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

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.

Comprehensive Somatic Copy Number Analysis Using Aqueous Humor Liquid Biopsy for Retinoblastoma

“…As in previous studies [19][20][21][22][23][24][25][26], the most common RB SCNAs included highly recurrent 6p gain (33 eyes, 48.5%), 1q gain (33 eyes, 48.5%), 16q loss (28 eyes, 41.2%), 2p gain (11 eyes, 16.2%), and 13q loss (8 eyes, 11.8%). The most common non-highly recurrent RB SCNAs were 12p loss (8 eyes; 11.8%), 16p loss (8 eyes; 11.8%), 5p gain (7 eyes; 10.3%), 17q gain (6 eyes; 8.8%), 18q gain (6 eyes; 8.8%), 17p loss (5 eyes; 7.4%), 20q gain (5 eyes; 7.4%), and 22p gain (5 eyes; 7.4%).…”

“…Hence, this study includes AH samples that have been analyzed and published previously, in addition to new samples taken after our most recent publications. Case numbers remained consistent with prior studies for comparison purposes [20][21][22]25,26]. As in previous studies [19][20][21][22][23][24][25][26], liquid biopsy specimens consisted of approximately 100 µL AH taken from the anterior chamber via clear corneal paracentesis for research purposes only.…”

“…Case numbers remained consistent with prior studies for comparison purposes [20][21][22]25,26]. As in previous studies [19][20][21][22][23][24][25][26], liquid biopsy specimens consisted of approximately 100 µL AH taken from the anterior chamber via clear corneal paracentesis for research purposes only. Treatment of all RB patients was carried out in a non-randomized manner per CHLA protocol [27][28][29], and treating physicians were blinded to the results of AH analyses.…”

“…This was due to previous inability to biopsy RB for risk of extraocular seeding [17,18]. Our group addressed this limitation by demonstrating that aqueous humor (AH) can be safely extracted and is a rich source of circulating tumor DNA (ctDNA) within the AH [19][20][21]. Using this platform, we have been able to establish relationships between specific genomic alterations and ocular prognosis that could not previously be evaluated in tumor tissue from enucleated eyes only.…”

“…Previous whole-genome analyses of RB SCNAs were limited to tumor tissue from enucleated eyes, so little was known about the genomic landscape of tumors in less advanced, salvaged eyes or new alterations identified during therapy [10,11,[14][15][16]. Additionally, although our group has since used the AH liquid biopsy to identify RB SCNAs in both salvaged and enucleated eyes, these studies focused primarily on a limited subset of highly recurrent SCNAs and did not evaluate for other less common SCNAs that might also be contributing to RB tumorigenesis, seeding, treatment failure, or progression of disease [19][20][21][22][23][24][25][26]. Herein we present a comprehensive (whole-genome) analysis of RB SCNAs using the AH liquid biopsy.…”