Retinoblastoma Research Articles

Abstract PS12-06: A phase 2 study of abemaciclib monotherapy for patients with retinoblastoma-positive (Rb+), triple-negative metastatic breast cancer

Abstract Background: Cyclin-dependent kinase (CDK) 4/6 inhibitors can significantly extend survival when given in combination with endocrine therapy as treatment for hormone receptor-positive metastatic breast cancer. Preclinical studies suggest CDK4/6 inhibitor monotherapy might also be effective in a subset of triple-negative breast cancers (TNBCs), including those that express a functional retinoblastoma (RB) protein and/or those of the Luminal Androgen Receptor (LAR) subtype. Currently, the clinical activity of CDK4/6 inhibitor therapy in TNBC has not been reported. Methods: We conducted a single-arm phase II study of abemaciclib monotherapy in patients with locally advanced or metastatic TNBC. Key eligibility criteria included: (i) Measurable disease by RECIST 1.1; (ii) Between 1-3 prior lines of systemic therapy for advanced TNBC; (iii) RB-positive tumor (defined as ≥ 50% of tumor cells staining positive for RB by immunohistochemistry [archival or fresh sample] on central testing); (iv) ECOG PS 0/1. Patients were treated with abemaciclib 200 mg orally (28-day cycles) twice daily until disease progression, unacceptable toxicity, withdrawal of consent, or death. Tumor biopsies were mandatory at baseline and C2D1 if tumor tissue was safely accessible. The primary outcome was objective response rate (ORR). Key secondary endpoints included progression-free survival (PFS), overall survival (OS), clinical benefit rate (CBR: CR + PR + SD ≥ 24 weeks), and safety and tolerability. The study had a two-stage design. Thirteen patients were enrolled in the first stage, with the plan to enrol a further 25 patients if at least one objective response was observed. Results: One unconfirmed partial response was observed in stage 1, and a total of 27 patients were enrolled before the trial was closed early due to slow accrual. The median age was 61 years and patients had received a median of 2 prior lines of systemic therapy for metastatic disease. Twelve patients had received prior immunotherapy. After a median follow-up of 28.5 months, the confirmed ORR was 0% and the CBR was 15%, with 4 of 27 patients experiencing stable disease for ≥ 24 weeks. The median PFS was 1.94 months (95% CI: 1.8 – 11.5 months), and the median OS was 8.44 months (95% CI: 4.6 – 15.6 months). There was no significant difference in PFS or OS between patients with PD-L1-positive versus PD-L1-negative disease, or Androgen Receptor (AR) positive versus negative tumors by immunohistochemistry. The most common adverse events of grade 2 or higher were diarrhea (41%), neutropenia (41%), anemia (30%), and nausea (30%). RNA-sequencing of baseline biopsies has been performed to identify biomarkers associated with clinical benefit, and results will be presented at the meeting. Conclusions: Abemaciclib monotherapy did not show clinical activity in patients with Rb+ metastatic TNBC. This finding suggests that future trials of CDK4/6 inhibition as monotherapy in TNBC are not warranted. Citation Format: Shom Goel, Bojana Jovanović, Xiangying Chu, Melissa Hughes, Ayesha Mohammed-Abreu, Julie Kasparian, Timothy Erick, Molly DiLullo, Eileen Wrabel, Rinath Jeselsohn, Nabihah Tayob, Stuart Schnitt, Nancy Lin, Sara Tolaney. A phase 2 study of abemaciclib monotherapy for patients with retinoblastoma-positive (Rb+), triple-negative metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS12-06.

An anti-GD2 aptamer-based bifunctional spherical nucleic acid nanoplatform for synergistic therapy targeting MDM2 for retinoblastoma

Retinoblastoma (RB) is a type of pediatric solid tumor in the fundus. The lack of precision therapies combined with the difficulty of delivering small interfering RNA (siRNA) into the eyes means that there is currently no nucleic acid-based therapy for RB in clinical practice. Here, we reported on anti-GD2 and glutathione-responsive spherical nucleic acids (SNAs), loaded with siRNA and the inhibitor NVP-CGM097, which jointly blocked the oncogenic factor n in RB cells (Y79 and WERI-RB-1). The SNAs were formed through the self-assembly of bifunctional cholesterol amphiphiles containing aptamers that specifically targeted GD2-positive RB cells, allowing for the formation of an SNA with a dense DNA shell. The aptamer/siRNA component functioned both as a carrier and a payload, enhancing the specific recognition and delivery of both components and constituting an active agent for MDM2 regulation. Following SNA endocytosis by RB cells, siRNA and NVP-CGM097 were released from the SNA particles by glutathione, which synergistically blocked the MDM2-p53 pathway, increasing p53 protein content and inducing cell apoptosis. This study showed a potent antitumor effect following intravitreal injection of SNAs in Y79 tumor-bearing mice through clinical manifestation and tumor pathological analysis. In hematological analysis and hepatotoxicity assays, SNAs were safer for mice than melphalan, the favored drug for treating RB in clinical practice. Our results illustrated the potential of intravitreally injected SNAs as a precision medicine for treating RB.

Gastric Inhibitory Polypeptide Receptor (GIPR) Overexpression Reduces the Tumorigenic Potential of Retinoblastoma Cells

Retinoblastoma (RB) is the most common malignant intraocular tumor in early childhood. Gene expression profiling revealed that the gastric inhibitory polypeptide receptor (GIPR) is upregulated following trefoil factor family peptide 1 (TFF1) overexpression in RB cells. In the study presented, we found this G protein-coupled transmembrane receptor to be co-expressed with TFF1, a new diagnostic and prognostic RB biomarker for advanced subtype 2 RBs. Functional analyses in two RB cell lines revealed a significant reduction in cell viability and growth and a concomitant increase in apoptosis following stable, lentiviral GIPR overexpression, matching the effects seen after TFF1 overexpression. In chicken chorioallantoic membrane (CAM) assays, GIPR-overexpressing RB cells developed significantly smaller CAM tumors. The effect of GIPR overexpression in RB cells was reversed by the GIPR inhibitor MK0893. The administration of recombinant TFF1 did not augment GIPR overexpression effects, suggesting that GIPR does not serve as a TFF1 receptor. Investigations of potential GIPR up- and downstream mediators suggest the involvement of miR-542-5p and p53 in GIPR signaling. Our results indicate a tumor suppressor role of GIPR in RB, suggesting its pathway as a new potential target for future retinoblastoma therapy.

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.

Management of Extraocular Retinoblastoma: ICMR Consensus Guidelines.

Retinoblastoma (RB) is the most common intraocular malignancy of childhood. Advanced stage presentation of RB is common in low middle-income countries (LMICs) due to lack of awareness, social taboos associated with enucleation, seeking alternative conservative treatment options, and poor accessibility to health care. Over the last few decades, there have been significant advancements in the management of extraocular RB (EORB) which have improved outcomes and helped in minimizing treatment-related toxicities. The incorporation of multimodality approaches including chemotherapy, surgery, and radiotherapy (RT) has shown promising results; however, prognosis remains poor especially in LMICs. In this article, authors have discussed the ICMR consensus guidelines on the management of EORB, including metastatic RB.

Therapeutic Strategies for RB1-Deficient Cancers: Intersecting Gene Regulation and Targeted Therapy.

The retinoblastoma (RB) transcriptional corepressor 1 (RB1) is a critical tumor suppressor gene, governing diverse cellular processes implicated in cancer biology. Dysregulation or deletion in RB1 contributes to the development and progression of various cancers, making it a prime target for therapeutic intervention. RB1's canonical function in cell cycle control and DNA repair mechanisms underscores its significance in restraining aberrant cell growth and maintaining genomic stability. Understanding the complex interplay between RB1 and cellular pathways is beneficial to fully elucidate its tumor-suppressive role across different cancer types and for therapeutic development. As a result, investigating vulnerabilities arising from RB1 deletion-associated mechanisms offers promising avenues for targeted therapy. Recently, several findings highlighted multiple methods as a promising strategy for combating tumor growth driven by RB1 loss, offering potential clinical benefits in various cancer types. This review summarizes the multifaceted role of RB1 in cancer biology and its implications for targeted therapy.

INDOCYANINE GREEN ANGIOGRAPHY-GUIDED PHOTOCOAGULATION OF LARGE MICROVASCULAR ABNORMALITIES ("TELCAPS") IN A PATIENT WITH PERSISTENT MACULA EDEMA IN THE CONTEXT OF RADIATION RETINOPATHY.

The purpose of this report is to describe a patient with radiation retinopathy who responded to indocyanine green angiography (ICGA)-guided photocoagulation as treatment for persistent macula edema secondary to TelCaps, a newly defined type of large microvascular abnormality. Retrospective case report. A 40-year-old man with a history of bilateral retinoblastoma and right enucleation presented with decreased vision in the left eye secondary to macula edema. Examination revealed radiation retinopathy with a cluster of ICGA-avid large microaneurysms temporal to the macula. The patient did not respond to treatment with intravitreal bevacizumab or triamcinolone. Complete resolution of macula edema was demonstrated after two rounds of ICGA-guided focal photocoagulation to the aforementioned microaneurysm cluster. No subsequent or adjunctive intravitreal injections of anti-VEGF were required, and there was no recurrence of the TelCaps lesions at follow-up to eight years. TelCaps are large microvascular abnormalities with high affinity for ICG. These lesions may not have been recognized because of difficulties in their detection on routine examination and with investigations including ocular coherence tomography angiogram and fluorescein angiogram. Early evidence supports their role in macula edema that is not responsive to intravitreal injections of anti-VEGF. ICGA-guided photocoagulation of TelCaps lesions can result in resolution of macula edema. To our knowledge, this is the first reported case of TelCaps secondary to radiation retinopathy.

Management of Intraocular Retinoblastoma: ICMR Consensus Guidelines.

Retinoblastoma (RB) is the most common childhood intraocular malignancy. Delayed presentation due to a lack of awareness and advanced intraocular tumors are a common scenario in low-middle income countries (LMICs). Remarkable treatment advances have been made in the past few decades allowing globe salvage in advanced intraocular RB (IORB) including systemic chemotherapy with focal consolidation and targeted treatments like intraarterial chemotherapy and intravitreal chemotherapy. However, a lack of availability and affordability limits the use of such advances in LMICs. External beam radiotherapy, despite risk of second cancers in RB with germline mutations, still remains useful for recalcitrant RB not responding to any other treatment. When choosing conservative treatment for advanced IORB, the cost and long duration of treatment, morbidity from multiple evaluation under anesthesias (EUAs), side effects of treatment and risk of treatment failure need to be taken into account and discussed with the parents. In this article, the authors discuss the ICMR consensus guidelines on the management of IORB.

The Impact of Tumor Laterality (Unilateral vs. Bilateral) on Presentation and Management Outcome in Patients with Retinoblastoma.

Background: This study compares the outcomes of managing retinoblastoma between patients with unilateral and bilateral presentations. Methods: The study, conducted at the King Hussein Cancer Center in Amman, Jordan, retrospectively analyzed cases of retinoblastoma treated between March 2003 and December 2019. Evaluation criteria included clinical features, disease stage, treatment methods, and overall management outcomes. Results: The study comprised 697 eyes from 478 patients with retinoblastoma, with 52% being males. Bilateral disease was observed in 70% of patients, and a family history of retinoblastoma was more prevalent in cases with bilateral disease (20%) compared to those with unilateral disease (4%). Unilateral cases had a median age at diagnosis of 28 months, whereas bilateral cases were diagnosed at a median age of 6 months. Extra-ocular retinoblastoma was detected in 1% of eyes. According to the International Intraocular Retinoblastoma Classification (IIRC), 88% of unilateral cases presented with advanced disease (IIRC group D/E), compared to 46% in bilateral cases. Primary enucleation was performed in 29% of unilateral cases and 16% of bilateral cases (p-value 0.0007). Eye salvage rates were 31% in unilateral cases and 68% in bilateral cases (p-value < 0.0001). At 120 months of follow-up, 5% of patients died from secondary neoplasms or metastases, 81% were alive, and 14% were lost to follow-up. There was no significant difference in metastasis, secondary neoplasms, or mortality between patients with unilateral and bilateral retinoblastoma. Conclusions: This study highlights the nuanced differences in clinical characteristics and outcomes between unilateral and bilateral retinoblastoma, emphasizing the necessity of customized management and early detection strategies. It demonstrates that while bilateral retinoblastoma benefits from earlier detection and has a higher rate of eye salvage, there is no significant difference in metastasis or mortality rates when compared to unilateral cases. The critical roles of primary enucleation in advanced cases, along with effective communication and patient education, are also underscored to improve treatment adherence. Overall, these findings point to the importance of tailored approaches in optimizing outcomes for the diverse patient population affected by retinoblastoma.

Assessment of Retinal Microvasculature and Choroidal Vascularity After Intra-arterial Chemotherapy for Retinoblastoma: Retinal and Choroidal Vascular Assessment After IAC for RB

PurposeTo evaluate changes in retinal microvascular density and choroidal vascularity in patients with retinoblastoma (RB) after intra-arterial chemotherapy (IAC). DesignRetrospective clinical cohort study. MethodsThis study included 12 unilateral RB eyes treated with IAC (RB tumour), 12 contralateral normal eyes (RB fellow), and 12 healthy controls. The macular retinal thickness and retinal microvascular structure, including foveal avascular zone (FAZ) area, the macular and peripapillary superficial vessel density (SVD) and deep vessel density (DVD), were measured by optical coherence tomography angiography (OCTA). The choroidal thickness (ChT) and choroidal vascularity, including total choroidal area (TCA), luminal area (LA), stromal area (SA) and Choroidal Vascularity Index (CVI), were measured by spectral-domain optical coherence tomography (SD-OCT). A comparison among the three groups was conducted, while the correlations among the parameters were analyzed. ResultsBetween the three cohorts, the foveal retinal thickness, the SVD, DVD, ChT, TCA, LA, SA and CVI were significantly lower in RB tumour compared to RB fellow and the control eyes (all P<0.01). There were no significant differences in the parameters between the contralateral and control eyes. The correlation analyses indicated a significant negative correlation between the total melphalan dose and foveal and parafoveal DVD, ChT, and LA. ConclusionsThe retinal microvascular density and choroidal vascularity were lower in unilateral RB treated with IAC, and seemed to be related to the total melphalan dose. Moreover there were no measurable changes in the contralateral eyes.

Abstract 3642: Phenotypic biomarkers of aqueous humor extracellular vesicles from retinoblastoma eyes

Abstract Purpose: Retinoblastoma (RB) is the most common pediatric intraocular cancer, yet tumor biopsies are contraindicated. This challenge emphasizes the need for investigation of non-tissue biomarkers. Extracellular vesicles (EVs), which are secretory vesicles containing bioactive molecules, have emerged as promising biomarker candidates due to their ability to be phenotyped using known tetraspanin markers. Aqueous humor (AH) provides an exciting liquid biopsy source for intraocular tumor information, with EVs having recently been identified in AH. Our laboratory has previously proposed that CD63/81+EVs are associated with retinoblastoma, but specific tumor-derived markers have not been linked to AH EVs using traditional tetraspanin assays. In this study, we employed MACSPlex, a high-dimensional magnetic bead-based immunoassay, to further explore RB-EV. Methods: 7 AH samples consisting of two congenital glaucoma (GLC) samples and five RB samples taken at the time of primary enucleation were analyzed. We utilized Macsplex, a multiplex bead-based flow cytometry assay, to analyze the expression profiles of surface markers found on EVs. With 39 capture beads coated with monoclonal antibodies for 37 different EV surface antigens and two negative controls, the mean fluorescence intensity (MFI) of APC conjugated antibody cocktail from three tetrapanins (CD9, CD63 and CD81) on EVs captured by each surface antigen could be measured by flow cytometry. Enrichment ratios were then calculated by normalizing the fluorescence of each signal to the mean fluorescence of CD9, CD63, and C81. Results: Macsplex analysis of the 2 GLC AH samples demonstrated a strong CD63 dominance compared with RB AH. The mean CD63 enrichment ratio for GLC samples was 2.6 (S.D. = 0.34), and 1.3 (S.D. = 0.27) in RB samples, with a p-value of 0.069. In comparison to the GLC patients, the analysis of EVs in the AH of the 5 RB samples exhibited a greater co-dominance of CD63/CD81 positivity consistent with past research on RB EVs. The mean CD81 enrichment ratio for the 5 RB samples was 1.5 (S.D. = 0.26), in contrast to 0.3 (S.D. = 0.24) in GLC AH, with a p-value of 0.03. Interestingly, 4 out of the 5 RB AH samples demonstrated very strong CD133 signals, which is a known cancer marker. Deeper investigation into one RB AH sample utilizing single tetraspanin detection revealed higher coexpression of CD63 and CD81 with CD133 than seen with CD9. The CD133 enrichment ratio for the CD81, CD63, and CD9 single channel experiments were 6.6, 3.4, and 1.5 respectively. Conclusions: Comparison between this study and previous work done by our group demonstrate a high concordance between results utilizing Macsplex and Exoview. However, this is the first study identifying the expression of CD133, a cancer stem cell marker, on retinoblastoma-derived EVs in AH. Macsplex technology illustrates an incredible clinical potential of AH EVs for characterizing retinoblastoma phenotype in vivo. Citation Format: Anne Amacker, Chen-Ching Peng, Bibiana J. Reiser, Jesse L. Berry, Liya Xu. Phenotypic biomarkers of aqueous humor extracellular vesicles from retinoblastoma eyes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3642.

Abstract 967: Clinical applicability of targeted panel sequencing in liquid biopsies for disease diagnosis and monitoring of patients with pediatric solid tumors

Abstract Liquid biopsy assays based on sequence analysis of cell-free DNA (cfDNA) have the potential to transform patient care. We recently validated a liquid biopsy platform, LBSeq4Kids, for patients with retinoblastoma, brain tumors and solid tumors using low pass whole genome sequencing (LP-WGS) of cfDNA from aqueous humor (AH), cerebrospinal fluid (CSF), and plasma (PL) to detect Copy Number Alterations (CNAs) in circulating tumor DNA (ctDNA). Herein, we present phase 2, a high-depth comprehensive Next-Generation Sequencing (NGS) panel using cfDNA that allows us to detect SNV’s, indels and gene fusions in clinically relevant genes for pediatric solid and brain tumors. Seventy-four cfDNA specimens have been analyzed to date, using plasma from 43 patients with solid tumors, CSF from 25 patients with brain tumors and AH from six patients with ocular tumors including retinoblastoma. cfDNA was extracted at diagnosis, during therapy, at remission and/or relapse. Sequencing libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of cfDNA. For the detection of recurrent mutations and fusions we designed a custom panel using a hybridization-based capture method (Twist Bioscience) that included full coverage of the exons of 136 genes harboring recurrent mutations and selected introns for five genes with recurrent rearrangement breakpoints including BRAF, EWSR1, SYT, FOXO1 and NTRK1. Hybridized libraries were paired-end sequenced (Illumina NextSeq 500) at an average 1000x collapsed coverage. Mutations identified in cfDNA were compared with those in the primary or metastatic tumors profiled with our OncoKids® NGS panel. A total of 35/43 (81%) plasma specimens were positive for either an SNV, deletion or fusion with the most common events being a somatic missense variant in TP53 (13/35, 37%) or translocation involving EWSR1 (9/35, 26%). A total of 16/25 (64%) CSF specimens were positive for SNVs and fusions, most often involving KIAA1549::BRAF (7/25, 28%) in low grade gliomas. A germline variant in RB1 was detectable in two AH specimens from the left and right eye of a patient with bilateral retinoblastoma while plasma from a patient with metastatic retinoblastoma demonstrated clinically relevant variants in RB1 and DICER1. Serial cfDNA profiling revealed a clinically reported TP53 c.746G&amp;gt;C missense variant in the plasma of a patient with osteosarcoma at multiple timepoints through the disease course, with evidence for relapse prior to standard imaging. These data suggest that clinical implementation of a second version of LBSeq4Kids that combines CNA and mutation analysis allows for diagnosis, residual disease detection and risk stratification for pediatric patients with diverse tumor types, regardless of histologic grade, with a variety of pediatric solid and brain tumors. Citation Format: Eirini Christodoulou, Venkata Yellapantula, Katrina O'Halloran, Liya Xu, Jesse L. Berry, Fariba Navid, Dejerianne Ostrow, Jaclyn A. Biegel. Clinical applicability of targeted panel sequencing in liquid biopsies for disease diagnosis and monitoring of patients with pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 967.

Epidemiology, Diagnosis and Genetics of Retinoblastoma: ICMR Consensus Guidelines.

Retinoblastoma (RB) is the most common intraocular tumor in childhood. It is mainly caused by mutations in both alleles of the RB1 tumor suppressor gene that is found on chromosome 13 and regulates the cell cycle. Approximately 8000 children are diagnosed with RB globally each year, with an estimated 1500 cases occurring in India. The survival rate of RB has improved to more than 90% in the developed world. Leukocoria and proptosis are the most common presenting features of RB in Asian Indian populations. Most cases of RB are diagnosed by fundus examination followed by ultrasound. The International Classification of Retinoblastoma is the most used scheme for the staging and classification of intraocular RB in India. Prenatal testing and preimplantation genetic testing for RB may be beneficial in high-risk families. Histopathologic risk factors such as massive choroidal invasion and post-laminar optic nerve help in predicting the occurrence of metastasis in children with RB, while presence of microscopic residual disease requires aggressive adjuvant treatment in eyes enucleated for group E RB. The review provides a consensus document on diagnosis and genetics of RB in India.

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

PurposeTo 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.MethodsImmunohistochemical (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.ResultsA 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.ConclusionThe 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.

Tumor DNA sampling from aqueous humor in retinoblastoma - A report from South Asia.

Retinoblastoma (RB) is the most common intraocular tumor in pediatric age group. The role of genetics has been explored in predicting survival prognosis, but its role in predicting globe salvage remains largely unexplored. We hereby aim to isolate cell-free DNA (cfDNA) from aqueous humor (AH) in RB eyes and validate its use for genetic studies. AH was obtained from 26 eyes undergoing enucleation (arm A) or intravitreal chemotherapy (arm B). Isolation of cfDNA was done using QIAamp® Circulating Nucleic Acid kit, and the cfDNA was utilized for targeted sequencing of RB1 gene. We could isolate cfDNA in all eyes (72% unilateral and 28% bilateral) with a distribution peak between 140 and 160 bp and a mean concentration of 27.75 ng/μl for arm A and 14 ng/μl for arm B. Targeted sequencing done on four samples showed RB1 gene mutations, namely, inframe deletion (c. 78-80del, p.Pro29del), start-loss mutation (c.1A>T, p.Met1?), nonsense mutations (c.2236G>T, p.Glu746Ter), (c.1659T>A, p.Cys553Ter), and (c.2065C>T, p.Gln689Ter), and novel missense mutations (c.672C>A, p.Asp224Glu) and c.692C>T (p.Pro231Leu). Genetic profile of cfDNA extracted from AH and genomic DNA from the tumor tissue was comparable. Our study supports the previous reports that AH may be used as a source of tumor-derived cfDNA. This is the first report from South Asia on isolation and genetic analysis of cfDNA from AH of RB eyes and, therefore, a big step forward in paving the role of tumor genetics in RB. Further studies are required to elucidate concordance between the tumor and AH genetic profile.

Caracterización de pacientes con retinoblastoma en un centro de referencia de la costa norte de Colombia

Purpose: The purpose of the following study was to describe the sociodemographic, clini-cal, and therapeutic characteristics of patients with retinoblastoma (RB), the most common intraocular tumor in children, receiving medical care in an eyecare institution in the Carib-bean region of Colombia.Methods: A case series between 2016 and 2021 was carried out. Eyes of patients with RB, receiving medical care in a single health care institution, and in compliance with the esta-blished follow-up protocol, were included. Descriptive analyzes of relevant variables were performed.99Characterization of Retinoblastoma Patients in a Referral Center on the Colombian North CoastAna María Álvarez Castillo, Cesar Atencia Niño,Diana Cifuentes Zapata, Katia Gutiérrez Brun,Jaime Moreno Chaparro, Javier Antonio Amaya Nieto, Javier Vásquez RamírezVol. 40(1), 2024, 97-108Barranquilla (Col.) Results: In this study, a totalof 21 eyes of 16 patients were examined. The average age at the time of diagnosis was 17.7 months, and 50% of children were male. Using the Interna-tional Classification for Intraocular Retinoblastoma, 42.9% of the total cases were classi-fied as group D. Out of the 21 cases, 71.4% went into total remission without the necessity for enucleation, and the most common treatment was chemotherapy, using Melphalan® via intraarterial or intravitreal administration.Conclusion: Specific findings of patients with RB in the Caribbean Coast of Colombia were identified. These findings imply that, despite the obstacles to healthcare access, an early diagnosis and a treatment plan using intraarterial chemotherapy may provide a satisfac-tory clinical outcome (remission).

LncRNA NEAT1 promotes angiogenesis of retinoblastoma cells through regulation of the miR-106a/HIF-1α axis

ObjectiveTo explore the role and mechanisms of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in angiogenesis of retinoblastoma (RB) cells. MethodsThis study investigated the roles of NEAT1 in RB progression. The RNA expression levels of NEAT1, miR-106a, and hypoxia-inducible factor-1alpha (HIF-1α) examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) were compared between RB cells and normal retinal pigment epithelial (RPE) cells. The binding sites between NEAT1 and miR-106a, and between miR-106a and HIF-1α were predicted by the TargetScan database and verified using the dual-luciferase reporter assay. By transfection of overexpression plasmid or shRNA of NEAT1, and/or treatment of miR-106a inhibitor or mimics, proliferation, invasion, and angiogenesis of RB cells (measured by the MTT assay, the Transwell assay, and the tube formation assay, respectively) were compared between groups. Group comparisons were analyzed using one-way analysis of variance (ANOVA), and Tukey's post-hoc test was employed for further statistical assessment. P-value less than 0.05 was considered statistically significant. ResultsThe RNA expression levels of NEAT1 and HIF-1α were upregulated in RB cells, whereas the expression level of miR-106a was downregulated compared with RPE cells. NEAT1 overexpression or miR-106a knockdown advanced proliferation, invasion, and tube formation of RB cells. As a target of NEAT1, miR-106a could sponge HIF-1α to downregulate HIF-1α expression level. Functional analyses indicated that miR-106a knockdown reversed the inhibitory effects of NEAT1 silencing on the proliferation, invasion, and tube formation of RB cells. Furthermore, miR-106a overexpression suppressed RB cell angiogenesis by downregulating HIF-1α expression level. ConclusionNEAT1 promoted proliferation, invasion, and angiogenesis of RB cells through upregulation of HIF-1α expression level by sponging miR-106a, demonstrating that NEAT1 may be a novel target for RB treatment.

IRF3 activates RB to authorize cGAS-STING-induced senescence and mitigate liver fibrosis.

Cytosolic double-stranded DNA surveillance by cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) signaling triggers cellular senescence, autophagy, biased mRNA translation, and interferon-mediated immune responses. However, detailed mechanisms and physiological relevance of STING-induced senescence are not fully understood. Here, we unexpectedly found that interferon regulatory factor 3 (IRF3), activated during innate DNA sensing, forms substantial endogenous complexes in the nucleus with retinoblastoma (RB), a key cell cycle regulator. The IRF3-RB interaction attenuates cyclin-dependent kinase 4/6 (CDK4/6)-mediated RB hyperphosphorylation that mobilizes RB to deactivate E2 family (E2F) transcription factors, thereby driving cells into senescence. STING-IRF3-RB signaling plays a notable role in hepatic stellate cells (HSCs) within various murine models, pushing activated HSCs toward senescence. Accordingly, IRF3 global knockout or conditional deletion in HSCs aggravated liver fibrosis, a process mitigated by the CDK4/6 inhibitor. These findings underscore a straightforward yet vital mechanism of cGAS-STING signaling in inducing cellular senescence and unveil its unexpected biology in limiting liver fibrosis.

Non-RB1 germline cancer predisposing variants found in retinoblastoma patients

PurposeIt is well known that individuals with hereditary retinoblastoma are at lifelong high risk for developing subsequent malignant neoplasms (SMN). However, the role that non-RB1 germline variants play in tumorigenesis and SMN risk has not yet been studied. The purpose of this study is to report the frequency and spectrum of non-RB1 germline cancer predisposing variants in individuals with retinoblastoma (RB). MethodsRetrospective data collection from institutional electronic medical records of 94 individuals seen at our institution with personal history of retinoblastoma, who had undergone next-generation sequencing germline analysis. ResultsThe prevalence of individuals with cancer predisposition was 57%, of which 45% had a pathogenic/likely pathogenic in the RB1 gene, 12% harbored a pathogenic/likely pathogenic variant in a non-RB1 gene, and 7% had both. No difference was found between patients with and without non-RB1 variants when comparing demographic and clinical characteristics, including time to SMN. Variants were found in 7 different genes, with only 1 variant repeating 3 times. ConclusionIn this small cohort of patients with retinoblastoma, non-RB1 variants did not appear to augment tumorigenesis or disease progression. Larger studies are required to determine associations between specific variants and development of SMN.

ACOT7 promotes retinoblastoma resistance to vincristine by regulating fatty acid metabolism reprogramming

The rate of vincristine (VCR) resistance in the treatment of retinoblastoma (RB) is relatively high, and the exact role and mechanism of autophagy and fatty acid (FA) metabolism in RB are still unknown. The aim of this study was to elucidate the molecular mechanism by which acyl-CoA thioesterase 7 (ACOT7) regulates FA metabolism and autophagy, which may lead to potential therapeutic strategies for RB. In the present study, the relationship between FA metabolism and cellular drug sensitivity was evaluated through ACOT7 overexpression or inhibition tests in RB-resistant cells. The lipase inhibitor orlistat and the autophagy inhibitor CQ were used to determine the effects of ACOT7 on FA metabolism, autophagy, and cellular drug sensitivity, as well as the therapeutic value of ACOT7 targeting. The results showed that ACOT7 was upregulated in VCR-resistant RB cells, significantly enhancing cell resistance and indicating that ACOT7 may serve as a biomarker for VCR resistance in RB cells. Knockdown of ACOT7 inhibited FA metabolism and reduced cell viability in VCR-resistant RB cells. The effect of ACOT7 overexpression was opposite to that of ACOT7 knockdown, and ACOT7 overexpression promoted autophagy in VCR-resistant RB cells. After treatment with orlistat or CQ, FA metabolism in VCR-resistant RB cells decreased, cell viability and autophagy were inhibited, EMT was inhibited, and the sensitivity of RB cells to VCR was increased. In conclusion, ACOT7 knockdown can mediate FA metabolism to inhibit autophagy and the migration of RB cells, thereby improving the sensitivity of RB cells to VCR.