RESUMEN
Context: Interpretation of calcitonin measurement to predict the prognosis of medullary thyroid carcinoma (MTC) requires multiple measurements over an extended time period, making it an imperfect biomarker for evaluating prognosis or disease behavior. Single circulating cell-free DNA (cfDNA) values have been shown to be a valuable prognostic marker for several solid tumors. Objective: We tested the hypothesis that cfDNA containing the RET M918T mutation could be detected in the blood of patients with advanced MTC whose tumor harbored an M918T mutation and would be able to predict overall survival more reliably than calcitonin. Design: The level of cfDNA containing RET M918T mutation was measured in the plasma of patients with MTC via droplet digital polymerase chain reaction. Patients: Patients had a confirmed sporadic MTC diagnosis, a serum calcitonin measurement >100 pg/mL, and tumor tissue biopsy results providing RET M918T mutation status. There were 75 patients included in this study, 50 of whom harbored an RET M918T mutation by tissue biopsy. Results: RET M918T cfDNA was detected in 16 of 50 patients (32%) with a positive tissue biopsy. The detection of RET M918T cfDNA strongly correlated with worse overall survival and more accurately predicted a worse outcome than calcitonin doubling time. Conclusions: Liquid biopsy is able to detect RET M918T mutations in patient plasma with high specificity but low sensitivity. In patients with established somatic RET M918T mutations, the allelic fraction of circulating tumor DNA is prognostic for overall survival and may play a role in monitoring response to treatment.
Asunto(s)
Carcinoma Neuroendocrino/genética , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias de la Tiroides/genética , Adulto , Anciano , Biopsia con Aguja , Carcinoma Neuroendocrino/mortalidad , Carcinoma Neuroendocrino/patología , Estudios de Cohortes , Análisis Mutacional de ADN , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/patología , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa/métodos , Valor Predictivo de las Pruebas , Pronóstico , Medición de Riesgo , Análisis de Supervivencia , Neoplasias de la Tiroides/mortalidad , Neoplasias de la Tiroides/patologíaRESUMEN
Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Marcación de Gen , Células Madre Pluripotentes Inducidas/metabolismo , Alelos , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Endonucleasas/genética , Endonucleasas/metabolismo , Expresión Génica , Marcación de Gen/métodos , Vectores Genéticos/genética , Genotipo , Recombinación Homóloga , Humanos , Células Madre Pluripotentes Inducidas/citología , Mutación , Reparación del ADN por Recombinación , Análisis de Secuencia de ADN , Dedos de Zinc/genéticaRESUMEN
The reasons underlying the occurrence of multiple revertant genotypes in Wiskott-Aldrich syndrome (WAS) patients remain unclear. We have identified more than 30 revertant genotypes in a C995T WAS patient having 10-15% revertant, WAS protein (WASp)-expressing circulating lymphocytes. Of 497 allospecific T-cell clones generated from the peripheral blood, 47.1% carried a revertant sequence. All revertant T-cell clones exhibited restoration of WASp expression. However, anti-CD3-induced proliferative responses varied greatly amongst revertants. Several revertant T-cell clones expressed an internally deleted WASp mutant lacking much of the proline-rich region. This potentially accounts for the reduced anti-CD3 proliferative responses of these T-cell clones. We found no evidence for an increased DNA mutation rate in this patient. We conclude that the diversity of revertant genotypes in our patient does not result from an extraordinary mutation rate and that the amino acid sequence space explored by WASp in revertant T-cells is significantly smaller than might have been predicted from the diversity of revertant genotypes.