RESUMEN
BACKGROUND: Resistance to osimertinib in advanced EGFR-mutated non-small cell lung cancer (NSCLC) constitutes a significant challenge for clinicians either in terms of molecular diagnosis and subsequent therapeutic implications. METHODS: This is a prospective single-centre study with the primary objective of characterising resistance mechanisms to osimertinib in advanced EGFR-mutated NSCLC patients treated both in first- and in second-line. Next-Generation Sequencing analysis was conducted on paired tissue biopsies and plasma samples. A concordance analysis between tissue and plasma was performed. RESULTS: Sixty-five advanced EGFR-mutated NSCLC patients treated with osimertinib in first- (n = 56) or in second-line (n = 9) were included. We managed to perform tissue and liquid biopsies in 65.5% and 89.7% of patients who experienced osimertinib progression, respectively. Acquired resistance mechanisms were identified in 80% of 25 patients with post-progression samples, with MET amplification (n = 8), EGFR C797S (n = 3), and SCLC transformation (n = 2) the most frequently identified. The mean concordance rates between tissue and plasma for the EGFR activating mutation and for the molecular resistance mechanisms were 87.5% and 22.7%, respectively. CONCLUSIONS: Resistance to osimertinib demonstrated to be highly heterogeneous, with MET amplification the main mechanism. Plasma genotyping is a relevant complementary tool which might integrate tissue analysis for the study of resistance mechanisms.
Asunto(s)
Acrilamidas , Carcinoma de Pulmón de Células no Pequeñas , Indoles , Neoplasias Pulmonares , Pirimidinas , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudios Prospectivos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Receptores ErbB/genética , Genotipo , Mutación , Resistencia a Antineoplásicos/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Compuestos de Anilina/uso terapéutico , Biopsia LíquidaRESUMEN
The most peculiar transcriptional property of eukaryotic tRNA genes, as well as of other genes served by RNA polymerase III, is their complete dependence on the intragenic interaction platform provided by transcription factor IIIC (TFIIIC) for the productive assembly of the TBP-containing initiation factor TFIIIB. The sole exception, in yeast, is the U6 RNA gene, which is able to exploit a TATAAATA element, 30 bp upstream of the transcription start site, for the TFIIIC-independent assembly of TFIIIB. To find out whether this extragenic core promoter organization and autonomous TFIIIB assembly capacity are unique features of the U6 gene or also apply to other genes transcribed by RNA polymerase III, we scanned the 5'-flanking regions (up to position -100) of the entire tRNA gene set of Saccharomyces cerevisiae searching for U6-like TATA motifs. Four tRNA genes harboring such a sequence motif around position -30 were identified and found to be transcribed in vitro by a minimal system only composed of TFIIIB and RNA polymerase III. In this system, start site selection is not at all affected by the absence of TFIIIC, which, when added, significantly stimulates transcription by determining an increase in the number, rather than in the efficiency of utilization, of productive initiation complexes. A specific TBP-TATA element interaction is absolutely required for TFIIIC-independent transcription, but the nearby sequence context also contributes to the efficiency of autonomous TFIIIB assembly. The existence of a TFIIIB assembly pathway leading to the faithful transcription of natural eukaryotic tRNA genes in the absence of TFIIIC provides novel insights into the functional flexibility of the eukaryotic tRNA gene transcription machinery and on its evolution from an ancestral RNA polymerase III system relying on upstream, TATA- centered control elements.
Asunto(s)
Genes Fúngicos/genética , ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción TFIII/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Secuencia de Bases , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Regulación Fúngica de la Expresión Génica/genética , Frecuencia de los Genes/genética , Genes de Plantas/genética , Cinética , Datos de Secuencia Molecular , Mutación/genética , ARN Polimerasa III/metabolismo , ARN de Hongos/análisis , ARN de Hongos/biosíntesis , ARN de Hongos/genética , ARN Nuclear Pequeño/genética , ARN de Transferencia/análisis , ARN de Transferencia/biosíntesis , Saccharomyces cerevisiae/enzimología , TATA Box/genética , Proteína de Unión a TATA-Box , Moldes Genéticos , Factor de Transcripción TFIIIBRESUMEN
The Ras-association domain family 1A (RASSF1A) tumour suppressor gene is inactivated in a variety of solid tumours, usually by epigenetic silencing of the promoter and/or allelic loss of its locus at 3p21.3. RASSF1A induces cell cycle arrest through inhibition of cyclin D1 accumulation. In this work, 62 endocrine tumours from different sites in the gut were investigated for methylation of the RASSF1A promoter using the polymerase chain reaction, the presence of 3p21.3 deletions by loss of heterozygosity analysis, and cyclin D1 expression by immunohistochemistry. Methylation was found in 20/62 (32%) cases and was restricted to foregut tumours; deletion at 3p21.3 was found in 15/58 (26%) informative cases and restricted to malignant foregut tumours; cyclin D1 hyper-expression was found in 31/58 (53%) cases and correlated with RASSF1A methylation. Our data suggest that RASSF1A is involved in the development of endocrine tumours derived from the foregut only, and that the presence of both RASSF1A methylation and 3p21.3 deletion is associated with malignancy. These results may provide a rationale for foregut-targeted therapy for aggressive endocrine carcinomas entailing the use of demethylating agents.
Asunto(s)
Carcinoma Neuroendocrino/genética , Neoplasias Gastrointestinales/genética , Pérdida de Heterocigocidad/genética , Proteínas Supresoras de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Apéndice/genética , Neoplasias del Apéndice/metabolismo , Carcinoma Neuroendocrino/metabolismo , Ciclina D1/análisis , Ciclina D1/genética , Neoplasias Duodenales/genética , Neoplasias Duodenales/metabolismo , Femenino , Neoplasias Gastrointestinales/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias del Íleon/genética , Neoplasias del Íleon/metabolismo , Neoplasias Intestinales/genética , Neoplasias Intestinales/metabolismo , Masculino , Metilación , Persona de Mediana Edad , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Regiones Promotoras Genéticas/genética , Neoplasias del Recto/genética , Neoplasias del Recto/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Structural studies of eukaryotic ribosomes are complicated by the tendency of their constituent proteins to be expressed at very low levels in Escherichia coli. We find that this is mainly due to their exceptionally high content of AGA/AGG arginine codons, which are poorly utilized by the bacterial translational machinery. In fact, we could overcome this limitation by the combined use of a T7 RNA polymerase expression vector and a plasmid carrying the E. coli gene argU, which encodes the minor tRNA(Arg) species that reads AGA/AGG codons. In this system, five cytoplasmic ribosomal proteins from three different eukaryotic lineages (Saccharomyces cerevisiae S8, L13, and L14; Arabidopsis thaliana L13; and Homo sapiens L7) could be overexpressed to up to 50% of total bacterial protein and were purified to homogeneity in tens of milligrams amounts. The purification procedure simply involved metal affinity chromatography followed, in some cases, by an additional heparin chromatography step. Recombinant polypeptides bound RNA with high affinity (K(d) between 50 and 300 nM). This novel overexpression/purification strategy will allow the production of high amounts of most eukaryotic ribosomal proteins in a form suitable for structural and functional studies. Coupled with recently completed and ongoing whole-genome sequencing projects, it will facilitate the molecular characterization of the eukaryotic ribosome.