Cell-Free DNA Tumor Fraction in the Aqueous Humor Is Associated With Therapeutic Response in Retinoblastoma Patients

Abstract

The aqueous humor (AH) liquid biopsy enables in vivo evaluation of tumorderived cell-free DNA (cfDNA) from retinoblastoma (RB) eyes. Herein, we test our hypothesis that longitudinal dynamics of AH cfDNA-including tumor fraction (TFx) and somatic copy number alteration (SCNA) amplitude-correspond to therapeutic response. Methods: Eyes with ≥3 AH extractions during intravitreal chemotherapy (IVM) or at secondary enucleation between 2015 to 2019 were included. AH cfDNA was sequenced to assess RB SCNA amplitude; ichorCNA software was used to estimate TFx. Eyes without SCNAs or with TFx < 0.10 across all samples were excluded. Therapeutic responses for each eye were determined from clinical records. Statistical analyses included Mann-Whitney U and Pearson correlation tests. Results: Twenty eyes of 20 patients underwent ≥3 AH extractions; 6 eyes lacked SCNAs or had TFx < 0.10 throughout sampling and were excluded. Clinical progression was associated with significantly higher SCNA amplitudes and TFx values than regression (P ≤ 0.04). Relative increases in TFx (TFx 1.86 ± 2.22) were associated with disease progression, whereas relative decreases in TFx (TFx 0.53 ± 0.36) were associated with disease regression (P < 0.00001). A ≥15% increase in TFx relative to baseline during treatment was associated with an over 90-fold increased likelihood of clinical progression (odds ratio = 90.67, 95% confidence interval = 8.30-990.16, P = 0.0002). TFx and SCNA amplitude were significantly positively correlated throughout sampling (P ≤ 0.002). Conclusions: Longitudinal changes in AH-derived cfDNA TFx and SCNA amplitude are concordant with clinical responses of intraocular RB during active therapy. Translational Relevance: Longitudinal evaluation of AH cfDNA may provide an objective, quantitative way to monitor therapeutic response and disease burden in RB patients.

Establishing the Clinical Utility of ctDNA Analysis for Diagnosis, Prognosis, and Treatment Monitoring of Retinoblastoma: The Aqueous Humor Liquid Biopsy

“…The workflow for the AH platform has been published extensively previously [ 12 , 13 , 15 , 16 , 17 , 18 , 19 ]. Further methods are available as a supplemental file (Methods S1) .…”

“…Thus, it is an ideal liquid biopsy platform for RB [ 12 , 13 , 14 ]. Multiple subsequent studies have connected RB genomics to clinically relevant outcomes, notably the ability to cure and save the eye with standard therapies [ 12 , 13 , 15 , 16 , 17 , 18 , 19 ]. Specifically, we demonstrated that chromosome 6p gain is associated with a nearly 10-fold increased odds of enucleation (surgical removal of the eye) [ 13 , 16 ], and that dynamics in cfDNA tumor fraction are closely associated with response to therapy [ 19 ].…”

“…Multiple subsequent studies have connected RB genomics to clinically relevant outcomes, notably the ability to cure and save the eye with standard therapies [ 12 , 13 , 15 , 16 , 17 , 18 , 19 ]. Specifically, we demonstrated that chromosome 6p gain is associated with a nearly 10-fold increased odds of enucleation (surgical removal of the eye) [ 13 , 16 ], and that dynamics in cfDNA tumor fraction are closely associated with response to therapy [ 19 ]. We also published a comprehensive workflow for the AH liquid biopsy that included simultaneous whole-genome SCNA analysis, RB1 pathogenic variant detection, and tumor fraction estimation from a single 100µL sample of AH from RB eyes [ 17 ].…”

“…Following technical and clinical validation, the AH is now established as an enriched liquid biopsy source of tumor-derived molecular information with potential clinical utility for the management of children with RB. An important distinction is that all previous studies of the AH liquid biopsy platform have focused on the safety and clinical research applications from eyes actively undergoing therapy, with retrospective analysis [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. An accepted protocol for obtaining AH at the time of diagnosis (i.e., prior to surgical or chemotherapeutic treatment) has not yet been developed.…”

“…An accepted protocol for obtaining AH at the time of diagnosis (i.e., prior to surgical or chemotherapeutic treatment) has not yet been developed. This initial step in prospective validation of previous findings could only be initiated once we established analytical and clinical validation, with prolonged follow-up [ 12 , 13 , 15 , 16 , 17 , 18 , 19 ]. Herein, we describe the initial series of treatment-naive RB patients from whom AH was taken as part of their diagnostic evaluation under an approved research protocol.…”