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

• Published in: Ophthalmic genetics 2020-12, Vol.41 (6), p.526-532
• Main Authors: Xu, Liya, Shen, Lishuang, Polski, Ashley, Prabakar, Rishvanth K, Shah, Rachana, Jubran, Rima, Kim, Jonathan W, Biegel, Jacklyn, Kuhn, Peter, Cobrinik, David, Hicks, James, Gai, Xiaowu, Berry, Jesse L
• Format: Article
• Language: English
• ISSN: 1381-6810; 1744-5094
• DOI: 10.1080/13816810.2020.1799417

Detection of germline 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 variants and SCNAs in the aqueous humor (AH) of RB eyes. 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 and . Illumina paired-end 2x150bp sequencing was used to characterize single-nucleotide variants (SNVs) and loss of heterozygosity (LOH). Results were compared to peripheral blood testing. Tumor fraction (TFx) was calculated using ichorCNA. Four of 7 AH samples contained clinically significant SCNAs. Of the 3 other samples, 1 showed focal amplification and 1 showed focal deletion. All 4 enucleated tumors contained SCNAs. Mutational analysis of tumor DNA identified all first hits (2 germline SNVs, 2 germline CNAs) and second hits (4 SNVs). variants in AH were concordant with those obtained from corresponding tumor tissue and blood. In AH samples without paired tumor, both hits were identified with high variant allele frequency, even in the absence of SCNAs. AH liquid biopsy is a minimally invasive, alternative to tissue analysis for the simultaneous identification of variants and SCNAs in RB eyes.