Liya Xu1,2, Lishuang Shen3, Ashley Polski1,4, Rishvanth K Prabakar5, Rachana Shah6, Rima Jubran6, Jonathan W Kim1,4, Jacklyn Biegel3, Peter Kuhn2,7,8,9, David Cobrinik1,4,7,10, James Hicks2,7,11, Xiaowu Gai3, Jesse L Berry1,4,7,10. 1. The Vision Center, Children's Hospital Los Angeles , Los Angeles, California, USA. 2. Department of Biological Sciences, Dornsife College of Letters, Arts, and Sciences, University of Southern California , Los Angeles, California, USA. 3. Center for Personalized Medicine, Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles , Los Angeles, California, USA. 4. USC Roski Eye Institute, Keck School of Medicine of USC , Los Angeles, California, USA. 5. Department of Molecular and Computational Biology, University of Southern California , Los Angeles, California, USA. 6. Center for Blood Disorders, Children's Hospital Los Angeles , Los Angeles, California, USA. 7. Norris Comprehensive Cancer Center, Keck School of Medicine of USC , Los Angeles, California, USA. 8. Department of Aerospace and Mechanical Engineering, Viterbi School of Engineering, University of Southern California , Los Angeles, California, USA. 9. Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California , Los Angeles, California, USA. 10. The Saban Research Institute, Children's Hospital Los Angeles , Los Angeles, California, USA. 11. Department of Biochemistry and Molecular Medicine, Keck School of Medicine of USC , Los Angeles, California, USA.
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.
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 RBpatients 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.
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Authors: Elyssa Y Wong; Liya Xu; Lishuang Shen; Mary E Kim; Ashley Polski; Rishvanth K Prabakar; Rachana Shah; Rima Jubran; Jonathan W Kim; Jaclyn A Biegel; Xiaowu Gai; Peter Kuhn; James Hicks; Jesse L Berry Journal: NPJ Precis Oncol Date: 2021-07-27