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

Ophthalmology. 2020 Jun;127(6):804-813. doi: 10.1016/j.ophtha.2019.12.005. Epub 2019 Dec 12.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Child
  • Child, Preschool
  • Cohort Studies
  • DNA Mutational Analysis
  • DNA, Neoplasm / genetics
  • Eye Enucleation
  • Female
  • Gene Silencing*
  • Germ-Line Mutation*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Infant
  • Male
  • Paraffin Embedding
  • Retinal Neoplasms / genetics*
  • Retinal Neoplasms / pathology*
  • Retinal Neoplasms / surgery
  • Retinoblastoma / genetics*
  • Retinoblastoma / pathology*
  • Retinoblastoma / surgery
  • Retinoblastoma Binding Proteins / genetics*
  • Tissue Fixation
  • Ubiquitin-Protein Ligases / genetics*


  • DNA, Neoplasm
  • RB1 protein, human
  • Retinoblastoma Binding Proteins
  • Ubiquitin-Protein Ligases