Next-Generation Sequencing of Retinoblastoma Identifies Pathogenic Alterations beyond RB1 Inactivation That Correlate with Aggressive Histopathologic Features

Affiliations

¹ Department of Ophthalmology, University of California, San Francisco, San Francisco, California; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California. Electronic address: Armin.Afshar@ucsf.edu.
² Department of Ophthalmology, University of California, San Francisco, San Francisco, California; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Department of Pathology, University of California, San Francisco, San Francisco, California.
³ Department of Ophthalmology, University of California, San Francisco, San Francisco, California; Department of Pathology, University of California, San Francisco, San Francisco, California.
⁴ Cancer Genetics and Prevention Program, University of California, San Francisco, San Francisco, California.
⁵ Department of Pathology, University of California, San Francisco, San Francisco, California.
⁶ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Division of Hematology-Oncology, Department of Pediatrics, University of California, San Francisco, San Francisco, California.
⁷ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Computational Biology and Informatics, University of California, San Francisco, San Francisco, California.
⁸ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Computational Biology and Informatics, University of California, San Francisco, San Francisco, California; Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California.
⁹ Department of Pathology, University of California, San Francisco, San Francisco, California; Clinical Cancer Genomics Laboratory, University of California, San Francisco, San Francisco, California.
¹⁰ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Department of Pathology, University of California, San Francisco, San Francisco, California; Clinical Cancer Genomics Laboratory, University of California, San Francisco, San Francisco, California; Department of Dermatology, University of California, San Francisco, San Francisco, California.
¹¹ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Department of Pathology, University of California, San Francisco, San Francisco, California; Clinical Cancer Genomics Laboratory, University of California, San Francisco, San Francisco, California.
¹² Department of Ophthalmology, University of California, San Francisco, San Francisco, California; Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom.

PMID: 32139107
PMCID: PMC7246167
DOI: 10.1016/j.ophtha.2019.12.005

Next-Generation Sequencing of Retinoblastoma Identifies Pathogenic Alterations beyond RB1 Inactivation That Correlate with Aggressive Histopathologic Features

Armin R Afshar et al. Ophthalmology. 2020 Jun.

. 2020 Jun;127(6):804-813.

doi: 10.1016/j.ophtha.2019.12.005. Epub 2019 Dec 12.

Authors

Affiliations

¹ Department of Ophthalmology, University of California, San Francisco, San Francisco, California; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California. Electronic address: Armin.Afshar@ucsf.edu.
² Department of Ophthalmology, University of California, San Francisco, San Francisco, California; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Department of Pathology, University of California, San Francisco, San Francisco, California.
³ Department of Ophthalmology, University of California, San Francisco, San Francisco, California; Department of Pathology, University of California, San Francisco, San Francisco, California.
⁴ Cancer Genetics and Prevention Program, University of California, San Francisco, San Francisco, California.
⁵ Department of Pathology, University of California, San Francisco, San Francisco, California.
⁶ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Division of Hematology-Oncology, Department of Pediatrics, University of California, San Francisco, San Francisco, California.
⁷ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Computational Biology and Informatics, University of California, San Francisco, San Francisco, California.
⁸ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Computational Biology and Informatics, University of California, San Francisco, San Francisco, California; Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California.
⁹ Department of Pathology, University of California, San Francisco, San Francisco, California; Clinical Cancer Genomics Laboratory, University of California, San Francisco, San Francisco, California.
¹⁰ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Department of Pathology, University of California, San Francisco, San Francisco, California; Clinical Cancer Genomics Laboratory, University of California, San Francisco, San Francisco, California; Department of Dermatology, University of California, San Francisco, San Francisco, California.
¹¹ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California; Department of Pathology, University of California, San Francisco, San Francisco, California; Clinical Cancer Genomics Laboratory, University of California, San Francisco, San Francisco, California.
¹² Department of Ophthalmology, University of California, San Francisco, San Francisco, California; Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom.

PMID: 32139107
PMCID: PMC7246167
DOI: 10.1016/j.ophtha.2019.12.005

Abstract

Purpose: To determine the usefulness of a comprehensive, targeted-capture next-generation sequencing (NGS) assay for the clinical management of children undergoing enucleation for retinoblastoma.

Design: Cohort study.

Participants: Thirty-two children with retinoblastoma.

Methods: We performed targeted NGS using the UCSF500 Cancer Panel (University of California, San Francisco, San Francisco, CA) on formalin-fixed, paraffin-embedded tumor tissue along with constitutional DNA isolated from peripheral blood, buccal swab, or uninvolved optic nerve. Peripheral blood samples were also sent to a commercial laboratory for germline RB1 mutation testing.

Main outcome measures: Presence or absence of germline RB1 mutation or deletion, tumor genetic profile, and association of genetic alterations with clinicopathologic features.

Results: Germline mutation or deletion of the RB1 gene was identified in all children with bilateral retinoblastoma (n = 12), and these NGS results were 100% concordant with commercial germline RB1 mutation analysis. In tumor tissue tested with NGS, biallelic inactivation of RB1 was identified in 28 tumors and focal MYCN amplification was identified in 4 tumors (2 with wild-type RB1 and 2 with biallelic RB1 inactivation). Additional likely pathogenic alterations beyond RB1 were identified in 13 tumors (41%), several of which have not been reported previously in retinoblastoma. These included focal amplifications of MDM4 and RAF1, as well as damaging mutations involving BCOR, ARID1A, MGA, FAT1, and ATRX. The presence of additional likely pathogenetic mutations beyond RB1 inactivation was associated with aggressive histopathologic features, including higher histologic grade and anaplasia, and also with both unilateral and sporadic disease.

Conclusions: Comprehensive NGS analysis reliably detects relevant mutations, amplifications, and chromosomal copy number changes in retinoblastoma. The presence of genetic alterations beyond RB1 inactivation correlates with aggressive histopathologic features.

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Figures

**Figure 1:**
Oncoprint summary table of the clinical, pathologic, and genetic features of the 32 retinoblastoma patients.

**Figure 2:**
Genome-wide copy number profiles for two representative retinoblastoma cases. Shown is inferred copy number calls (log2 scale) along each of the chromosomes. ***Top***: Retinoblastoma case with focal homozygous/biallelic deletion on chromosome 13q14 that encompasses the 5’ portion of the *RB1* tumor suppressor gene **(blue asterisk)**. Additional chromosomal copy number variations include gains of proximal 1q, 5p, distal 6p, 7, 9q, portions of 17q, and distal Xp, as well as losses of distal 2p, distal 5q, distal 10q, distal 11q, 16q, and interstitial 17q. ***Bottom***: Retinoblastoma case with focal high-level amplification of the *MYCN* oncogene on chromosome 2p24 **(red asterisk)**. Additional chromosomal copy number variations include gains of 17q and 18q, as well as losses of distal 1p, 2, 3, interstitial 4q, 8p, distal 8q, 9p, 10, distal 11q, 14q, 15q, 16, 17p, and 19q.

See this image and copyright information in PMC

References

1. Kooi IE, Mol BM, Massink MP, et al. Somatic genomic alterations in retinoblastoma beyond RB1 are rare and limited to copy number changes. Sci Rep. 2016;6:25264. - PMC - PubMed
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Next-Generation Sequencing of Retinoblastoma Identifies Pathogenic Alterations beyond RB1 Inactivation That Correlate with Aggressive Histopathologic Features

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Next-Generation Sequencing of Retinoblastoma Identifies Pathogenic Alterations beyond RB1 Inactivation That Correlate with Aggressive Histopathologic Features

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