RESUMO
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.
Assuntos
Acrilamidas , Carcinoma Pulmonar de Células não Pequenas , Indóis , Neoplasias Pulmonares , Pirimidinas , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos Prospectivos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Receptores ErbB/genética , Genótipo , Mutação , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Compostos de Anilina/uso terapêutico , Biópsia LíquidaRESUMO
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.
Assuntos
Carcinoma Neuroendócrino/genética , Neoplasias Gastrointestinais/genética , Perda de Heterozigosidade/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Apêndice/genética , Neoplasias do Apêndice/metabolismo , Carcinoma Neuroendócrino/metabolismo , Ciclina D1/análise , Ciclina D1/genética , Neoplasias Duodenais/genética , Neoplasias Duodenais/metabolismo , Feminino , Neoplasias Gastrointestinais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias do Íleo/genética , Neoplasias do Íleo/metabolismo , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Masculino , Metilação , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Regiões Promotoras Genéticas/genética , Neoplasias Retais/genética , Neoplasias Retais/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
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.
Assuntos
Genes Fúngicos/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição TFIII/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Sequência de Bases , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Regulação Fúngica da Expressão Gênica/genética , Frequência do Gene/genética , Genes de Plantas/genética , Cinética , Dados de Sequência Molecular , Mutação/genética , RNA Polimerase III/metabolismo , RNA Fúngico/análise , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Nuclear Pequeno/genética , RNA de Transferência/análise , RNA de Transferência/biossíntese , Saccharomyces cerevisiae/enzimologia , TATA Box/genética , Proteína de Ligação a TATA-Box , Moldes Genéticos , Fator de Transcrição TFIIIBRESUMO
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.