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Purpose: Primary enucleation is a well-established method to achieve cure for advanced intraocular retinoblastoma. Recent treatment advances have induced a trend toward trial eye salvage using chemotherapy or other modalities. We investigated how pre-enucleation/postenucleation systemic chemotherapy and the resulting delayed enucleation affect patient survival after failed trial eye salvage., Design: Multicenter, retrospective cohort study., Participants: Children with Group D and E retinoblastoma primarily or secondarily enucleated at 29 Chinese treatment centers., Methods: Data reviewed included clinical staging, time from diagnosis to enucleation, numbers of cycles of carboplatin, etoposide/teniposide and vincristine chemotherapy, disease-specific survival (DSS), histopathology, and follow-up., Main Outcome Measures: Primary outcome was DSS. Secondary outcome was histopathology of enucleated eyes., Results: Primarily enucleated eyes had significantly shorter delay from diagnosis to enucleation than eyes treated with pre-enucleation chemotherapy (P < 0.001). Delay between diagnosis and enucleation >3.5 months (Group D) and >2 months (Group E) decreased survival (Group D: P = 0.018; Group E: P = 0.017). Compared with primarily enucleated children, children with 1 to 3 cycles of pre-enucleation chemotherapy for Group E eyes had no significant difference in survival (P = 0.74), but those who received ≥4 cycles had worse survival (P = 0.025). After pre-enucleation chemotherapy, more children with Group E (but not Group D) eyes had high-risk histopathology (pT3/pT4) (Group D: P = 0.076; Group E: P < 0.001) and worse survival than those primarily enucleated (P < 0.001). Postenucleation chemotherapy improved survival of children with high-risk histopathology (pT3/pT4) (P = 0.001) but did not change survival of children with low-risk histopathology (pT1/pT2) (P = 0.52)., Conclusions: We observed that pre-enucleation chemotherapy offered no survival benefit and timely enucleation minimized risk of metastatic death. Postenucleation chemotherapy improved survival of children with high-risk histopathology but was not useful for those with low-risk histopathology. These findings facilitate informed discussion on the risks and benefits of delayed enucleation, the use of systemic chemotherapy for trial salvage of eyes with advanced intraocular retinoblastoma, and the specific children who benefit from postenucleation chemotherapy., (Copyright © 2020 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Objective: To review the effectiveness and toxicity of periocular topotecan hydrochloride in fibrin sealant (Tisseel) for the control of intraocular retinoblastoma., Methods: Retrospective medical record review of visually threatening or recurrent intraocular retinoblastoma treated with periocular topotecan., Results: Eight children (10 eyes) received 1 to 4 injections of periocular topotecan in fibrin sealant, without or with concomitant laser and/or single freeze-thaw prechemotherapy cryotherapy. Median dose was 0.18 mg/kg (3.72 mg/m(2)). The 6 children who responded to treatment had small discrete tumors (8 International Intraocular Retinoblastoma Classification group A or B eyes). Of these, prior primary treatment for 3 children (3 eyes) was laser; for 1 child (2 eyes), systemic chemotherapy with focal laser; and for 2 children (3 eyes), periocular topotecan. In 4 children (4 eyes), tumor regression was sufficient for effective focal therapy, but in 2 children (4 eyes), long-term control required systemic chemotherapy. The 2 children who did not respond each had an International Intraocular Retinoblastoma Classification group D eye treated primarily with systemic chemotherapy, focal laser, and cryotherapy and recurrent disease that was not controlled by periocular topotecan; both eyes were eventually enucleated. No ocular and minimal hematological toxic effects were observed. At 11 months' median follow-up after topotecan treatment (18 months since diagnosis), all 8 group A and B eyes were retained with ongoing focal therapy required in only 1 group B eye; the 2 group D eyes were enucleated., Conclusion: Periocular topotecan in fibrin sealant can achieve volume reduction of small and recurrent retinoblastoma sufficient to allow successful focal therapy.

Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.

Purpose: To develop and use improved Semliki Forest vectors (SFVs) for functional and structural analyses of the retinoblastoma protein (RB) in developing retina and retinoblastoma cells., Methods: Virus was harvested from cells transfected with replicon and helper plasmids. Combinations of producer and target cells were tested for optimal virus production and protein expression. The replicon was improved by adding an expanded multiple cloning site, translational enhancer, and FLAG and HIS10 epitope and affinity tags. Affinity chromatography was used to purify beta-galactosidase or RB. RB function was assessed through interaction with E2F1. The efficacy of SFV as a retinal delivery system was tested on mouse explants and cultured human retinoblastoma cells., Results: The optimal producer and target cell lines were an HEK-293 derivative (Phoenix Eco) and BHK-21, respectively. Stable expression of structural proteins in the BHK-21 helper line simplified virus production and amplified virus yield 100-fold. The translational enhancer improved expression three- to eightfold. Full-length, functional RB was produced without the truncated products characteristic of bacterially produced RB and was purified using the affinity tags. SFVs efficiently transduced mouse retinal explants and multiple hard-to-transduce retinoblastoma tumor lines., Conclusions: This study describes a simple, rapid, SFV vector system to produce recombinant proteins, such as RB, in mammalian cells. These SFV vectors represent an efficient approach to purification of proteins and protein complexes and transduction of retinal or retinoblastoma cells for the purpose of in vivo analysis of protein functions., (Copyright Association for Research in Vision and Ophthalmology)

Murine models can provide valuable insight into mechanisms of tumorigenesis. Tumor size is often used to assess the impact of genetic insult or therapeutic treatment, usually using in vivo imaging of advanced tumors. We now describe a highly sensitive method to quantify tumor volume in a mouse model of retinoblastoma, from the earliest stages of tumor initiation to large, advanced tumors. This methodology combines immunohistochemistry, digital slide scanning and computer image analysis, and can be applied to quantitatively assess and characterize early tumor development in other models. [ABSTRACT FROM AUTHOR]

Trilateral retinoblastoma is characterized by retinal tumors in one or both eyes, as well as tumors of the pineal gland or parasellar region of the brain. Here we describe a 4-month-old girl, presenting with pituitary dysfunction, hypothalamic overgrowth syndrome and central blindness, in addition to suprasellar and bilateral retinal tumors. Biopsy of the suprasellar tumor confirmed the diagnosis of trilateral retinoblastoma. After biopsy, cerebrospinal fluid (CSF) metastasis was discovered. Overgrowth persisted, but blindness and pituitary dysfunction resolved when the suprasellar tumor and CSF metastasis responded to cyclosporine-modulated systemic chemotherapy with intraventricular chemotherapy, consolidated with marrow-ablative chemotherapy and stem cell rescue of the bone marrow. Twenty months after diagnosis and 12 months after transplant, an unusual pattern of tumor recurrence was observed along the catheter of the Ommaya reservoir used for delivering intraventricular chemotherapy, which was also at the site of the previous suprasellar needle biopsy. Salvage therapy consisted of resection, stereotactic radiation, and further systemic and intraventricular chemotherapy. At 25 months after diagnosis, the patient was developing well and seeing better. However, she died 32 months after diagnosis despite the salvage therapy. This case highlights the possibility of tumor dissemination after needle biopsy of a suprasellar tumor. Biopsy may be avoided if a characteristic clinicoradiological picture of trilateral retinoblastoma is recognized. We recommend that if a pineal or suprasellar tumor is observed in a child, the eyes should be examined for retinoblastoma, thereby avoiding biopsies of the intracranial tumor, which may track difficult-to-treat tumor cells through the brain, and disseminate tumor cells into the CSF. [ABSTRACT FROM AUTHOR]

Background: Most human cancers show inactivation of both pRB- and p53-pathways. While retinoblastomas are initiated by loss of the RB1 tumor suppressor gene, TP53 mutations have not been found. High expression of the p53-antagonist MDM2 in human retinoblastomas may compromise p53 tumor surveillance so that TP53 mutations are not selected for in retinoblastoma tumorigenesis. We previously showed that p14ARF protein, which activates p53 by inhibiting MDM2, is low in retinoblastomas despite high mRNA expression.Methods: In human fetal retinas, adult retinas, and retinoblastoma cells, we determined endogenous p14ARF mRNA, ARF protein, and miR-24 expression, while integrity of p53 signalling in WERI-Rb1 cells was tested using an adenovirus vector expressing p14ARF. To study p14ARF biogenesis, retinoblastoma cells were treated with the proteasome inhibitor, MG132, and siRNA against miR-24.Results: In human retinoblastoma cell lines, p14ARF mRNA was disproportionally high relative to the level of p14ARF protein expression, suggesting a perturbation of p14ARF regulation. When p14ARF was over-expressed by an adenovirus vector, expression of p53 and downstream targets increased and cell growth was inhibited indicating an intact p14ARF-p53 axis. To investigate the discrepancy between p14ARF mRNA and protein in retinoblastoma, we examined p14ARF biogenesis. The proteasome inhibitor, MG132, did not cause p14ARF accumulation, although p14ARF normally is degraded by proteasomes. miR-24, a microRNA that represses p14ARF expression, is expressed in retinoblastoma cell lines and correlates with lower protein expression when compared to other cell lines with high p14ARF mRNA. Transient over-expression of siRNA against miR-24 led to elevated p14ARF protein in retinoblastoma cells.Conclusions: In retinoblastoma cells where high levels of p14ARF mRNA are not accompanied by high p14ARF protein, we found a correlation between miR-24 expression and low p14ARF protein. p14ARF protein levels were restored without change in mRNA abundance upon miR-24 inhibition suggesting that miR-24 could functionally repress expression, effectively blocking p53 tumor surveillance. During retinal tumorigenesis, miR-24 may intrinsically compromise the p53 response to RB1 loss. [ABSTRACT FROM AUTHOR]