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.
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