinoblastoma disease burden. Cut-off values for analyte concentration that maximizes sensitivity and specificity are circled in red. Disease burden was defined as: Highest disease burden (Diagnostic: diagnostic or primarily enucleated samples), Moderate disease burden (During treatment: at secondary enucleation, systemic chemotherapy, intravitreal melphalan injection, or intra-arterial chemotherapy), Lowest disease burden (End of treatment and during bevacizumab injection after the end of treatment). A, double-stranded DNA (dsDNA), (B) single-stranded DNA (ssDNA), (C) micro-RNA (miRNA), and (D) protein concentration models. CI = confidence interval.

Figure 5

Analyte concentrations and somatic copy number alteration (SCNA) profiles from the same eye at different clinical times. A, AH analyte (double-stranded DNA [dsDNA], single-stranded DNA [ssDNA], micro-RNA [miRNA], RNA, and protein) concentrations from Case 14 undergoing intravitreal melphalan (IVM) and the same eye at the end of treatment receiving bevacizumab (BEV) injections. B, Case 14 IVM, retinoblastoma SCNAs of 6p gain and 16q loss are indicated with an ∗. C, Case 14 BEV, a flat SCNA profile.