Abstract 3553: Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes
Authors: Xu Liya , Schmidt Mike J. , Prakabar Rishvanth K. , Kuhn Peter , Hicks James , Berry Jesse 
Published: April 2023

Abstract
Introduction: Retinoblastoma (Rb) is the most common childhood intraocular cancer. Tissue biopsy of Rb can cause tumor spread, so it is contraindicated. We demonstrated that aqueous humor (AH), an ocular fluid, is a high-yield liquid biopsy enabling in vivo detection of tumor-derived cell-free DNA (cfDNA) thus overcoming the contraindication of biopsy. Somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) on RB1 gene, have been able to be identified in the same clinical samples but with two separate sequencing runs. In this study, we first developed a single targeted sequencing method to identify both SCNAs and RB1 SNVs. With this method, we further investigated the degree of genomic concordance between paired tumor and AH samples from the same Rb eye.
Materials and Methods: 11 paired AH and Rb tumor samples were included in the study. cfDNA of AH and tumor DNA of enucleated Rb eyes were isolated with QIAgen commercial kits. DNAs were constructed into whole genome libraries followed by hybridization target enrichment (Agilent SureSelect). The enrichment assay covers the whole length of RB1 and MycN genes, the all-exon regions of BCOR and CREBBP genes, and a whole genome CNV backbone. Libraries were submitted to Illumina HiSeq 4000 platform for fastq data generation with 2 × 150bp mode. The bioinformatics pipeline was optimized to generate SCNA profiles from targeted sequencing reads, along with SNV calling.
Results: For SCNA profiles, 11/11 AH samples (100%) and 8/11 tumor samples (72.72%) have positive RB-SCNA signatures. Strong concordances were observed between AH and tumor SCNA profiles (median = 90.1%) with targeted sequencing reads. In total, 9 disease-driving RB1 SNVs were identified in 6 patients (54.5%). 7/9 (77.8%) of the variants were shared between AH and tumor samples, while the AH and tumor each contained one unique SNV. In all SNVs, the AH displayed a higher allele frequency. Notably, 4/11 samples have focal RB1 gene deletion detected with SCNA profiling, which may explain the difficulties of RB1 SNV detection in some samples. Further, one case was driven by a MYCN gene amplification with no RB1 alterations.
Conclusions: This study presented highlights the utility of a single method to call both SVNs and SCNAs in a single clinical sample, with enriched tumor information detected in AH compared with tumor. This study further strengthens the utility of AH as a liquid biopsy platform for Rb eyes.
Citation Format: Liya Xu, Mike J. Schmidt, Rishvanth K. Prakabar, Peter Kuhn, James Hicks, Jesse Berry. Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3553.