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Simple Summary: Relapse outside the eye of retinoblastoma (the most common eye cancer in children) is an uncommon event in developed countries, however it is the main cause of death in patients with retinoblastoma worldwide. The genomic features of this population are not known. We studied 23 cases from four countries and found a characteristic pattern in chromosomal copy number alterations that could help guide future clinical management of these patients. Most reports about copy number alterations (CNA) in retinoblastoma relate to patients with intraocular disease and features of children with extraocular relapse remain unknown, so we aimed to describe the CNA in this population. We evaluated 23 patients and 27 specimens from 4 centers. Seventeen cases had extraocular relapse after initial enucleation and six cases after an initial preservation attempt. We performed an analysis of CNA and BCOR gene alteration by SNP array (Single Nucleotide Polymorfism array), whole-exome sequencing, IMPACT panel and CGH array (Array-based comparative genomic hybridization). All cases presented CNA at a higher prevalence than those reported in previously published studies for intraocular cases. CNA previously reported for intraocular retinoblastoma were found at a high frequency in our cohort: gains in 1q (69.5%), 2p (60.9%) and 6p (86.9%), and 16q loss (78.2%). Other, previously less-recognized, CNA were found including loss of 11q (34.8%), gain of 17q (56.5%), loss of 19q (30.4%) and BCOR alterations were present in 72.7% of our cases. A high number of CNA including 11q deletions, 17q gains, 19q loss, and BCOR alterations, are more common in extraocular retinoblastoma. Identification of these features may be correlated with a more aggressive tumor warranting consideration for patient management. [ABSTRACT FROM AUTHOR]

Simple Summary: We present two cases of a subtype of retinoblastoma, a rare ocular tumor in children, presenting without the typical mutation in the RB1 gene but showing amplification of the transcription factor MYCN frequently reported in pediatric malignancies. Even though previous reports suggested that this tumor sybtype could present metastases, patients with metastatic disease were not reported. Our cases had metastasis to the orbit and lymph nodes and poor sensitivity to standard chemotherapy but no dissemination to the central nervous system, as described for patients with mutations in the RB1. We were able to grow the cells of one of our patients in vitro, perform comprehensive genomic analysis that showed previously not reported mutations and other chromosomal alterations. In an animal model, we could reproduce the clinical dissemination and we identified an innovative active drug combination that could help for the treatment of these children with poor prognosis. An uncommon subgroup of unilateral retinoblastomas with highly aggressive histological features, lacking aberrations in RB1 gene with high-level amplification of MYCN (MCYN a m p l RB1 + / + ) has only been described as intra-ocular cases treated with initial enucleation. Here, we present a comprehensive clinical, genomic, and pharmacological analysis of two cases of MCYN a m p l RB1 + / + with orbital and cervical lymph node involvement, but no central nervous system spread, rapidly progressing to fatal disease due to chemoresistance. Both patients showed in common MYCN high amplification and chromosome 16q and 17p loss. A somatic mutation in TP53, in homozygosis by LOH, and high chromosomal instability leading to aneuploidy was identified in the primary ocular tumor and sites of dissemination of one patient. High-throughput pharmacological screening was performed in a primary cell line derived from the lymph node dissemination of one case. This cell line showed resistance to broad spectrum chemotherapy consistent with the patient's poor response but sensitivity to the synergistic effects of panobinostat–bortezomib and carboplatin–panobinostat associations. From these cells we established a cell line derived xenograft model that closely recapitulated the tumor dissemination pattern of the patient and served to evaluate whether triple chemotherapy significantly prolonged survival of the animals. We report novel genomic alterations in two cases of metastatic MCYN a m p l RB1 + / + that may be associated with chemotherapy resistance and in vitro/in vivo models that serve as basis for tailoring therapy in these cases. [ABSTRACT FROM AUTHOR]

A preclinical model could aid in understanding retinoblastoma vitreous seeds behavior, drug penetration, and response to chemotherapy to optimize patient treatment. Our aim was to develop a tridimensional in vitro model of retinoblastoma vitreous seeds to assess chemotherapy penetration by means of live-cell imaging. Cell cultures from patients with retinoblastoma who underwent upfront enucleation were established and thoroughly characterized for authentication of human tumor origin. The correlation of the in vitro tridimensional structures resembling human spheres and dusts vitreous seeds was established. Confocal microscopy was used to quantify real-time fluorescence of topotecan as a measure of its penetration into different sizes of spheres. Cell viability was determined after chemotherapy penetration. The in vitro spheres and dusts models were able to recapitulate the morphology, phenotype, and genotype of patient vitreous seeds. The larger the size of the spheres, the longer the time required for the drug to fully penetrate into the core (p < 0.05). Importantly, topotecan penetration correlated with its cytotoxic activity. Therefore, the studied tridimensional cell model recapitulated several characteristics of vitreous seeds observed in patients with retinoblastoma and were successfully used to assess live-cell imaging of chemotherapy penetration for drug distribution studies. [ABSTRACT FROM AUTHOR]