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Simultaneous identification of clinically relevant RB1 mutations and copy number alterations in aqueous humor of retinoblastoma eyes

ABSTRACT Background Detection of germline RB1 mutations is critical for risk assessment of retinoblastoma (RB) patients. Assessment of somatic copy number alterations (SCNAs) is also critically important because of their prognostic significance. Herein we present a refined approach for the simultaneous identification of RB1 variants and SCNAs in the aqueous humor (AH) of RB eyes. Materials and Methods Subjects included 7 eyes of 6 RB patients that underwent AH extraction, and 4 matched tumor samples. Cell-free DNA (cfDNA) was isolated and sequenced to assess genome-wide SCNAs. The same sequencing libraries then underwent targeted resequencing and mutation detection using a custom hybridization panel that targets RB1 and MYCN. Illumina paired-end 2x150bp sequencing was used to characterize single-nucleotide variants (SNVs) and loss of heterozygosity (LOH). Results were compared to peripheral blood RB1 testing. Tumor fraction (TFx) was calculated using ichorCNA. Results Four of 7 AH samples contained clinically significant SCNAs. Of the 3 other samples, 1 showed focal MYCN amplification and 1 showed focal RB1 deletion. All 4 enucleated tumors contained SCNAs. Mutational analysis of tumor DNA identified all first hits (2 germline RB1 SNVs, 2 germline CNAs) and second hits (4 RB1 SNVs). RB1 variants in AH were concordant with those obtained from corresponding tumor tissue and blood. In AH samples without paired tumor, both RB1 hits were identified with high variant allele frequency, even in the absence of SCNAs. Conclusions AH liquid biopsy is a minimally invasive, in vivo alternative to tissue analysis for the simultaneous identification of RB1 variants and SCNAs in RB eyes.