Focused cancer pathway analysis revealed unique therapeutic targets in retinoblastoma.

Retinoblastoma (RB) is a pediatric cancer of the eye that occurs in 1/15000 live births worldwide. Albeit RB is initiated by the inactivation of RB1 gene, the disease progression relies largely on transcriptional alterations. Therefore, evaluating gene expression is vital to unveil the therapeutic targets in RB management. In this study, we employed an RT2 Profiler™ PCR array for a focused analysis of 84 cancer-specific genes in RB. An interaction network was built with gene expression data to identify the dysregulated pathways in RB. The key transcript alterations identified in 13 tumors by RT2 Profiler™ PCR array was further validated in 15 tumors by independent RT-qPCR. Out of 84 cancer-specific genes, 68 were dysregulated in RB tumors. Among the 68 genes, 23 were chosen for further analysis based on statistical significance and abundance across multiple tumors. Pathway analysis of altered genes showed the frequent perturbations of cell cycle, angiogenesis and apoptotic pathways in RB. Notably, upregulation of MCM2, MKI67, PGF, WEE1, CDC20 and downregulation of COX5A were found in all the tumors. Western blot confirmed the dysregulation of identified targets at protein levels as well. These alterations were more prominent in invasive RB, correlating with the disease pathogenesis. Our molecular analysis thus identified the potential therapeutic targets for improving retinoblastoma treatment. We also suggest that PCR array can be used as a tool for rapid and cost-effective gene expression analysis.

Heterozygous RB1 mutation enhanced ATP production in human iPSC-derived retinal organoids.

BACKGROUND: Recent in vitro studies using RB1+/- fibroblasts and MSCs have shown molecular and functional disruptions without the need for biallelic loss of RB1. However, this was not reflected in the recent in vitro studies employing RB1+/- retinal organoids. To gain further insights into the molecular disruptions in the RB1+/- retinal organoids, we performed a high throughput RNA sequencing analysis. METHODS AND RESULTS: iPSCs were generated from RB1+/+ and RB1+/- OAMSCs derived from retinoblastoma patients. RB1+/+ and RB1+/- iPSCs were subjected to a step-wise retinal differentiation protocol. Retinal differentiation was evaluated by Real-time PCR and flow cytometry analysis of the retinal markers. To gain further insights into the molecular differences in RB1+/- retinal organoids, a high throughput RNA sequencing followed by differential gene expression analysis and gene set enrichment analysis (GSEA) was performed. The analysis revealed a shift from the regular metabolic process of glycolysis to oxidative phosphorylation in the RB1+/- retinal organoids. To investigate further, we performed assays to determine the levels of pyruvate, lactate and ATP in the retinal organoids. The results revealed significant increase in ATP and pyruvate levels in RB1+/- retinal organoids of day 120 compared to that of the RB1+/+. The results thus revealed enhanced ATP production in the RB1+/- retinal organoids. CONCLUSION: The study provides novel insights into the metabolic phenotype of heterozygous RB1 mutant suggesting dysregulation of energy metabolism and glycolytic pathways to be first step even before the changes in cellular proliferation or other phenotypic consequences ensue.

Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma.

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

Advancements in Nanosystems for Ocular Drug Delivery: A Focus on Pediatric Retinoblastoma.

The eye's complex anatomical structures present formidable barriers to effective drug delivery across a range of ocular diseases, from anterior to posterior segment pathologies. Emerging as a promising solution to these challenges, nanotechnology-based platforms-including but not limited to liposomes, dendrimers, and micelles-have shown the potential to revolutionize ophthalmic therapeutics. These nanocarriers enhance drug bioavailability, increase residence time in targeted ocular tissues, and offer precise, localized delivery, minimizing systemic side effects. Focusing on pediatric ophthalmology, particularly on retinoblastoma, this review delves into the recent advancements in functionalized nanosystems for drug delivery. Covering the literature from 2017 to 2023, it comprehensively examines these nanocarriers' potential impact on transforming the treatment landscape for retinoblastoma. The review highlights the critical role of these platforms in overcoming the unique pediatric eye barriers, thus enhancing treatment efficacy. It underscores the necessity for ongoing research to realize the full clinical potential of these innovative drug delivery systems in pediatric ophthalmology.

Retinoblastoma - A comprehensive review, update and recent advances.

Retinoblastoma is the most common pediatric ocular malignancy. It is triggered by a biallelic mutation in the RB1 gene or MYCN oncogene amplification. Retinoblastomas can be unilateral (60%-70%) or bilateral (30%-40%); bilateral tumors are always heritable and present at an earlier age as compared to unilateral ones (18-24 months vs. 36 months in India). High prevalence rates, delayed presentation, and inaccessibility to healthcare lead to worse outcomes in developing countries. The past few decades have seen a paradigm change in the treatment of retinoblastomas, shifting from enucleation and external beam radiotherapy to less aggressive modalities for eye salvage. Multimodality treatment is now the standard of care and includes intraarterial or intravenous chemotherapy along with focal consolidation therapies such as transpupillary thermotherapy, cryotherapy, and laser photocoagulation. Intravitreal and intracameral chemotherapy can help in controlling intraocular seeds. Advanced extraocular or metastatic tumors still have a poor prognosis. Genetic testing, counseling, and screening of at-risk family members must be incorporated as essential parts of management. A better understanding of the genetics and molecular basis of retinoblastoma has opened up the path for potential targeted therapy in the future. Novel recent advances such as liquid biopsy, prenatal diagnosis, prognostic biomarkers, tylectomy, and chemoplaque point to promising future directions.

Travel burdens to access care among children with cancer between 2016 and 2019: Analysis of a national population-based cancer registry in Japan.

BACKGROUND: Centralization of cancer care increases survival but increases the travel burden (i.e., travel durations, distances, and expenditures) in visiting hospitals. This study investigated the travel burdens to access cancer care for children aged 18 years and younger in Japan. METHODS: The study population comprised 10,709 patients diagnosed between 2016 and 2019 obtained from a national population-based cancer registry in Japan. Their residences were classified as urban or rural. We counted the number of patients treated at specialized hospitals and investigated the treatment centralization across diagnostic groups by Pareto plot. Travel burdens to access care were estimated using a route-planner web service and summarized using median values. A multivariable logistic model was performed to investigate factors associated with the events of car travel duration exceeding 1 h. RESULTS: Of the patients, 76.7% lived in urban areas, and 82.5% received treatment in designated hospitals for childhood cancer. The Pareto plot suggested that the top five hospitals treated 63.5% of patients with retinoblastoma. The estimated travel burdens for all patients were 0.62 h (0.57 h in urban areas and 1.00 h in rural areas), 16.9 km, and 0.0 dollars of toll charges. Regarding travel duration, 21.7% of patients had travel exceeding 1 h, and rural areas, retinoblastoma, malignant bone tumors, and childhood cancer-hub hospitals were associated with travel duration exceeding 1 h (adjusted odds ratios of 6.93, 3.59, 1.94, and 1.91, respectively). CONCLUSIONS: Most patients were treated in specialized hospitals and the treatments for specific diseases were centralized. However, most patients were estimated to travel less than 1 h, and the travel burden tended to increase for patients in rural areas, those with specific diseases, and those going to specialized hospitals. Cancer control measures in Japan have steadily improved centralized treatment while keeping the travel burden relatively manageable.

Update on chemotherapy modalities for retinoblastoma: Progress and challenges.

Retinoblastoma stands as a paradigm of success in treating malignancies among pediatric patients. Over recent decades, the approach to managing retinoblastoma has evolved significantly, transitioning from the preservation of patients' lives to the preservation of eyes and vision while minimizing treatment-related complications. Chemotherapy, administered through diverse routes, has solidified its role as the cornerstone of retinoblastoma treatment. In addition to intravenous chemotherapy (IVC), alternative administration routes, including intraarterial (IAC), intravitreal, intracameral, and periocular delivery, have emerged as promising modalities for retinoblastoma management. Numerous studies have demonstrated outstanding outcomes, achieving nearly 100% salvage rates for eyes classified under groups A-C. However, for advanced intraocular retinoblastoma (groups D and E eyes), IAC appears to offer superior local control rates compared to IVC. Intravitreal injection of chemotherapeutic agents, when administered in a controlled and secure manner, holds promise in averting the need for enucleation and radiotherapy in advanced retinoblastoma cases presenting with vitreous seeds. The optimal chemotherapy strategy remains meticulously tailored based on numerous factors. This review provides a comprehensive update on chemotherapy across various routes, encompassing key considerations, dosages, administration methods, treatment outcomes, and potential complications. Furthermore, it explores emerging potential treatments and outlines future directions aimed at enhancing treatment outcomes.

The Clinicopathological Significance of the Cyclin D1/E1-Cyclin-Dependent Kinase (CDK2/4/6)-Retinoblastoma (RB1/pRB1) Pathway in Epithelial Ovarian Cancers.

Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.

Differences in Childhood Growth Parameters Between Patients With Somatic and Heritable Retinoblastoma.

Purpose: Little is known regarding differences in childhood growth between somatic and heritable retinoblastoma (Rb) populations. We aimed to compare childhood growth parameters between somatic and heritable Rb cohorts at birth and at time of diagnosis with Rb. Methods: A multinational, longitudinal cohort study was conducted with patients from 11 centers in 10 countries who presented with treatment naïve Rb from January to December 2019. Variables of interest included age, sex, and size characteristics at birth and at time of presentation, as well as germline mutation status. After Bonferroni correction, results were statistically significant if the P value was less than 0.005. Results: We enrolled 696 patients, with 253 analyzed after exclusion criteria applied. Between somatic (n = 39) and heritable (n = 214) Rb cohorts, with males and females analyzed separately, there was no significant difference in birth weight percentile, weight percentile at time of diagnosis, length percentile at time of diagnosis, weight-for-length percentile at time of diagnosis, or change of weight percentile from birth to time of diagnosis. Patients with heritable Rb had a smaller mean weight percentile at birth and smaller mean weight and length percentiles at time of diagnosis with Rb, although this difference was not statistically significant. All cohorts experienced a slight negative change of weight percentile from birth to time of diagnosis. No cohort mean percentiles met criteria for failure to thrive, defined as less than the 5th percentile. Conclusions: Children with Rb seem to have normal birth and childhood growth patterns. There is no definitive evidence that somatic or heritable Rb has a biological or environmental impact on childhood growth parameters.

Strategies to improve diagnosis and access to treatment of retinoblastoma in low- and middle-income countries: A systematic review.

Retinoblastoma, the most common intraocular tumor in childhood, still faces challenges in diagnosis and treatment, particularly in low- and middle-income countries. Identifying strategies to improve the time to diagnosis and access to treatment is crucial to enhance survival rates and preserve ocular health. We conducted a systematic review to identify interventions that have demonstrated potential in addressing these challenges. We performed a comprehensive search across databases until March 2023. Out of the studies reviewed, 21 met the inclusion criteria and were categorized into five main areas: surveillance strategies, genetic counseling, education, public assistance, and international partnership. Despite the obstacles faced, the initiatives identified in this review present acts toward improving the time to diagnosis and access to treatment for retinoblastoma. Based on the extracted data, we propose a comprehensive chain of initiatives. We firmly believe that implementing this chain of initiatives can lead to improved clinical outcomes for retinoblastoma patients.

Recent progress in retinoblastoma: Pathogenesis, presentation, diagnosis and management.

Retinoblastoma, the primary ocular malignancy in pediatric patients, poses a substantial threat to mortality without prompt and effective management. The prognosis for survival and preservation of visual acuity hinges upon the disease severity at the time of initial diagnosis. Notably, retinoblastoma has played a crucial role in unraveling the genetic foundations of oncogenesis. The process of tumorigenesis commonly begins with the occurrence of biallelic mutation in the RB1 tumor suppressor gene, which is then followed by a cascade of genetic and epigenetic alterations that correspond to the clinical stage and pathological features of the tumor. The RB1 gene, recognized as a tumor suppressor, encodes the retinoblastoma protein, which plays a vital role in governing cellular replication through interactions with E2F transcription factors and chromatin remodeling proteins. The diagnosis and treatment of retinoblastoma necessitate consideration of numerous factors, including disease staging, germline mutation status, family psychosocial factors, and the resources available within the institution. This review has systematically compiled and categorized the latest developments in the diagnosis and treatment of retinoblastoma which enhanced the quality of care for this pediatric malignancy.

Role of Protein Tyrosine Phosphatase Receptor Type E (PTPRE) in Chemoresistant Retinoblastoma.

Protein tyrosine phosphatase receptor type E (PTPRE) is a member of the "classical" protein tyrosine phosphatase subfamily and regulates a variety of cellular processes in a tissue-specific manner by antagonizing the function of protein tyrosine kinases. PTPRE plays a tumorigenic role in different human cancer cells, but its role in retinoblastoma (RB), the most common malignant eye cancer in children, remains to be elucidated. Etoposide-resistant RB cell lines and RB patients display significant higher PTPRE expression levels compared to chemosensitive counterparts and the healthy human retina, respectively. PTPRE promotor methylation analyses revealed that PTPRE expression in RB is not regulated via this mechanism. Lentiviral PTPRE knockdown (KD) induced a significant decrease in growth kinetics, cell viability, and anchorage-independent growth of etoposide-resistant Y79 and WERI RB cells. Caspase-dependent apoptosis rates were significantly increased and a re-sensitization for etoposide could be observed after PTPRE depletion. In vivo chicken chorioallantoic membrane (CAM) assays revealed decreased tumor formation capacity as well as reduced tumor size and weight following PTPRE KD. Expression levels of miR631 were significantly downregulated in etoposide-resistant RB cells and patients. Transient miR631 overexpression resulted in significantly decreased PTPRE levels and concomitantly decreased proliferation and increased apoptosis levels in etoposide-resistant RB cells. These impacts mirror PTPRE KD effects, indicating a regulation of PTPRE via this miR. Additionally, PTPRE KD led to altered phosphorylation of protein kinase SGK3 and-dependent on the cell line-AKT and ERK1/2, suggesting potential PTPRE downstream signaling pathways. In summary, these results indicate an oncogenic role of PTPRE in chemoresistant retinoblastoma.

'It's not meant to be for life, but it carries on': a qualitative investigation into the psychosocial needs of young retinoblastoma survivors.

OBJECTIVE AND DESIGN: Retinoblastoma (Rb) is a rare childhood eye cancer, with 45% of individuals impacted by heritable disease and the remainder impacted non-heritably. The condition can leave survivors with life-long psychological and social challenges. This qualitative study examined the psychosocial needs of teenagers and young adults living beyond Rb. SETTING: A qualitative, exploratory study was conducted using focus groups with teenagers and interviews with young adults. Participants were recruited via the Childhood Eye Cancer Trust and the two national Rb treatment centres in the UK. Reflexive thematic analysis was used to analyse data using exploratory and inductive methods. PARTICIPANTS: 32 young survivors of Rb (10 heritable, 21 non-heritable, 1 unknown; 23 unilateral, 9 bilateral) aged between 13 and 29 years (12 male, 20 female). RESULTS: Data were rich and spanned the life course: three key themes were generated, containing eight subthemes. Theme 1 describes participants' experiences of childhood and trauma, including survivor guilt, memories from treatment and impact on personality. Theme 2 focuses on the challenges of adolescence, including the psychological impact of Rb, the impact on identity, and the sense of normality and adaptation to late effects. The third theme considered adulthood and the development of acceptance, a state of being widely considered unachievable during childhood, as well as the 'work' needed to feel supported, including seeking out information, peer support and therapeutic strategies. CONCLUSIONS: This study provides in-depth insight into the experiences of life beyond Rb. Findings highlight the need for specific psychosocial interventions informed by codesign.

Low expression of NR1D1 and NR2E3 is associated with advanced features of retinoblastoma.

PURPOSE: To investigate the expression of nuclear receptor subfamily 1 group D member 1 (NR1D1) and nuclear receptor subfamily 2 group E Member 3 (NR2E3) in retinoblastoma (RB) and their correlation with the clinical and pathological features of RB. METHODS: Immunohistochemical (IHC) assays were performed to detect and evaluate the expression levels of NR1D1 and NR2E3 in paraffin-embedded tissue samples. The relationship between the expression levels and clinicopathological characteristics of RB patients was analyzed using the χ2 test or Fisher exact test. RESULTS: A total of 51 RB patients were involved in this research. The expression levels of NR1D1 (P = 0.004) and NR2E3 (P = 0.024) were significantly lower in RB tumor tissues than in normal retina. The expression levels of NR1D1 and NR2E3 were less positive in RB patients with advanced stages (P = 0.007, P = 0.015), choroidal infiltration (P = 0.003, P = 0.029), and optic nerve infiltration (P = 0.036, P = 0.003). In addition, a low expression level of NR2E3 was associated with high-risk pathology (P = 0.025) and necrosis (P = 0.035) of RB tissues. CONCLUSION: The expression levels of NR1D1 and NR2E3 were decreased in RB and closely associated with the clinical stage and high invasion of the disease. These findings provide new insights into the mechanism of RB progression and suggest that NR1D1 and NR2E3 could be potential targets for treatment strategies.

Strabismus management in retinoblastoma survivors.

PURPOSE: To report the result of strabismus surgery in eye-salvaged retinoblastoma (Rb) patients. METHODS: A retrospective case series including 18 patients with Rb and strabismus who underwent strabismus surgery after completing tumor treatment by a single pediatric ophthalmologist. RESULTS: A total of 18 patients (10 females and 8 males) were included with a mean age of 13.3 ± 3.0 (range, 2-39) months at the time tumor presentation and 6.0 ± 1.5 (range, 4-9) years at the time of strabismus surgery. Ten (56%) patients had unilateral and 8(44%) had bilateral involvement and the most common worse eye tumor's group was D (n = 11), C (n = 4), B (n = 2) and E (n = 1). Macula was involved by the tumors in 12 (67%) patients. The tumors were managed by intravenous chemotherapy (n = 8, 47%), intra-arterial chemotherapy (n = 7, 41%) and both (n = 3, 17%). After complete treatment, the average time to strabismus surgery was 29.9 ± 20.5 (range, 12-84) months. Except for one, visual acuity was equal or less than 1.0 logMAR (≤ 20/200) in the affected eye. Seven (39%) patients had exotropia, 11(61%) had esotropia (P = 0.346) and vertical deviation was found in 8 (48%) cases. The angle of deviation was 42.0 ± 10.4 (range, 30-60) prism diopter (PD) for esotropic and 35.7 ± 7.9 (range, 25-50) PD for exotropic patients (P = 0.32) that after surgery significantly decreased to 8.5 ± 5.3 PD in esotropic cases and 5.9 ± 6.7 PD in exotropic cases (P < 0.001). The mean follow-up after surgery was 15.2 ± 2.0 (range, 10-24) months, in which, 3 (17%) patients needed a second surgery. CONCLUSION: Strabismus surgery in treated Rb is safe and results of the surgeries are acceptable and close to the general population. There was not associated with tumor recurrence or metastasis.