Primary Retinoblastoma Research Articles

MDM2 promotes cancer cell survival through regulating the expression of HIF-1α and pVHL in retinoblastoma.

Hypoxia is an important tumor feature and hypoxia-inducible factor 1 (HIF-1) is a master regulator of cell response to hypoxia. Mouse double minute 2 homolog (MDM2) promotes cancer cell survival in retinoblastoma (RB), with the underlying mechanism remaining elusive. In this study, we investigated the role of MDM2 and its relation to HIF-1α in RB. Expression analysis on primary human RB samples showed that MDM2 expression was positively correlated with that of HIF-1α while negatively correlated with von Hippel-Lindau protein (pVHL), the regulator of HIF-1α. In agreement, RB cells with MDM2 overexpression showed increased expression of HIF-1α and decreased expression of pVHL, while cells with MDM2 siRNA knockdown or MDM2-specific inhibitor showed the opposite effect under hypoxia. Further immuno-precipitation analysis revealed that MDM2 could directly interact with pVHL and promotes its ubiquitination and degradation, which consequently led to the increase of HIF-1α. Inhibition of MDM2 and/or HIF-1α with specific inhibitors induced RB cell death and decreased the stem cell properties of primary RB cells. Taken together, our study has shown that MDM2 promotes RB survival through regulating the expression of pVHL and HIF-1α, and targeting MDM2 and/or HIF-1α represents a potential effective approach for RB treatment.

Addition of intravitreal carboplatin with melphalan for management of vitreous seeding in retinoblastoma.

To evaluate the efficacy and toxicity of intravitreal carboplatin plus melphalan for the treatment of vitreous seeds in eyes with retinoblastoma (RB). This retrospective series at a tertiary referral center included 22 consecutive RB patients who had received intravitreal carboplatin (16μg per 0.05ml) combined with melphalan (30μg in 0.03ml) [IVi (Ca-Me)] for treatment of vitreous seeds. Tumor control and drug toxicities were recorded. There were 22 eyes of 22 patients, divided into primary group (n = 13) without history of previous intravitreal chemotherapy (IViC) and refractory group (n = 9) with history of previous IViC using melphalan and/or topotecan. The demographics and clinical findings of the primary and refractory groups did not differ significantly. The 6-month follow-up revealed complete vitreous seed control (77% vs. 89%, p = 0.47). Vitreous seed recurrence was detected in 1 eye of each group at 6months. During the next 18-month follow-up period, no recurrence of seed was observed. The response to IVi (Ca-Me) was not significantly influenced by previous IViC (p = 0.70), primary systemic or intra-arterial chemotherapy (p = 0.45), or the type of regression (p = 0.35). The most common tumor treatment complications were retinal detachment (RD) (n = 2), early hypotony (n = 2) and late hypotony (n = 4, unrelated), cataract (n = 2), and severe pigment dispersion (n = 1). Enucleation was performed in 8 eyes, for total RD (n = 1), phthisis bulbi (n = 5), and extensive solid tumor recurrence (n = 2). There was no case of orbital invasion, systemic metastasis, or death. Based on this interventional case series for primary and refractory vitreous RB seeds, carboplatin plus melphalan therapy may be effective with few toxic side effects.

Primary Macular Retinoblastoma: Clinical Presentation and Treatment Outcomes.

Purpose: To describe the clinical features and treatment outcomes in eyes with primary macular retinoblastoma (RB). Methods: Patients with primary macular RB were analyzed. Results: Of 41 patients (47 eyes), 20 (49%) were boys and 21 (51%) were girls. The mean age at diagnosis was 16 months (range, 1-60 months). The RB was bilateral in 6 patients (15%). At presentation, the macula was completely covered with the tumor in 22 eyes (47%) and partially covered with the fovea spared in 13 eyes (28%) and the fovea involved 12 eyes (25%). Based on the International Classification of Intraocular Retinoblastoma, 25 (53%), 15 (32%), and 7 (15%) of tumors were in Group B, Group C, and Group D, respectively. The tumor had exophytic features in 36 eyes (77%). The mean tumor basal diameter was 10.0 mm and the mean thickness, 5.6 mm. Associated features included subretinal seeds (10 eyes; 21%) and surrounding subretinal fluid (16 eyes; 34%). Forty-three eyes (92%) were treated with intravenous chemotherapy, 2 (4%) with intra-arterial chemotherapy, and 2 (4%) with transpupillary thermotherapy. Local tumor control was achieved in 45 eyes (96%), with 33 eyes (70%) showing a type III regression pattern. Over a mean follow-up of 23 months (range, 3-48 months), the macular tumor recurred in 5 eyes (11%), the globe was salvaged in all eyes with associated foveal atrophy (36 eyes; 77%), and 1 patient (2%) died. Conclusions: Macular RB has a good prognosis for globe salvage while vision salvage might be compromised as a result of associated foveal atrophy.

Genetic testing in adult survivors of retinoblastoma in Denmark: A study of the experience and impact of genetic testing many years after initial diagnosis

BackgroundSurvivors with heritable retinoblastoma (RB) face a high risk for second primary cancer and RB in their children. Knowledge of heredity can support second cancer surveillance, convey reproductive options or early diagnosis of RB in their offspring. Currently, all newly diagnosed Danish patients with RB are offered genetic testing, as opposed to a minority of survivors diagnosed before available DNA testing. ObjectiveTo examine RB survivors' response to unsolicited contact, uptake of genetic testing, and RB1 variant detection rate, and to qualitatively evaluate the experience and overall impact of genetic testing for heritable RB. MethodsGenetically untested adult RB survivors were invited to receive genetic counseling, undergo genetic testing for heritable RB and complete an eye examination. The number of responses, uptake of genetic testing and genetic results are descriptively reported.Additionally, responding survivors participated in a qualitative interview study of the perceived impact of genetic testing. Interviews were audio-recorded, transcribed verbatim and thematically analyzed. ResultsAmong invited RB survivors, 58% responded. Of these, 88% opted for genetic counseling and genetic testing. A diagnosis of heritable RB was established in 23% of RB survivors. Interestingly, all of these survivors were unilaterally affected. Analysis of data from the interviews revealed three recurring themes regarding the impact of genetic counseling and testing several years after initial diagnosis: ‘Risk of what?‘, ‘Knowledge is important’ and ‘Impact of the result’. The possible risk ofsecond cancer and RB in their children was new knowledge for several participants; however, in general, the participants appreciated receiving genetic information and certainty about heredity. Accordingly, the impact of genetic counseling and testing was perceived in a positive way. ConclusionOverall, RB survivors valued the opportunity to receive genetic counseling and undergo genetic testing many years after diagnosis. Responding RB survivors appreciated the invitation to test, felt well-informed and described little decisional conflict regarding their decision-making, valuing the genetic information and certainty. Heritable RB was confirmed in 23% of the previously untested RB survivors. These individuals emphasized the value of knowing and being proactive regarding both reproduction and cancer risk.

Object Detection and Statistical Analysis of Microscopy Image Sequences

Confocal microscope images are wide useful in medical diagnosis and research. The automatic interpretation of this type of images is very important but it is a challenging endeavor in image processing area, since these images are heavily contaminated with noise, have low contrast and low resolution. This work deals with the problem of analyzing the penetration velocity of a chemotherapy drug in an ocular tumor called retinoblastoma. The primary retinoblastoma cells cultures are exposed to topotecan drug and the penetration evolution is documented by producing sequences of microscopy images. It is possible to quantify the penetration rate of topotecan drug because it produces fluorescence emission by laser excitation which is captured by the camera.In order to estimate the topotecan penetration time in the whole retinoblastoma cell culture, a procedure based on an active contour detection algorithm, a neural network classifier and a statistical model and its validation, is proposed.This new inference model allows to estimate the penetration time. Results show that the penetration mean time strongly depends on tumorsphere size and on chemotherapeutic treatment that the patient has previously received.

RB1-Negative Retinal Organoids Display Proliferation of Cone Photoreceptors and Loss of Retinal Differentiation.

Simple SummaryRetinoblastoma is a tumor of the eye’s retina, which is the very specialized tissue responsible for vision. In 98% of cases, the tumor is caused by inactivation of the RB1 gene. Due to lack of material and models, the understanding of RB1 mutations in tumor development is still unsatisfactory. We aimed to establish a human laboratory model for retinoblastoma. While differentiating stem cells with a mutation in RB1 into retina, we observed reduced differentiation potential but enhanced proliferation—general hallmarks of tumor development. The gene expression signature in the model resembled that of tumor material. This approach now enables research on retinoblastoma and probably therapy in the correct tissue, the human retina.Retinoblastoma is a tumor of the eye in children under the age of five caused by biallelic inactivation of the RB1 tumor suppressor gene in maturing retinal cells. Cancer models are essential for understanding tumor development and in preclinical research. Because of the complex organization of the human retina, such models were challenging to develop for retinoblastoma. Here, we present an organoid model based on differentiation of human embryonic stem cells into neural retina after inactivation of RB1 by CRISPR/Cas9 mutagenesis. Wildtype and RB1 heterozygous mutant retinal organoids were indistinguishable with respect to morphology, temporal development of retinal cell types and global mRNA expression. However, loss of pRB resulted in spatially disorganized organoids and aberrant differentiation, indicated by disintegration of organoids beyond day 130 of differentiation and depletion of most retinal cell types. Only cone photoreceptors were abundant and continued to proliferate, supporting these as candidate cells-of-origin for retinoblastoma. Transcriptome analysis of RB1 knockout organoids and primary retinoblastoma revealed gain of a retinoblastoma expression signature in the organoids, characterized by upregulation of RBL1 (p107), MDM2, DEK, SYK and HELLS. In addition, genes related to immune response and extracellular matrix were specifically upregulated in RB1-negative organoids. In vitro retinal organoids therefore display some features associated with retinoblastoma and, so far, represent the only valid human cancer model for the development of this disease.

Poly ADP Ribose Polymerase Inhibitor Olaparib Targeting Microhomology End Joining in Retinoblastoma Protein Defective Cancer: Analysis of the Retinoblastoma Cell-Killing Effects by Olaparib after Inducing Double-Strand Breaks.

Retinoblastoma is the most common intraocular cancer in childhood. Loss of function in both copies of the RB1 gene is the causal mutation of retinoblastoma. Current treatment for retinoblastoma includes the use of chemotherapeutic agents, such as the DNA damaging agent etoposide, which is a topoisomerase II poison that mainly generates DNA double-strand breaks (DSBs) and genome instability. Unfaithful repairing of DSBs could lead to secondary cancers and serious side effects. Previously, we found that RB knocked-down mammalian cells depend on a highly mutagenic pathway, the micro-homology mediated end joining (MMEJ) pathway, to repair DSBs. Poly ADP ribose polymerase 1 (PARP1) is a major protein in promoting the MMEJ pathway. In this study, we explored the effects of olaparib, a PARP inhibitor, in killing retinoblastoma cells. Retinoblastoma cell line Y79 and primary retinoblastoma cells expressed the cone-rod homeobox protein (CRX), a photoreceptor-specific marker. No detectable RB expression was found in these cells. The co-treatment of olaparib and etoposide led to enhanced cell death in both the Y79 cells and the primary retinoblastoma cells. Our results demonstrated the killing effects in retinoblastoma cells by PARP inhibitor olaparib after inducing DNA double-strand breaks. The use of olaparib in combination with etoposide could improve the cell-killing effects. Thus, lower dosages of etoposide can be used to treat retinoblastoma, which would potentially lead to a lower level of DSBs and a relatively more stable genome.

Experience in the treatment of primary malignant tumors (retinoblastoma and osteosarcoma): analysis of the clinical case

Background. Retinoblastoma is a malignant intraocular tumor developing from the retinal neuroectoderm and diagnosed primarily in young children. This type of cancer is associated with a high risk of multiple primary tumors emerging after treatment completion. Multiple primary tumors are two or more independent tumors developing in one patient. Treatment of this disease is challenging.Objective – to evaluate the impact of risk factors on the efficacy of therapy for multiple primary tumors and to analyze treatment outcomes.Materials and methods. A 2-year-old boy was diagnosed with bilateral retinoblastoma (OD – stage T3bN0M0 and OS – stage T3cN0M0). He received special treatment from September 2005 to November 2006. In 2012, the patient underwent cataract surgery: the lens was removed, then an intraocular lens was installed, and laser dissection of the posterior capsule of the lens was performed. Six years later, in August 2018, the patient was diagnosed with osteosarcoma. The boy received combination organ-sparing therapy according to the EURAMOS-1 treatment protocol for osteosarcoma. During therapy, he developed a secondary tumor, namely osteoblastic osteosarcoma. Both the boy and his father were found to have a mutation in the RB1 gene.Results. Currently, patient’s condition is satisfactory; he has no complains. The boy is in remission for 2 years.Conclusion. The development of secondary tumors depends on the genetic factors, type of treatment for primary tumor, and environmental factors. Therefore, it is extremely important to assess risk factors for multiple primary tumors at the moment of primary retinoblastoma detection. The results of such assessment will help to choose an optimal treatment strategy.

Abstract 3027: Role of BCOR in retinoblastoma

Abstract Retinoblastoma, which is usually initiated by the biallelic mutational inactivation of RB1, is the most common primary eye cancer in children and is fatal if left untreated. While retinoblastoma is initially sensitive to chemotherapy, it demonstrates a strong propensity for therapeutic escape, which is a leading cause of ocular morbidity, eye loss, and reduced survival. This problem is exacerbated by a poor understanding of how retinoblastoma progresses after the initial loss of RB1. Genomic analysis of primary retinoblastoma tumors has revealed the corepressor BCOR as the second most common target of mutations in retinoblastoma. Here, we explored the role of BCOR in normal retinal development to provide insights into its role in retinoblastoma. Mass spectrometry following immunoprecipitation of wildtype BCOR in primary retinoblastoma cells revealed novel BCOR interacting proteins in the Polycomb and MiDAC epigenetic complexes. Subsequent studies revealed potential functional consequences of BCOR mutation that may promote retinoblastoma progression. This work reveals new insights the role of epigenetic deregulation in retinoblastoma progression, and they nominate new candidates for therapeutic intervention. Citation Format: Michelle Garying Zhang, Dawn Owens, Stefan Kurtenbach, Jeffim Kuznetsov, Matthew Fields, Daniel Pelaez, James W. Harbour. Role of BCOR in retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3027.

Characteristics and Survival Outcomes of Second Primary Cancers in Long-term Retinoblastoma Survivors.

Retinoblastoma (RB) is the most common intraocular cancer and is associated with lifelong risks of developing a second primary cancer, especially in patients with hereditary RB and/or childhood exposure to radiotherapy. The study included all consecutive patients with a history of RB treated for a second primary cancer during 1994-2018. Patient demographics and characteristics of the primary RB and second primary cancer were examined. The associations among radiation or chemotherapy exposure as a treatment for RB, unilateral versus bilateral status, types and multiplicity of second primary cancers, and survival after diagnosis of second primary cancer were analyzed. A wide spectrum of second primary cancer types was identified from 62 eligible patients (30 males and 32 females), including sarcoma, breast cancer, various skin cancers, gastrointestinal and genitourinary cancers, and endocrine cancers. Of all patients who had second primary cancers, 40 patients (65%) had bilateral RB and 17 patients (27%) had unilateral RB. Thirty-five patients (56%) who developed second primary cancers received radiation therapy during childhood as the treatment of RB, and 17 patients (27%) received chemotherapy for the treatment of RB. The 5-year and 10-year survival rates for RB patients diagnosed with a second primary cancer were 54.0% and 36.0%, respectively. The median age of onset of second primary cancer among RB survivors was 36.6 years. In contrast to previous studies, we found a broader spectrum of second primary cancer types. All RB survivors, regardless of unilateral or bilateral status, should undergo strict cancer surveillance particularly as they approach the fourth decade of life.

Aurora Kinase B Expression, Its Regulation and Therapeutic Targeting in Human Retinoblastoma.

PurposeAurora kinase B (AURKB) plays a pivotal role in the regulation of mitosis and is gaining prominence as a therapeutic target in cancers; however, the role of AURKB in retinoblastoma (RB) has not been studied. The purpose of this study was to determine if AURKB plays a role in RB, how its expression is regulated, and whether it could be specifically targeted.MethodsThe protein expression of AURKB was determined using immunohistochemistry in human RB patient specimens and immunoblotting in cell lines. Pharmacological inhibition and shRNA-mediated knockdown were used to understand the role of AURKB in cell viability, apoptosis, and cell cycle distribution. Cell viability in response to AURKB inhibition was also assessed in enucleated RB specimens. Immunoblotting was employed to determine the protein levels of phospho-histone H3, p53, p21, and MYCN. Chromatin immunoprecipitation–qPCR was performed to verify the binding of MYCN on the promoter region of AURKB.ResultsThe expression of AURKB was found to be markedly elevated in human RB tissues, and the overexpression significantly correlated with optic nerve and anterior chamber invasion. Targeting AURKB with small-molecule inhibitors and shRNAs resulted in reduced cell survival and increased apoptosis and cell cycle arrest at the G2/M phase. More importantly, primary RB specimens showed decreased cell viability in response to pharmacological AURKB inhibition. Additional studies have demonstrated that the MYCN oncogene regulates the expression of AURKB in RB.ConclusionsAURKB is overexpressed in RB, and targeting it could serve as a novel therapeutic strategy to restrict tumor cell growth.

Retinoblastoma Is Characterized by a Cold, CD8+ Cell Poor, PD-L1- Microenvironment, Which Turns Into Hot, CD8+ Cell Rich, PD-L1+ After Chemotherapy.

PurposeTo investigate the impact of chemotherapy (CHT) on human retinoblastoma (RB) tumor microenvironment (TME).Cases and MethodsNinety-four RBs were studied, including 44 primary RBs treated by upfront surgery (Group 1) and 50 primary RBs enucleated after CHT (CHT), either intra-arterial (IAC; Group 2, 33 cases) or systemic (S-CHT; Group 3, 17 cases). Conventional and multiplexed immunohistochemistry were performed to make quantitative comparisons among the three groups, for the following parameters: tumor-infiltrating inflammatory cells (TI-ICs); programmed cell death protein 1 (PD-1) positive TI-ICs; Ki67 proliferation index; gliosis; PD-1 ligand (PD-L1) protein expression; vessel number. We also correlated these TME factors with the presence of histological high-risk factors (HHRF+) and RB anaplasia grade (AG).ResultsAfter CHT, a decrease in both RB burden and Ki67 positivity was observed. In parallel, most subsets of TI-ICs, PD-1+ TI-ICs, gliosis, and PD-L1 protein expression significantly increased (P < 0.001, P = 0.02, P < 0.001, respectively). Vessel number did not significantly vary. Age, HHRFs+ and AG were significantly different between primary and chemoreduced RBs (P < 0.001, P = 0.006, P = 0.001, respectively) and were correlated with most TME factors.ConclusionsCHT modulates host antitumor immunity by reorienting the RB TME from anergic into an active, CD8+, PD-L1+ hot state. Furthermore, some clinicopathological characteristics of RB correlate with several factors of TME. Our study adds data in favor of the possibility of a new therapeutic scenario in human RB.

Allostery governs Cdk2 activation and differential recognition of CDK inhibitors.

Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding, and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit, and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates, and comparative specialization toward its primary substrate Retinoblastoma (RB).

International travel to obtain medical treatment for primary retinoblastoma: A global cohort study.

Early diagnosis and treatment of retinoblastoma (Rb), the most common intraocular malignancy, can save both the child's life and vision. However, access to services and hence chances for survival and preserving the eye and its vision vary widely across the globe. Some families have to, or make a choice to, leave their home country to seek planned medical treatment abroad. We aimed to investigate how frequently this cross-border travel occurs and the factors associated with it. A total of 278 Rb centres in 153 countries were recruited to participate in a global cross-sectional analysis of newly diagnosed Rb patients in 2017. Number and proportions of children who travelled from their home country for treatment were analysed by country, continent, socioeconomic stratum and clinical and demographic features. The cohort included 4351 new patients of whom 223 [5.1%, 95% confidence interval 4.5-5.8] were taken across country borders for planned medical treatment. Independently significant predictors of travelling across borders included: being from a country with a smaller population, being from a country classified as low socioeconomic status, having bilateral Rb and having intraocular disease without extraocular spread. The factors that determine international travel for Rb treatment are complex and deserve further investigation. We may need to rethink the way services are delivered in the light of the threat of severe curtailment of international travel from pandemics like corona virus disease 2019.

Survivin silencing improved the cytotoxicity of carboplatin and melphalan in Y79 and primary retinoblastoma cells.

Survivin stands out as one of the most specific cancer targets discovered to date. Although single inhibition, e.g. through small interfering RNA (siRNA), has shown modest results in clinical trials, its combination with drugs holds promise to sensitize cancer cells to chemotherapeutics. In this study, we propose a sequential treatment of siRNA survivin followed by chemotherapy. Firstly, we demonstrated that siRNA-loaded switchable lipid nanoparticles (siLNP) silence survivin in a panel of cancer cell lines. Subsequently, we selected retinoblastoma (RB) as our model to screen four chemotherapeutic agents: carboplatin, topotecan, melphalan or teniposide. The effect of drugs on survivin expression and caspase-3 was investigated by RT-qPCR. The best drug combination was selected measuring the viability, survivin expression and the selectivity of the treatment. Our stepwise method revealed that siRNA delivery by switchable LNP sensitized Y79, but not the healthy APRE-19 cell line, to carboplatin and melphalan cytotoxicity. This ability was validated on primary human RB cells. Finally, the distinct behavior of the drugs demonstrated that a diligent screening of drugs should be envisioned when looking for synergy with survivin. Our sequential approach highlighted carboplatin and melphalan as agents to be investigated in future survivin-associated in vivo testing to tackle RB.

Serum exosomal miRNA as biomarkers for Retinoblastoma

Retinoblastoma (RB) is a childhood eye tumor, caused by RB1 mutation. Though diagnosing RB is easier, prognosticating RB is limited to examining the patient under anesthesia and imaging technique. The aim of the study is to find exosomal miRNA biomarkers to prognosticate RB. Exosomes were isolated from one control – MIO-M1 and two RB cell lines – WERI-Rb-1 and NCC-RbC-51. Small RNA sequencing was performed on exosomal miRNA isolated from the three cell lines. miRNAs specific to each cell line were shortlisted. A total of 243, 606 and 400 miRNAs were identified in MIO-M1, WERI-Rb-1 and NCC-RbC-51 cell lines respectively. Nine miRNAs were shortlisted based on adjusted p value and literature, MIO-M1 specific (n = 1), WERI-RB-1 specific (n = 2), NCC-RbC-51 specific (n = 2) and miRNAs common to both RB cell lines (n = 4) were chosen. Validation was done using specific Taqman miRNA assays.miRNA validation was carried out on cell lines, cell line derived exosomes, primary RB tissues and exosomes isolated from serum of the RB patients. Validation of the miRNAs in cell lines and exosomes derived from the cell lines, confirmed the sequencing data. However, only 2 miRNAs – hsa-miR-301b-3p and hsa-miR-216b-5p were upregulated in the primary RB tissues. None of the miRNAs had significant expression in the serum exosomes of RB patients. Therefore, serum exosomal miRNA may not be ideal for prognosticating RB.Further research on other body fluids like CSF and vitreous could serve as potential source for biomarkers for prognosticating RB.

Combined association of massive choroidal and optic nerve invasion as a prognostic relevance in primary retinoblastoma: A 10-year study.

To determine the significance of both massive choroidal invasion and optic nerve invasion (retrolaminar [(RL]+cut end [CE]) as a criterion for classifying high metastatic potential retinoblastoma and their relationship with other known histopathological high-risk features. A retrospective review of 650 eyes diagnosed as retinoblastoma over a 10-year period. In our study, there is male predominance and a higher percentage of the poorly differentiated tumors. The age of most of the patients ranges from 1 month to 8years with a median age of 2years. There were 24% of eyes with massive choroidal invasion and 18% of eyes with optic nerve invasion up to the cut end. On performing Cox-proportional hazard analysis, it was found that massive choroidal invasion in association with optic nerve invasion up to the cut end was an independent prognostic parameter. On Kaplan-Meier analysis, overall survival had reduced in patients having both massive choroidal invasion and an optic nerve cut end invasion along with orbital invasion (P<.05). The presence of massive choroidal invasion in association with optic nerve cut end invasion (RL+CE) could be used as a better prognostic predictor in assessing retinoblastoma patients with high metastatic potential and need to be kept for longer follow up.

Development of In Vivo Model of Intraocular Retinoblastoma.

Here we describe creation of a model of intraocular retinoblastoma on 4 mature immunodeficient BALB/c nude mice of both sexes. A suspension of the primary retinoblastoma culture was injected into each of the 8 eyes of the test animals. The injections were performed under the control of an operating microscope using insulin syringes with a diameter of 29G transsclerally into the subretinal space in a volume of 0.3 ml at a rate of 10,000 cells in 25 μl physiological saline. The culture of Rb10 cells derived from a patient with retinoblastoma passed 12 passages at the time of the experiments. After 9-week follow-up, a clinical (ophthalmoscopy) and instrumental (ultrasound) study was performed with visualization of the tumor mass on the fundus. After enucleation, a morphological study was performed. The retinoblastoma tumor nodes were identified in two eyes. The proposed model can be used for further research and for testing new chemotherapeutic drugs and treatment regimens for retinoblastoma.

Clinical relevance of B7H3 expression in retinoblastoma

Retinoblastoma (RB) is the most common paediatric intraocular tumour. Currently, chemotherapy is widely used to reduce the chance of metastasis as well as for vision salvage. The limitations of chemotherapy for RB include chemoresistance and cytotoxicity. Recently, immunotherapy is considered for treating chemoresistant cancers. Although, several molecular targets are available for immunotherapy in different cancers, we were interested in B7H3, as it was differentially expressed between retinoblastoma and retina in our earlier proteomics study. Hence, in this study we validated the previous finding by Western blotting and immunohistochemistry on primary RB tumor samples. The results suggest significantly increased expression of B7H3 in RB tumor samples compared to retina by western blotting. Immunohistochemistry revealed spatial, inter and intratumoral heterogeneity in the primary RB tumor sections. Correlation of the B7H3 expression with clinical and histopathological data revealed significantly increased expression of B7H3 in poorly differentiated, non-neural invasive tumors and lower expression in neural invasion and severe anaplastic areas of the tumors. B7H3 expression did not significantly vary between low-risk and high-risk tumors. The study also revealed considerably reduced infiltration of T lymphocytes in RB. We conclude that B7H3 is prominently expressed in primary RB tumors and could be used for targeted therapy.

Discovery of a transcriptomic core of genes shared in 8 primary retinoblastoma with a novel detection score analysis.

Expression microarrays are powerful technology that allows large-scale analysis of RNA profiles in a tissue; these platforms include underexploited detection scores outputs. We developed an algorithm using the detection score, to generate a detection profile of shared elements in retinoblastoma as well as to determine its transcriptomic size and structure. We analyzed eight briefly cultured primary retinoblastomas with the Human transcriptome array 2.0 (HTA2.0). Transcripts and genes detection scores were determined using the Detection Above Background algorithm (DABG). We used unsupervised and supervised computational tools to analyze detected and undetected elements; WebGestalt was used to explore functions encoded by genes in relevant clusters and performed experimental validation. We found a core cluster with 7,513 genes detected and shared by all samples, 4,321 genes in a cluster that was commonly absent, and 7,681 genes variably detected across the samples accounting for tumor heterogeneity. Relevant pathways identified in the core cluster relate to cell cycle, RNA transport, and DNA replication. We performed a kinome analysis of the core cluster and found 4 potential therapeutic kinase targets. Through analysis of the variably detected genes, we discovered 123 differentially expressed transcripts between bilateral and unilateral cases. This novel analytical approach allowed determining the retinoblastoma transcriptomic size, a shared active transcriptomic core among the samples, potential therapeutic target kinases shared by all samples, transcripts related to inter tumor heterogeneity, and to determine transcriptomic profiles without the need of control tissues. This approach is useful to analyze other cancer or tissue types.