RESUMEN
OBJECTIVES: To report the benefits of genetic diagnosis in patients with retinoblastoma. METHOD: Observational study. Patients with retinoblastoma and their families were included. Demographic and clinical data were recorded. Blood and tumour samples were obtained. Next generation sequencing was performed on the samples. When deletion 13 q syndrome was suspected, cytogenetics microarray was performed (Cytoscan® HD, Affymetrix, Santa Clara, CA, USA), with a high density chip of 1.9 million of non-polymorphic probes and 750 thousand SNP probes. RESULTS: Of the 7 cases were analysed 4 were male. The mean age at diagnosis was 21 months (range 5-36). Three cases had bilateral retinoblastoma, and 4 unilateral. None had family history. In all patients, blood was analysed, and a study was performed on the tissue from 2 unilateral enucleated tumours, in which 6 mutations were identified, all de novo. Just one was novel (c.164delC; case 1). One case of unilateral tumour revealed blood mosaicism, showing that his condition was inheritable, and that there is a high risk of developing retinoblastoma in the unaffected eye. The patient also has an increased risk of presenting with other primary tumours. CONCLUSION: Molecular diagnosis of RB1 in patients with retinoblastoma impacts on the decision process, costs, treatment, and prognosis of patients, as well as their families.
Asunto(s)
ADN de Neoplasias/genética , Neoplasias del Ojo/genética , Genes de Retinoblastoma , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Unión a Retinoblastoma/genética , Retinoblastoma/genética , Ubiquitina-Proteína Ligasas/genética , Preescolar , Chile , Deleción Cromosómica , Trastornos de los Cromosomas/diagnóstico , Trastornos de los Cromosomas/genética , Cromosomas Humanos Par 13/genética , Análisis Mutacional de ADN , ADN de Neoplasias/sangre , ADN de Neoplasias/aislamiento & purificación , Neoplasias del Ojo/sangre , Neoplasias del Ojo/química , Neoplasias del Ojo/diagnóstico , Femenino , Humanos , Lactante , Masculino , Mosaicismo , Mutación , Neoplasias Primarias Múltiples/sangre , Neoplasias Primarias Múltiples/química , Neoplasias Primarias Múltiples/diagnóstico , Neoplasias Primarias Múltiples/genética , Polimorfismo de Nucleótido Simple , Retinoblastoma/sangre , Retinoblastoma/química , Retinoblastoma/diagnóstico , Análisis de Secuencia de ADN/métodosRESUMEN
INTRODUCTION: The identification of molecules expressed selectively on the surface of retinoblastoma cells would allow applying targeted therapies. The Ganglioside, N-Glycolyl-GM3 (NeuGc-GM3), is an attractive candidate, as it has been detected in other paediatric neuroectodermic tumours, and it is not expressed in human normal tissues. The 14F7 antibody recognizes specifically the ganglioside NeuGc-GM3. PURPOSE: To characterize the expression of NeuGc-GM3 in retinoblastoma cell lines and in retinoblastoma tumours using the 14F7 monoclonal antibody. METHODS: We studied WERI-Rb1 and Y79 cell lines, 24 retinoblastoma primary tumours from unilateral and bilateral cases and two bone marrow biopsies from metastatic retinoblastoma. Tumours were classified into three groups: non-invasive (n = 13), invasive (n = 9) and metastatic (n = 2). Three eyes enucleated because of non-tumoural conditions were used as controls. Cell lines and tumour sections were studied by immunohistochemistry using the 14F7 antibody. NeuGc-GM3 expression was evaluated by analysing the percentage of positive tumoural cells and the staining intensity. These parameters were analysed comparatively among the three groups. RESULTS: Both retinoblastoma cell lines showed immunoreactivity to NeuGc-GM3 but WERI-Rb1 presented higher intensity than Y79. All the tumours studied showed strong immunoreactivity to NeuGc-GM3 with no significant differences among groups. In both bone marrow specimens, NeuGc-GM3 immunoreactivity was observed in retinoblastoma cells. In bilaterally enucleated cases, NeuGc-GM3 immunoreactivity was not altered before and after chemotherapy. Non-tumoural retinas were negative. CONCLUSIONS: NeuGc-GM3 is highly expressed in retinoblastoma cell lines, tumours and metastatic cells to the bone marrow, and it is not detectable in control eyes. There were no significant differences in the immunoreactivity to 14F7 among tumours from different disease stages. Its immunoreactivity did not change after chemotherapy.
Asunto(s)
Autoantígenos/análisis , Gangliósido G(M3)/análogos & derivados , Neoplasias de la Retina/química , Retinoblastoma/química , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Gangliósido G(M3)/análisis , Gangliósido G(M3)/inmunología , Humanos , Técnicas para InmunoenzimasRESUMEN
Constitutional RB1 gene mutations were studied in a series of 21 families with unilateral and bilateral retinoblastoma patients. Peripheral blood lymphocytes were analyzed by "exon by exon" PCR-heteroduplex and sequencing. Mutations were identified in 6 (29%) of the patients. One mutation corresponded to an intronic polymorphism in g.174351T > A. The other five mutations resulted C to T exonic transitions, four were CGA sequences (g.65386, g.150037 in two patients, and g.162237), creating stop codons and presumably truncated proteins. The fifth one was new and resulted in alanine to valine substitution (g.73774). Two patients had the same the germline truncated mutation (g.150037C > T), one with a familial bilateral early onset retinoblastoma and one with a sporadic unilateral late onset retinoblastoma. The later type has not been previously described. This finding is discussed in the genotype/phenotype correlation context. Additionally, a single nucleotide change was found in six studied samples, where a C to T homozygous transversion was identified in intron 26 (IVS26 + 28). It is worthy the non concordance of the nucleotide with the published sequence. This analysis proved to be a useful method for the detection of mutations in the RB1 gene, and contributed to the adequate genetic counseling to patients and relatives.