Simultaneous copy number alteration and variant detection in paired aqueous humor and tumor samples from retinoblastoma eyes

Abstract

Purpose : Tissue biopsy of retinoblastoma (Rb) is contraindicated due to risk of extraocular spread. We previously demonstrated that the aqueous humor (AH) is a high yield liquid biopsy enabling in vivo detection of tumor-derived cell free DNA (cfDNA), thus overcoming the contraindication to tissue biopsy. Somatic genomic alterations, including somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) on the RB1 gene, have been previously identified in clinical samples with the use of two separate sequencing runs. In this study, we developed a single targeted sequencing method to simultaneously identify both SCNAs and RB1 SNVs. Using this method, we further investigated the degree of genomic concordance between paired tumor and AH samples from the same Rb eye.

Methods : 11 paired AH and Rb tumor samples were included in this study. cfDNA of AH and tumor DNA of enucleated Rb eyes were isolated using Qiagen kits and constructed into whole genome libraries, followed by hybridization target enrichment (Agilent SureSelect). The enrichment assay covers the whole length of RB1 and MYCN genes and the whole genome SCNA backbone. Libraries were sequenced using Illumina HiSeq 4000 platform (2x150bp). 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%) using targeted sequencing reads. In total, the causative mechanism of disease was identified in 10/11 cases (91%). 9 disease driving RB1 SNVs were identified in 6 patients, 3 patients had focal RB1 deletion, and 1 case was driven by MYCN amplification. 7/9 (77.8%) of the SNVs 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.

Conclusions : The method developed in this study of simultaneous SCNA and SNV profiling using the AH provides clinicians a more efficient pipeline to obtain a thorough understanding of the genomic landscape of the tumor in Rb patients at the time of diagnosis and throughout treatment. Strong concordance in genomic profiles between tumor and AH samples further strengthens the utility of the AH as a surrogate tumor biopsy in Rb eyes.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.