microRNA-144 functions as a diagnostic and prognostic marker for retinoblastoma

OBJECTIVES: Retinoblastoma (RB) is a highly malignant eye tumor with a low survival rate and a high metastatic rate. The current work was designed to investigate the potential roles of microRNA-144 (miR-144) in the diagnosis and prognosis of RB. METHODS: miR-144 expression levels in RB tissues and adjacent normal tissues, as well as serum samples from RB patients and healthy controls were measured. The association between miR-144 expression levels and clinical features were analyzed. Moreover, diagnostic and prognostic values of miR-144 in RB were verified by receiver operating characteristic analysis and Kaplan-Meier survival assays. RESULTS: The expression level of miR-144 was markedly decreased in tumor tissues of RB patients, and the expression level of miR-144 was positively associated with tumor size and metastasis in RB patients. Moreover, miR-144 can distinguish tumor tissues from normal tissues with high specificity and sensitivity, and RB patients with lower miR-144 expression have shorter overall and disease-free survival rates than those with higher miR-144 expression. Alternatively, miR-144 also decreased in the serum of RB patients in comparison with healthy subjects, and miR-144 expression levels in the tissue samples and serum were positively correlated. Furthermore, miR-144 levels in the serum of RB patients sensitively distinguished RB patients from healthy controls. CONCLUSIONS: miR-144 expression was downregulated in serum and tissue samples of RB patients and may function as a diagnostic and prognostic marker for RB.

Uncommon RB1 somatic mutations in a unilateral retinoblastoma patient

Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a developing retina is a crucial event in the initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed the DNA from tumor tissue and peripheral blood of a unilateral retinoblastoma patient to determine the RB1 mutation status and to provide an accurate genetic counseling. A comprehensive approach, based on our previous experience, was used to identify the causative RB1 mutations. Screening for RB1 mutations was performed by PCR direct sequencing, multiplex ligation-dependent probe amplification (MLPA) and Real Time-PCR analyses. Three different mutations were identified in the tumor DNA, which were absent in blood DNA. The somatic origin of these mutations was vital to rule out the heritable condition in this patient.

El retinoblastoma (RB) es el cáncer ocular más común de la niñez. La inactivación somática de ambos alelos del gen supresor de tumores RB1 en la retina en desarrollo es un evento crucial en la iniciación de la tumorigénesis en la mayoría de los casos de retinoblastoma unilateral. Nosotros analizamos el ADN de tumor y de sangre periférica de un paciente con retinoblastoma unilateral para identificar las mutaciones y así proveer un asesoramiento genético a la familia. Para ello utilizamos un protocolo basado en nuestra previa experiencia para identificar todas las mutaciones en el gen RB1 que causaron el RB. El rastreo de mutaciones se realizó por medio de los siguientes análisis: PCR-secuenciación, amplificación multiplex de sondas ligadas (MLPA) y PCR-Tiempo Real. Se encontraron tres mutaciones diferentes en el ADN del tumor, las cuales estaban ausentes en el ADN de la sangre. El origen somático de estas mutaciones es importante para indicar que la enfermedad no es hereditaria.

Treatment of Retinoblastoma: The Role of External Beam Radiotherapy

The risk of radiotherapy-related secondary cancers in children with constitutional retinoblastoma 1 (RB1) mutations has led to reduced use of external beam radiotherapy (EBRT) for RB. Presently, tumor reduction with chemotherapy with or without focal surgery (chemosurgery) is most commonly undertaken; EBRT is avoided as much as possible and is considered only as the last treatment option prior to enucleation. Nevertheless, approximately 80% of patients are diagnosed at a locally advanced stage, and only 20-25% of early stage RB patients can be cured with a chemosurgery strategy. As a whole, chemotherapy fails in more than two-thirds of eyes with advanced stage disease, requiring EBRT or enucleation. Radiotherapy is still considered necessary for patients with large tumor(s) who are not candidates for chemosurgery but who have visual potential. When radiation therapy is indicated, the lowest possible radiation dose combined with systemic or local chemotherapy and focal surgery may yield the best clinical outcomes in terms of local control and treatment-related toxicity. Proton beam therapy is one EBRT method that can be used for treatment of RB and reduces the radiation dose delivered to the adjacent orbital bone while maintaining an adequate dose to the tumor. To maximize the therapeutic success of treatment of advanced RB, the possibility of integrating radiotherapy at early stages of treatment may need to be discussed by a multidisciplinary team, rather than considering EBRT as only a last treatment option.

Identification of three new mutations in the RB1 gene in patients with sporadic retinoblastoma in Colombia / Identificación de tres nuevas mutaciones en el gen RB1 en pacientes con retinoblastoma esporádico en Colombia

Introduction. Retinoblastoma is a childhood cancer of the retina originated by altered or null retinoblastoma protein (pRb) expression. Genetic alterations in both RB1 alleles in the retinal cells are required for the development of retinoblastoma. In the sporadic form, non-hereditary RB1 gene mutations take place in a single retinoblast cell, and are therefore only present in tumor DNA (somatic mutations). Sporadic retinoblastoma is primarily unilateral, lacks family history and has no risk of transmission to descendants. Genetic tests for detection of RB1 mutation has improved the identification of carriers and facilitated accurate genetic counseling. Objective. To identify mutations in the RB1 gene in Colombian patients with sporadic retinoblastoma by PCR-SSCP followed by sequence. Materials and methods. Four patients with sporadic retinoblastoma were analyzed by PCR-SSCP, followed by DNA sequencing to identify variations in the RB1 gene. Results. We identified five variations in RB1 gene: three new mutations (one germline and two somatic mutations), one new polymorphism and one already reported somatic mutation. Four mutations were found in three patients with unilateral retinoblastoma and one mutation was found in a patient with bilateral retinoblastoma. One of these was a germline mutation in a sporadic unilateral retinoblastoma that was not present in the parents or three siblings analyzed. Conclusions. Our results emphasize the importance of identifying mutations for genetic counseling and clinical management of sporadic retinoblastoma patients. Description of a new RB1 gene variant is interesting since there have been a small number of polymorphisms reported for this gene.

Introducción. El retinoblastoma es un cáncer pediátrico de la retina originado por la expresión alterada o ausente de la proteína del retinoblastoma (pRb). Se requiere la alteración genética de ambos alelos RB1 en las células de la retina para el desarrollo del retinoblastoma. En la forma esporádica, las mutaciones no hereditarias del gen RB1 ocurren en un solo retinoblasto y están presentes sólo en el ADN del tumor (mutaciones somáticas). El retinoblastoma esporádico es generalmente unilateral, no tiene historia familiar y no tiene riesgo de transmisión a la descendencia. Las pruebas genéticas para la detección de mutaciones en RB1 han mejorado la identificación de portadores y han facilitado la precisión de la asesoría genética. Objetivo. Detectar mutaciones en el gen RB1 en pacientes colombianos con retinoblastoma esporádico mediante PCR-SSCP seguido de secuenciación. Materiales y métodos. Se analizaron cuatro pacientes con retinoblastoma esporádico para la detección de variaciones en el gen RB1 mediante PCR-SSCP, seguida de secuenciación. Resultados. Se identificaron cinco variaciones del gen RB1 : tres mutaciones nuevas (una de línea germinal y dos somáticas), un polimorfismo nuevo y una mutación somática ya reportada. Las cuatro mutaciones se encontraron en tres pacientes con retinoblastoma unilateral y uno con bilateral. La mutación germinal se detectó en un paciente con compromiso unilateral y no se encontró en los padres ni en los tres hermanos analizados. Conclusión. Estos resultados enfatizan la importancia, para asesoría genética y manejo clínico, de identificar mutaciones del gen RB1 en pacientes con retinoblastoma esporádico. La descripción de una nueva variante en RB1 es interesante, dado el muy bajo número de polimorfismos reportados para este gen.

Consideraciones clínicas, diagnósticas y de tratamiento en retinoblastoma / Clinical, diagnostic and therapeutic considerations in retinoblastoma

El retinoblastoma es el tumor intraocular primari o más frecuente en l a infancia. Su detección temprana y el inici o del tratamiento adecuado permi te mejorar dramáticamente l a sobrevida en estos niños. En este artícul o se hace una revisión general de l a enfermedad. Se empleó PubMed y se revisaron artículos representativos del tema, que permi tieran dar una idea general de los di ferentes avances alcanzados. Dada su cl ínica característica, el médico de atención primaria, es pieza fundamental en l a captación inicial del paciente. [Vi l lami l JF , Quintero LM, Serrano RA, Moreno IA. Consideraciones cl ínicas, diagnósticas y de tratamiento en retinoblastoma. MedUNAB 201 1; 14:180-187].

Retinoblastoma is the most common primary intraocular tumor in childhood. Its early detection and initiation of appropriate therapy , can dramatically improve the life expectancy in these children. This article is a general review of the disease. PubMed was employed and representative articles about the topic were selected in order to given us a general idea about the advances achieved. Due to clinical features, primary care physician is a fundamental part in the initial catchment of patient. [Villamil JF , Quintero LM, Serrano RA, Moreno IA. Clinical, diagnostic and therapeutic considerations in retinoblastoma. MedUNAB 2011; 14:180-187].

Ki-67 cell proliferation in familial and in esporadic unilateral retinoblastoma: case report

PURPOSE: Ki-67 is a nuclear protein that is expressed at all phases of the cell cycle except the resting phase. This study is a clinicopathologic observational case report that aims to report on the cell proliferation rates, as measured by the Ki-67 antigen, in two enucleated retinoblastoma eyes. METHODS: One unilateral familial (mother with unilateral disease - patient 1) and one unilateral sporadic retinoblastoma (patient 2) patients were submitted to enucleation without previous treatment. The tumor cell proliferation rate was assessed by the Ki-67 antigen labeling index (stained cells / 100 cells) in five different fields of the tumor. RESULTS: Patient 1 was 23 months old and the tumor was exophytic with associated neovascularization of the iris; patient 2 was 6 years old and the tumor was endophytic with coarse vitreous seeds. Both enucleated eyes presented optic nerve with free surgical margins. Positive Ki-67 cell index in patient 1 varied from 75 to 90 (MD ± SD: 79.5 ± 6.61) and in patient 2 from 38 to 60 (MD ± SD: 46.6 ± 8.2). CONCLUSIONS: The familial retinoblastoma, besides the earlier age presentation, showed 45.8 percent more Ki-67 positive cells than the same stage sporadic one. This proliferation rate may explain the earlier presentation age of the tumor in the inherited disease.

OBJETIVO: O Ki-67 é antígeno nuclear que se expressa em todas as fases do ciclo celular, exceto no período de repouso. Este é um estudo de casos com correlação clínico-patológica que visa avaliar a taxa de proliferação celular, medida pelo antígeno Ki-67, em 2 olhos enucleados com retinoblastoma. MÉTODOS: Um paciente com retinoblastoma unilateral familiar (mãe com doença unilateral - paciente 1) e outro com retinoblastoma unilateral esporádico (paciente 2) foram submetidos à enucleação ocular sem outro tratamento prévio. A taxa de proliferação celular foi avaliada segundo índice obtido pela contagem de células marcadas com Ki-67, em 5 campos sob microscópia óptica (células marcadas/100 células). RESULTADOS: O paciente 1, com 23 meses de idade, apresentou tumor exofítico com neovascularização de íris associada; o paciente 2, de 6 anos, apresentou tumor de crescimento endofítico, com sementes vítreas importantes. Ambos os olhos enucleados apresentaram margens cirúrgicas do nervo óptico livres de neoplasia. O índice de células positivas no paciente 1 variou de 75 a 90 (Média ± DP: 79,5 ± 6,61), e no paciente 2, de 38 a 60 (Média ± DP: 46,6 ± 8,2). O retinoblastoma familiar, além de sua manifestação em idade mais precoce, apresentou 45,8 por cento mais células positivas que o retinoblastoma esporádico com o mesmo estadiamento. CONCLUSÃO: O retinoblastoma familiar, além de surgimento mais precoce, apresentou 45,8 por cento mais células em proliferação que o retinoblastoma esporádico em mesmo estádio. Essa taxa de proliferação pode explicar a menor idade de aparecimento do tumor nos casos de doença herdada.

Karyotyping in retinoblastoma--a statistical approach.

PURPOSE: Karyotype analysis in hereditary retinoblastoma is considered to be of marginal value in risk prediction due to uncertainties in the assessment of 13q14 deletions. However, it is a low cost genetic test for retinoblastoma in developing countries. In the present study, the results of karyotype analysis were refined by a statistical method to overcome limitations. METHODS: Karyotype analysis was performed by trypsin-Giemsa banding and naked eye karyotyping for 33 bilateral, 25 unilateral and one regressed retinoblastoma patients. The percentage of metaphases with 13q14 deletions in each case was plotted on a scatter diagram. Normalization of the data was achieved by log transformation and the results were statistically analyzed by one-sample 't' test using SPSS version 9.0. RESULTS: Seven samples had 13q14 deletion percentages above the cutoff value. One-sample 't' test showed significance (p< 0.001). By this method, two unilateral and five bilateral patients had 13q14 deletions, constituting 11.8 % of cases. CONCLUSION: For accuracy, statistical analysis should be considered as an adjunct in karyotyping.

Perspectivas en la genómica del retinoblastoma: Implicaciones del gen supresor de tumor RB1 / Genomic retinoblastoma perspectives: Implications of supressor gene of tumor RB1

In order to define the molecular and cellular bases of the development of retinoblastomas it is necessary to know its etiology, and to apply the advances in genome technology to this kind of neoplasia. Retinoblastomas are childhood tumors of the eye with an average incidence of one case in every 15,000-20,000 live births, which occur in sporadic and hereditary forms. The sporadic form appears regularly as a unilateral tumor, while in the familial form of the disease, tumors may be unilateral and bilateral. This neoplasia is characterized by leukocoria, strabism, and heterochromia. The retinoblastoma gene (RBl) is a molecular marker of retinoblastoma tumors. This gene is located in chromosome 13q14.2 and encodes a nuclear phosphoprotein (pRB) of 110 KDa, which plays a major role in cell proliferation control through cell cycle-regulated phosphorylation/dephosphorylation cycles of this protein. The RBl gene is mainly affected by point mutations, which occur most frequently in exons 3, 8, 18 and 20. At the end of the last century, DNA technology has improved notably, allowing for its application to the study of a vast array of diseases. The aim of this work is to show the molecular aspects involved in retinoblastoma which are currently deciphering; this is possible thanks to new technology platforms that have been developed. This will allow us in a near future, to offer tests for the early diagnoses, prognoses, and the determination of individual predisposition towards this neoplasia.

El retinoblastoma es una neoplasia embrionaria que se manifiesta en dos formas: esporádica (no heredada) o familiar (heredada). En los casos esporádicos el tumor es unilateral y en la forma familiar puede presentarse de manera unilateral o bilateral. Esta neoplasia tiene una incidencia promedio de 1/15,000 nacidos vivos, presentando signos y síntomas que incluyen leucocoria, estrabismo, midriasis unilateral y heterocromía. El gen que predispone al desarrollo de retinoblastoma es RBl y se localiza en el cromosoma 13 en la región ql4.2. El gen RBl codifica para una fosfoproteína nuclear que participa de manera importante en la regulación del ciclo celular. De acuerdo con la hipótesis de Knudson, para que se desarrolle la neoplasia se deben presentar dos mutaciones en el gen RBl. Las mutaciones puntuales son las que más frecuentemente se presentan en el gen RBl; la mayoría de los estudios indican que los exones 3, 8, 18, 19 y 20 son las regiones de mutación preferencial. En la áltima década ha habido un gran avance en la tecnología del DNA, lo cual hace posible su aplicación en diferentes enfermedades. Estas herramientas moleculares podrían ser de gran utilidad en el diagnóstico o conocimiento de la predisposición a desarrollar un retinoblastoma. Entre estas valiosas herramientas se cuenta con la hibridación fluorescente realizada in situ, hibridación genómica comparativa, las microhileras y por áltimo la identificación de polimorfismos de un sólo nucleótido. En conclusión, actualmente se están descifrando los aspectos moleculares que están relacionados con el retinoblastoma, gracias a la aplicación de nuevas plataformas tecnológicas. Esto permitirá en un futuro próximo ofrecer pruebas para un diagnóstico temprano o para conocer el pronóstico y la predisposición de individuos a desarrollar esta patología. Con el fin de entender las bases celulares y moleculares del desarrollo del retinoblastoma, el objetivo del presente trabajo es mostrar el estado del arte del conocimiento de esta neoplasia, así como su origen y los avances en la genómica aplicada al retinoblastoma.

Información sobre las secuelas y futuros riesgos para los sobrevivientes de retinoblastoma / Information on late effects and future risks for retinoblastoma survivors

Los índices de sobrevida de los pacientes con cáncer en la infancia han aumentado en las últimas décadas. Se observan secuelas como consecuencia de esta enfermedad y su tratamiento. Los sobrevivientes de retinoblastoma presentan efectos adversos como consecuencia de la cirugía, la radioterapia y la quimioterapia. Las formas bilaterales irradiadas presentan un riesgo aumentado de desarrollar un segundo cáncer. Tanto los casos que presentaron retinoblastoma unilateral o bilateral pueden transmitir esta enfermedad a su descendencia. El diagnóstico de este tumor es excepcional después de los 4 años de edad y no se sabe claramente qué conocimiento tienen estos sobrevivientes del diagnóstico, del tratamiento y de los futuros riesgos. El consentimiento informado y el posterior resumen de historia clínica no aclaran demasiado a los padres. Los pacientes curados de retinoblastoma se verían beneficiados con un seguimiento prolongado en las instituciones en que fueron tratados, ya que les permitiría recibir información acerca de lo padecido, del tratamiento, de los riesgos y se podrían identificar las consecuencias posteriores de la enfermedad y su tratamiento.

Osteosarcoma como segunda neoplasia de retinoblastoma / Osteosarcoma as a second neoplasm in a children with retinoblastoma

Introducción. Es bien conocido que el osteosarcoma se presenta frecuentemente como segunda neoplasia del retinoblastoma congénito, así como otro tipo de carcinomas, melanomas y tumores neuroepiteliales. Todos los pacientes con retinoblastoma bilateral congéntio presentan una alteración del gen RB1 localizado en el cromosoma 13q14. Caso clínico. Se presenta el caso de una paciente con retinoblastoma bilateral congénito diagnosticado a la edad de 1 año 11 meses, quien recibió tratamiento con ciclofosfamida, epirrubicina y VP 16, entre otros agentes; y que desarrolló osteosarcoma peroneo con metástasis pulmonares tras una latencia de 10 años 6 meses. En esta paciente es conocido el uso de alquilantes, antracíclicos y etopósido, así como los antecedentes familiares de cáncer por ambas ramas. Conclusión. El retinoblastoma bilateral conlleva factores de riesgo para el desarrollo de segundas neoplasias. Los antecedentes familiares constituyen razones suficientes para catalogarlo como un síndrome de cáncer familiar, el uso de agentes alquilantes, antraciclicos y etopósidos, aumentan este riesgo acortando el período de latencia

Familial cancers

Although cancer is common in the general population, only a small proportion is thought to be due to highly penetrant cancer genes. Families with a highly penetrant cancer predisposing gene may be recognised by the presence of a high number of individuals within the family with a particular cancer type, or individuals with features associated with rare inherited cancers, such as retinal changes or multiple colonic polyps. It is likely that there are many other less penetrant genes, which, in combination with environmental influences, will confer a greater lifetime predisposition to cancers. These genes are currently being identified through epidemiological studies

Detección clínica de casos con susceptibilidad genética al retinoblastoma: en focos geográficos venezolanos / Clinical detection of cases with genetic susceptibility a the retinoblastoma: in venezuelan geographics focus

El retinoblastoma en Venezuela tiene un origen exclusivamente hereditario, con aparición de casos esporádicos por ausencia de antecedentes familiares en la mayoría de los casos. La agregación geográfica en el retinoblastoma para nuestro estudio no puede explicarse por agregación de neomutaciones, por lo tanto apoya la hipótesis de un gen ancestral

Retinoblastoma cromossômico / Cromossomal abnormalities and retinoblastoma

Os autores descrevem três casos de retinoblastoma associados a retardo neuro-psico-motor pesquisados quanto à análise cariotípica com detecçäo da deleçäo parcial do braço longo do cromossomo 13 já descrita como característica dessa condiçäo. As características clínicas dos pacientes e da condiçäo retinoblastoma com alteraçäo cromossômica säo relatadas e enfatizadas na discussäo, ressaltando-se os cuidados especiais quanto à rotina de exame, a modalidades terapêutica a serem empregadas ou evitadas e à necessidade de exame genético dos pais para orientaçäo familiar adequada

Oncologia ocular / Ocular tumors

O carcinoma da conjuntiva, o melanoma de coróide e o retinoblastoma säo neoplasias familiares ao oftalmologista. O primeiro é de fácil detecçäo e tratamento. Em compensaçäo, o segundo é de um diagnóstico mais difícil, e o seu tratamento tem sido um enervante quebra-cabeça para os especialistas, permanecendo controverso até os dias de hoje. O retinoblastoma é encontrado na criança, trazendo a possibilidade de acometimento bilateral. É uniformemente maligno, e o seu tratamento, que se centraliza na Radioterapia, depende de um estadiamento criterioso.

Retinoblastoma familial associado a carcinoma primário de plexo coróide. Relato de caso / Familial retinoblastoma associated to primary choroid plexus carcinoma. Report of a case

Retinoblastoma éum tumor originado de células neuropiteliais da retina, que pode ocorrer de forma esporádica ou familial e está relacionado com o gene RB-1, do braço longo do cromossomo 13 (regiäo 13q14). Este gene é um supressor tumoral e quando sofre deleçäo promove crescimento celular. O retinoblastoma relaciona-se com a mutaçäo dos dois alelos RB-1: quando o indivíduo sofre duas mutaçöes durante o desenvolvimento somático da retina, ele tem a forma esporádica ou näo hereditária da doença; quando uma das mutaçöes é herdada de células germinativas, ele tem a familial ou hereditária. Apresentamos um caso de retinoblastoma bilateral, familial, associado com carcinoma primário de plexo coróide. Após revisäo, näo encontramos associaçäo semelhante na literatura.