RESUMO
Retinoblastoma is a rare childhood tumor caused by the inactivation of both copies of the RB1 gene. Early diagnosis and identification of heritable RB1 mutation carriers can improve the disease outcome and management via genetic counseling. We used the Multiplex Ligation-dependent Probe Amplification (MLPA) method to analyze the RB1 gene and flanking regions in blood samples from 159 retinoblastoma patients previously negative for RB1 point mutations via Sanger sequencing. We detected a wide spectrum of germline chromosomal alterations, ranging from partial loss or duplication of RB1 to large deletions spanning RB1 and adjacent genes. Mutations were validated via karyotyping, fluorescent in situ hybridization (FISH), SNP-arrays (Single Nucleotide Polymorphism-arrays) and/or quantitative relative real-time PCR. Patients with leukocoria as a presenting symptom showed reduced death rate (p = 0.013) and this sign occurred more frequently among carriers of two breakpoints within RB1 (p = 0.05). All unilateral cases presented both breakpoints outside of RB1 (p = 0.0075). Patients with one breakpoint within RB1 were diagnosed at earlier ages (p = 0.017). Our findings characterize the mutational spectrum of a Brazilian cohort of retinoblastoma patients and point to a possible relationship between the mutation breakpoint location and tumor outcome, contributing to a better prospect of the genotype/phenotype correlation and adding to the wide diversity of germline mutations involving RB1 and adjacent regions in retinoblastoma.
Assuntos
Neoplasias da Retina , Retinoblastoma , Humanos , Retinoblastoma/diagnóstico , Retinoblastoma/genética , Retinoblastoma/patologia , Hibridização in Situ Fluorescente , Brasil/epidemiologia , Genes do Retinoblastoma/genética , Mutação , Neoplasias da Retina/patologia , Análise Mutacional de DNARESUMO
BACKGROUND: Retinoblastoma (Rb) most frequently presents as a unilateral sporadic disease up to 40% of cases, however, arise from a monoallelic germline pathogenic variant. Only 10% of the germline mutations are inherited, and high penetrance is seen in up to 90% of these cases. As an effort to optimize counseling and screening, mutations are classified according to inheritance patterns. However, RB1 spectrum is highly heterogeneous, and information for unaffected carriers remains scarce. MATERIALS AND METHODS: The Mexican family of a 5-month-old patient diagnosed with Rb was studied. The family consisted of five individuals (father, mother, and three siblings). Genetic testing using a next-generation sequencing assay targeting RB1 with oligonucleotide baits designed to capture its exons and 20 bases flanking intronic sequences was performed in every family member. Clinical history and a complete ophthalmological examination (best-corrected visual acuity, slit-lamp biomicroscopy, macular optical coherence tomography, fundus autofluorescence, optical coherence tomography angiography, and electrophysiological studies) were performed in members testing positive to RB1 mutation. RESULTS: The father and her five-month-old daughter tested positive for a non-synonymous RB1 mutation c.459del (p.Lys154Serfs*21). The girl presented with bilateral retinoblastoma, successfully treated with cryotherapy and intravenous chemotherapy. The father had no relevant findings on imaging studies or ophthalmologic evaluation. CONCLUSIONS: This report describes a rare case of a novel low-penetrance RB1 germline mutation. Long-term follow-up of the father will include periodic evaluation of the eyes and orbits, and surveillance for systemic sarcoma and secondary malignancies. Implications for unaffected individuals need to be further studied.
Assuntos
Neoplasias da Retina , Retinoblastoma , Análise Mutacional de DNA , Éxons , Feminino , Mutação em Linhagem Germinativa , Humanos , Lactente , Masculino , Mutação , Penetrância , Neoplasias da Retina/diagnóstico , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Retinoblastoma/diagnóstico , Retinoblastoma/genética , Retinoblastoma/patologia , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
OBJECTIVES: Accumulating research have reported that microRNAs (miRNAs) play important roles in Retinoblastoma (RB). Nonetheless, the function and underlying mechanism of miR-181a-5p in RB remain ambiguous. METHODS: The relative expression levels of miR-181a-5p and NRAS mRNA were detected by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). RB cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) and 5'-Bromo-2'-deoxyuridine (BrdU) assays. Transwell assays and flow cytometry were performed to detect the migration, invasion, and apoptosis of RB cells. The interaction between miR-181a-5p and NRAS was explored using luciferase experiments, western blotting, and qRT-PCR. RESULTS: miR-181a-5p expression was found to be decreased in RB tissues and cell lines, and its expression was correlated with unfavorable pathological features of the patients. In vitro experiments revealed that miR-181a-5p reduced RB cell proliferation, migration, and invasion while enhancing apoptosis. Further research confirmed that NRAS is a direct target of miR-181a-5p. miR-181a-5p inhibited NRAS expression at both the mRNA and protein levels. Co-transfection of pcDNA-NRAS or NRAS small interfering RNA (siRNA) reversed the effects of miR-181a-5p mimics or miR-181a-5p inhibitors on RB cells. CONCLUSION: miR-181a-5p was significantly downregulated during the development of RB, and it suppressed the malignant behaviors of RB cells by targeting NRAS.
Assuntos
MicroRNAs , Neoplasias da Retina , Retinoblastoma , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas de Membrana/genética , MicroRNAs/genética , Invasividade Neoplásica/genética , Proto-Oncogenes , Neoplasias da Retina/genética , Retinoblastoma/genética , Retinoblastoma/patologiaRESUMO
INTRODUCTION: To report a case of a Hispanic girl with late-onset Retinoblastoma (Rb) who was misdiagnosed as a pars planitis prior to referral. Nearly 95% of all Rb cases are detected before age 5, and this patient was 8 years-old. METHODS: Case report of a late-onset Retinoblastoma with anterior chamber (AC) involvement plus the presence of an Ahmed valve. The patient had a history of a couple of months of topical therapy comprising medication for glaucoma, systemic steroids, and a filtration surgery (Ahmed valve), after that a biopsy was performed prior to referral. Upon arrival at our clinic, we performed an examination under anesthesia (EUA) and a B-scan ultrasound (US). RESULTS: Unilateral Retinoblastoma with an Ahmed valve in an AC filled with Rb seeds was diagnosed with the EUA and US in the left eye. An orbital exenteration with map biopsies of the left orbital cavity was performed with confirmation by histopathology of a poorly differentiated endophytic retinoblastoma with Bruch's membrane invasion. Follow-up sessions were then arranged as well as subsequent systemic chemotherapy cycles. CONCLUSION: Given the rare incidence of retinoblastoma in children older than 5 years old, it can be easily mistaken for other differential diagnoses and treated with filtration surgeries that could put the patient's life at risk. In this report, late-onset Rb diagnosis is highlighted as a differential diagnosis in children and adults with atypical uveitis, which required a multidisciplinary approach.
Assuntos
Pars Planite , Neoplasias da Retina , Retinoblastoma , Uveíte Intermediária , Câmara Anterior/patologia , Criança , Pré-Escolar , Feminino , Hispânico ou Latino , Humanos , Neoplasias da Retina/diagnóstico , Neoplasias da Retina/patologia , Retinoblastoma/diagnóstico , Retinoblastoma/patologia , Estudos RetrospectivosRESUMO
The transcription factor Zinc finger E-box binding 1 (ZEB1) displays a range of regulatory activities in cell function and embryonic development, including driving epithelial-mesenchymal transition. Several aspects of ZEB1 function can be regulated by its functional interactions with noncoding RNA types, namely microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). Increasing evidence indicates that ZEB1 importantly influences cancer initiation, tumor progression, metastasis, and resistance to treatment. Cancer is the main disease-related cause of death in children and adolescents. Although the role of ZEB1 in pediatric cancer is still poorly understood, emerging findings have shown that it is expressed and regulates childhood solid tumors including osteosarcoma, retinoblastoma, neuroblastoma, and central nervous system tumors. Here, we review the evidence supporting a role for ZEB1, and its interplays with miRNAs and lncRNAs, in pediatric cancers.
Assuntos
MicroRNAs/genética , Neoplasias/genética , Neoplasias/metabolismo , RNA Longo não Codificante/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Carcinogênese , Criança , Transição Epitelial-Mesenquimal , Humanos , Neoplasias/patologia , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
BACKGROUND: Prognosis for pediatric metastatic Retinoblastoma (Rb) is poor and current therapies are limited by high systemic toxicity rates and insufficient therapeutic efficacy for metastatic Rb. Tumor dissemination to the brain is promoted by the heterogeneous adhesive and invasive properties of Rb cells within the tumor. In this study we evaluate, for the first time, the expression, and roles of the ELTD1 and GPR125 adhesion G protein-coupled receptors (GPCRs) in Rb cell migration, viability and invasion. METHODS: We characterized the RNA expression of adhesion-GPCRs in 64 Rb tumors compared to 11 fetal retinas using the database from the Childhood Solid Tumor Network from St Jude Children's Research Hospital. The role of ELTD1 and GPR125 in Rb were investigated ex vivo by microarray analysis, in vitro by cell viability, Western blot and migration assays, in addition to imaging of the subcellular localization of the GPCRs. To elucidate their role in vivo we utilized siRNA technology in an established Rb orthotopic xenograft murine model. RESULTS: Our investigation demonstrates, for the first time, that ELTD1 but not GPR125, is significantly increased in Rb tumors compared to fetal retinas. We utilized established the Rb cell lines Y79 and Weri-Rb-1, which represent an aggressive, metastatic, and non-metastatic phenotype, respectively, for the in vitro analyses. The studies demonstrated that ELTD1 is enriched in Weri-Rb-1 cells, while GPR125 is enriched in Y79 cells. The measured differences extended to their subcellular localization as ELTD1 labeling displayed punctate clusters in cell-to-cell adhesion sites of Weri-Rb-1 cells, while GPR125 displayed a polarized distribution in Y79 cells. Lastly, we demonstrated the lack of both adhesion receptors does not affect Rb cell viability, yet inhibition of ELTD1 decreases Y79 cell migration in vitro and invasion in vivo. CONCLUSION: Taken together, our data suggest that ELTD1, is a potential target to prevent extraocular Rb. The results within establish ELTD1 as a potential therapeutic target for metastatic Rb.
Assuntos
Biomarcadores Tumorais/metabolismo , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Invasividade Neoplásica , Receptores Acoplados a Proteínas G/genética , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Retinoblastoma/genética , Retinoblastoma/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Intraocular retinoblastoma treatment has changed radically over the last decade, leading to a notable improvement in ocular survival. However, eyes that relapse remain difficult to treat, as few alternative active drugs are available. More challenging is the scenario of central nervous system (CNS) metastasis, in which almost no advancements have been made. Both clinical scenarios represent an urgent need for new drugs. Using an integrated multidisciplinary approach, we developed a decision process for prioritizing drug selection for local (intravitreal [IVi], intrathecal/intraventricular [IT/IVt]), systemic, or intra-arterial chemotherapy (IAC) treatment by means of high-throughput pharmacological screening of primary cells from two patients with intraocular tumor and CNS metastasis and a thorough database search to identify clinical and biopharmaceutical data. This process identified 169 compounds to be cytotoxic; only 8 are FDA-approved, lack serious toxicities and available for IVi administration. Four of these agents could also be delivered by IT/IVt. Twelve FDA-approved drugs were identified for systemic delivery as they are able to cross the blood-brain barrier and lack serious adverse events; four drugs are of oral usage and six compounds that lack vesicant or neurotoxicity could be delivered by IAC. We also identified promising compounds in preliminary phases of drug development including inhibitors of survivin, antiapoptotic Bcl-2 family proteins, methyltransferase, and kinesin proteins. This systematic approach may be applied more broadly to prioritize drugs to be repurposed or to identify novel hits for use in retinoblastoma treatment.
Assuntos
Descoberta de Drogas/organização & administração , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Infusões Intraventriculares , Injeções Espinhais , Injeções Intravítreas , Metástase Neoplásica , Recidiva Local de Neoplasia , Neoplasias da Retina/patologia , Retinoblastoma/patologiaRESUMO
BACKGROUND: Little is known about socioeconomic status (SES) and its effects in childhood cancer survival. This study aims to discuss the association between SES and survival of patients with retinoblastoma (RB) from a tertiary treatment center. PROCEDURE: A retrospective cohort study was conducted, including all patients with RB referred to the Brazilian National Institute of Cancer in Rio de Janeiro (January 2000-December 2016). RESULTS: Data from 160 patients were analyzed with mean age at diagnosis of 22.85 months (SD ± 14.29). Eighty-three patients (51.9%) had an interval to diagnosis equal to or longer than six months, and 13 children (8.1%) abandoned treatment. Five-year overall survival rate for all patients was 78.8% (95% CI, 72.4%-85.9%). In a multivariate model, patients whose fathers had more than nine years of study had a lower death risk. Patients from families having more than one child under five years had a 213% higher risk of death compared with those living with no other small child. Treatment abandonment also had a profound effect on death risk. CONCLUSION: Childhood cancer is notably important considering the potential years of life lost. RB has even more important elements, as the possibility of vision loss in cases with delayed diagnosis. Family characteristics seem to be highly related to RB survival, especially in low- and middle-income countries, where inequalities are still a public health issue. Strategies to improve survival should focus not only on large-scale settings such as improving national healthcare systems but also on more personalized actions that might help to mitigate disparities.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Retina/mortalidade , Retinoblastoma/mortalidade , Classe Social , Centros de Atenção Terciária/estatística & dados numéricos , Protocolos de Quimioterapia Combinada Antineoplásica/economia , Brasil , Pré-Escolar , Atenção à Saúde , Feminino , Seguimentos , Humanos , Lactente , Masculino , Prognóstico , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/economia , Neoplasias da Retina/patologia , Retinoblastoma/tratamento farmacológico , Retinoblastoma/economia , Retinoblastoma/patologia , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
BACKGROUND: Bilateral retinoblastoma (Rb) treatment remains a challenge for ophthalmologists and pediatric oncologists despite new therapeutic strategies for eye preservation. The purpose of this work is to evaluate treatment outcomes in patients who underwent eye salvage treatment at a single-center prior to the chemotherapy in situ era. PROCEDURE: We followed a cohort of 88 consecutive Rb patients diagnosed at Hospital Infantil de México between November 2000 and June 2014. Eye salvage treatment consisted of systemic chemotherapy plus focal therapy planned by a multidisciplinary team. Unresponsive tumors were treated with episcleral brachytherapy and external beam radiotherapy (EBRT). RESULTS: A total of 96 eyes underwent eye salvaging therapy. Seventy-eight eyes (81%) were salvaged. Seven patients (8%) required brachytherapy and 34 patients (39%) underwent EBRT. Thirty-three of 78 preserved eyes (42%) achieved normal visual acuity: 5/27 (20%) in radiated patients and 28/51 (61%) in nonradiated patients. Eight patients developed secondary primary malignancies; however, those treated with EBRT did not have a significantly increased risk when compared with nonirradiated patients (OR: 1.66; P = 0.492). The overall survival rate was 86% (95% CI, 76%-92%) after a mean follow-up of 10 years. CONCLUSIONS: Eye preservation, long-term tumor control, and functional visual acuity could be maintained in many child and adolescent Rb survivors. Our data suggest that ocular radiotherapy can be used as consolidation treatment when other recently developed therapies with potentially fewer side effects are not available. Multidisciplinary management of Rb is mandatory to obtain cancer control during eye salvage treatment.
Assuntos
Olho/efeitos dos fármacos , Olho/efeitos da radiação , Neoplasias Orbitárias/terapia , Tratamentos com Preservação do Órgão/métodos , Neoplasias da Retina/terapia , Retinoblastoma/terapia , Terapia de Salvação , Quimiorradioterapia/métodos , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Masculino , México , Neoplasias Orbitárias/patologia , Prognóstico , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Acuidade VisualRESUMO
miRNAs regulate post-transcriptional gene expression in metazoans, and thus are involved in many fundamental cellular biological processes. Extracellular miRNAs are also found in most human biofluids including plasma. These circulating miRNAs constitute a long distance inter cellular communication system and are potentially useful biomarkers. High throughput technologies like microarrays are able to scan a complete miRNome providing useful detection scores that are underexplored. We proposed to answer how many and which miRNAs are detectable in plasma or extracellular vesicles as these questions have not yet been answered. We set out to address this knowledge gap by analyzing the mirRNome in plasma and corresponding extracellular vesicles (EVs) from 12 children affected by retinoblastoma (Rb) a childhood intraocular malignant tumor, as well as from 12 healthy similarly aged controls. We calculated an average of 537 detectable miRNAs in plasma and 625 in EVs. The most miRNA enriched compartment were EVs from Rb cases with an average of 656 detectable elements. Using hierarchical clustering with the detection scores, we generated broad detection mirnome maps and identified a plasma signature of 19 miRNAs present in all Rb cases that is able to discriminate cases from controls. An additional 9 miRNAs were detected in all the samples; within this group, miRNA-5787 and miRNA-6732-5p were highly abundant and displayed very low variance across all the samples, suggesting both are good candidates to serve as plasma references or normalizers. Further exploration considering participant's sex, allowed discovering 5 miRNAs which corresponded only to females and 4 miRNAs corresponding only to males. Target and pathway analysis of these miRNAs revealed hormonal function including estrogen, thyroid signaling pathways and testosterone biosynthesis. This approach allows a comprehensive unbiased survey of a circulating miRNome landscape, creating the possibility to define normality in mirnomic profiles, and to locate where in these miRNome profiles promising and potentially useful circulating miRNA signatures can be found.