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

Liya Xu, Mary E Kim, Ashley Polski, Rishvanth K Prabakar, Lishuang Shen, Chen-Ching Peng, Mark W Reid, Patricia Chévez-Barrios, Jonathan W Kim, Rachana Shah, Rima Jubran, Peter Kuhn, David Cobrinik, Jaclyn A Biegel, Xiaowu Gai, James Hicks, Jesse L Berry, Liya Xu, Mary E Kim, Ashley Polski, Rishvanth K Prabakar, Lishuang Shen, Chen-Ching Peng, Mark W Reid, Patricia Chévez-Barrios, Jonathan W Kim, Rachana Shah, Rima Jubran, Peter Kuhn, David Cobrinik, Jaclyn A Biegel, Xiaowu Gai, James Hicks, Jesse L Berry

Abstract

Because direct tumor biopsy is prohibited for retinoblastoma (RB), eye-specific molecular biomarkers are not used in clinical practice for RB. Recently, we demonstrated that the aqueous humor (AH) is a rich liquid biopsy source of cell-free tumor DNA. Herein, we detail clinically-relevant molecular biomarkers from the first year of prospective validation data. Seven eyes from 6 RB patients who had AH sampled at diagnosis and throughout therapy with ≥12 months of follow-up were included. Cell-free DNA (cfDNA) from each sample was isolated and sequenced to assess genome-wide somatic copy number alterations (SCNAs), followed by targeted resequencing for pathogenic variants using a RB1 and MYCN custom hybridization panel. Tumoral genomic information was detected in 100% of diagnostic AH samples. Of the seven diagnostic AH samples, 5/7 were positive for RB SCNAs. Mutational analysis identified RB1 variants in 5/7 AH samples, including the 2 samples in which no SCNAs were detected. Two eyes failed therapy and required enucleation; both had poor prognostic biomarkers (chromosome 6p gain or MYCN amplification) present in the AH at the time of diagnosis. In the context of previously established pre-analytical, analytical, and clinical validity, this provides evidence for larger, prospective studies to further establish the clinical utility of the AH liquid biopsy and its applications to precision oncology for RB.

Keywords: SCNA; aqueous humor; ctDNA; liquid biopsy; precision oncology; retinoblastoma.

Conflict of interest statement

Drs. Berry, Xu, and Hicks have filed a provisional patent application entitled, Aqueous Humor Cell Free DNA for Diagnostic and Prognostic Evaluation of Ophthalmic Disease. Otherwise, the authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1
Diagnostic information from AH liquid biopsy with graph showing the tumor fraction (TFx) of SCNA (dark blue bar), variant allele frequency (VAF) of RB1 SNV (light blue bar), and cfDNA quantification (orange dots) for each case’s AH sample at time of diagnosis. NA: not available; ND: not detected.
Figure 2
Figure 2
Genomic profiles for the 4 eyes with detectable large-scale SCNAs (cases 33, 45, 47, 48) and the 1 eye with a focal RB1 gene deletion (Case 44 OS).
Figure 3
Figure 3
Prognostic information from AH liquid biopsy. Genomic profiles from the blood, AH sample at time of diagnosis, and tumor tissue at time of enucleation for (a) Case 33, asterisk showing 6p gain with an amplitude of 1.5 ratio to the median and (b) Case 48, asterisk showing MYCN amplification. Genomic profiles from the blood were without SCNAs, compared to the genomic profiles from the AH at time of diagnosis, which were highly concordant with those obtained from the tumor tissue. Due to admixing with normal retinal tissue, SCNAs from tumor tissue may show lower amplitude compared to AH due to diluted TFx.
Figure 4
Figure 4
Longitudinal information from AH liquid biopsy for Case 33. Fundus photos, tumor fraction (TFx), cfDNA quantification, estimated clinical tumor volume from B-scan measurements, and genomic profiles for each clinical timepoint at which an AH sample was evaluated. This eye was non-responsive to treatment, and ultimately required secondary enucleation (ES) due to an apical tumor recurrence with persistently active seeds. A dramatic decrease in cfDNA quantity was observed over time, consistent with the decrease in the main tumor volume. This indicates that the total number of tumor cells that were actively shedding cfDNA into the AH decreased. However, TFx remained at a high level throughout treatment, reflecting the persistent seeding that still contributes to tumor-derived cfDNA in the AH. Without much physiological cfDNA to dilute the tumor-derived cfDNA, the TFx remained high. Genomic profiles consistently demonstrated the same 3 SCNAs that were present at diagnosis, as well as a new large-scale 2p gain (*) and 19q loss in the AH of the enucleated eye, suggesting clonal evolution at the time of recurrence.
Figure 5
Figure 5
Longitudinal information from AH liquid biopsy for Case 47. Fundus photos, tumor fraction (TFx), cfDNA quantification, estimated clinical tumor volume from B-scan measurements, and genomic profiles for each clinical timepoint at which an AH sample was evaluated. This eye was responsive to treatment and remained salvaged at 19 months of follow-up. A decrease in cfDNA quantity was observed over time, consistent with the decrease in the main tumor volume. TFx also decreased, reflecting resolution of seeding. Genomic profiles completely normalized over treatment, as would be seen with clinical regression of disease.

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