RESUMEN
BACKGROUND: Circulating cell-free tumor DNA (ctDNA)-based mutation profiling, if sufficiently sensitive and comprehensive, can efficiently identify genomic targets in advanced lung adenocarcinoma. Therefore, the authors investigated the accuracy and clinical utility of a commercially available digital next-generation sequencing platform in a large series of patients with non-small cell lung cancer (NSCLC). METHODS: Plasma-based comprehensive genomic profiling results from 8388 consecutively tested patients with advanced NSCLC were analyzed. Driver and resistance mutations were examined with regard to their distribution, frequency, co-occurrence, and mutual exclusivity. RESULTS: Somatic alterations were detected in 86% of samples. The median variant allele fraction was 0.43% (range, 0.03%-97.62%). Activating alterations in actionable oncogenes were identified in 48% of patients, including EGFR (26.4%), MET (6.1%), and BRAF (2.8%) alterations and fusions (ALK, RET, and ROS1) in 2.3%. Treatment-induced resistance mutations were common in this cohort, including driver-dependent and driver-independent alterations. In the subset of patients who had progressive disease during EGFR therapy, 64% had known or putative resistance alterations detected in plasma. Subset analysis revealed that ctDNA increased the identification of driver mutations by 65% over standard-of-care, tissue-based testing at diagnosis. A pooled data analysis on this plasma-based assay demonstrated that targeted therapy response rates were equivalent to those reported from tissue analysis. CONCLUSIONS: Comprehensive ctDNA analysis detected the presence of therapeutically targetable driver and resistance mutations at the frequencies and distributions predicted for the study population. These findings add support for comprehensive ctDNA testing in patients who are incompletely tested at the time of diagnosis and as a primary option at the time of progression on targeted therapies.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN Tumoral Circulante/genética , Neoplasias Pulmonares/genética , Mutación , Adenocarcinoma del Pulmón/sangre , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Alelos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , ADN Tumoral Circulante/sangre , Estudios de Cohortes , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Oncogenes , Inhibidores de Proteínas Quinasas/uso terapéutico , Análisis de Secuencia de ADN , Transducción de Señal/genética , Resultado del TratamientoRESUMEN
A comprehensive understanding of RNA structure will provide fundamental insights into the cellular function of both coding and non-coding RNAs. Although many RNA structures have been analysed by traditional biophysical and biochemical methods, the low-throughput nature of these approaches has prevented investigation of the vast majority of cellular transcripts. Triggered by advances in sequencing technology, genome-wide approaches for probing the transcriptome are beginning to reveal how RNA structure affects each step of protein expression and RNA stability. In this Review, we discuss the emerging relationships between RNA structure and the regulation of gene expression.
Asunto(s)
Estudio de Asociación del Genoma Completo , Genoma , ARN Mensajero/genética , Transcriptoma , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The hepatitis C viral RNA genome forms a complex with liver-specific microRNA (miR-122) at the extreme 5' end of the viral RNA. This complex is essential to stabilize the viral RNA in infected cultured cells and in the liver of humans. The abundances of primary and precursor forms of miR-122, but not the abundance of mature miR-122, are regulated in a circadian rhythm in the liver of animals, suggesting a possible independent function of precursor molecules of miR-122 in regulating viral gene expression. Modified precursor molecules of miR-122 were synthesized that were refractory to cleavage by Dicer. These molecules were found to enhance the abundance of HCV RNA. Furthermore, they diminished the expression of mRNAs that contained binding sites for miR-122 in their 3' noncoding regions. By use of duplex and precursor miR-122 mimetic molecules that carried mutations in the passenger strand of miR-122, the effects on viral and reporter gene expression could be pinpointed to the action of precursor miR-122 molecules. Targeting the circadian expression of precursor miR-122 by specific compounds likely provides novel therapeutic strategies.
Asunto(s)
Hepacivirus/genética , MicroARNs/genética , Interferencia de ARN , Precursores del ARN/genética , ARN Viral/metabolismo , Animales , Proteínas Argonautas/metabolismo , Secuencia de Bases , Ritmo Circadiano , Fibroblastos/metabolismo , Fibroblastos/virología , Expresión Génica , Células HeLa , Humanos , Secuencias Invertidas Repetidas , Ratones , MicroARNs/metabolismo , Precursores del ARN/metabolismo , Estabilidad del ARN , ARN Viral/genéticaRESUMEN
MicroRNAs (miRNAs) typically downregulate protein expression from target mRNAs through limited base-pairing interactions between the 5' 'seed' region of the miRNA and the mRNA 3' untranslated region (3'UTR). In contrast to this established mode of action, the liver-specific human miR-122 binds at two sites within the hepatitis C viral (HCV) 5'UTR, leading to increased production of infectious virions. We show here that two copies of miR-122 interact with the HCV 5'UTR at partially overlapping positions near the 5' end of the viral transcript to form a stable ternary complex. Both miR-122 binding sites involve extensive base pairing outside of the seed sequence; yet, they have substantially different interaction affinities. Structural probing reveals changes in the architecture of the HCV 5'UTR that occur on interaction with miR-122. In contrast to previous reports, however, results using both the recombinant cytoplasmic exonuclease Xrn1 and liver cell extracts show that miR-122-mediated protection of the HCV RNA from degradation does not correlate with stimulation of viral propagation in vivo. Thus, the miR-122:HCV ternary complex likely functions at other steps critical to the viral life cycle.
Asunto(s)
Regiones no Traducidas 5' , Hepacivirus/genética , MicroARNs/metabolismo , ARN Viral/química , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Exorribonucleasas/metabolismo , Genoma Viral , Humanos , Datos de Secuencia Molecular , ARN Viral/metabolismoRESUMEN
The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome.
Asunto(s)
Factor 3 de Iniciación Eucariótica/metabolismo , Hepacivirus/metabolismo , Biosíntesis de Proteínas , ARN Viral/metabolismo , Sitios de Unión , Codón Iniciador/genética , Codón Iniciador/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Secuencias Hélice-Asa-Hélice , Hepacivirus/genética , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , ARN Viral/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Conformación de Ácido Nucleico , ARN/química , ARN/genética , Análisis de Secuencia de ARN/métodos , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Secuencia de Bases , Biología Computacional , Código de Barras del ADN Taxonómico , Cartilla de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Modelos Moleculares , Sondas Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación Puntual , ARN Catalítico/química , ARN Catalítico/genética , Ribonucleasa P/química , Ribonucleasa P/genética , Análisis de Secuencia de ARN/estadística & datos numéricosRESUMEN
Sequence census methods reduce molecular measurements such as transcript abundance and protein-nucleic acid interactions to counting problems via DNA sequencing. We focus on a novel assay utilizing this approach, called selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), that can be used to characterize RNA secondary and tertiary structure. We describe a fully automated data analysis pipeline for SHAPE-Seq analysis that includes read processing, mapping, and structural inference based on a model of the experiment. Our methods rely on the solution of a series of convex optimization problems for which we develop efficient and effective numerical algorithms. Our results can be easily extended to other chemical probes of RNA structure, and also generalized to modeling polymerase drop-off in other sequence census-based experiments.
Asunto(s)
Conformación de Ácido Nucleico , ARN/química , ARN/genética , Análisis de Secuencia de ARN/métodos , Algoritmos , Automatización , Biología Computacional , Funciones de Verosimilitud , Modelos Moleculares , Plásmidos/química , Plásmidos/genética , ARN Bacteriano/química , ARN Bacteriano/genética , Análisis de Secuencia de ARN/estadística & datos numéricos , Staphylococcus aureus/química , Staphylococcus aureus/genéticaRESUMEN
A striking and widespread observation is that higher-order folding for many RNAs is very slow, often requiring minutes. In some cases, slow folding reflects the need to disrupt stable, but incorrect, interactions. However, a molecular explanation for slow folding in most RNAs is unknown. The specificity domain of the Bacillus subtilis RNase P ribozyme undergoes a rate-limiting folding step on the minute time-scale. This RNA also contains a C2'-endo nucleotide at A130 that exhibits extremely slow local conformational dynamics. This nucleotide is evolutionarily conserved and essential for tRNA recognition by RNase P. Here we show that deleting this single nucleotide accelerates folding by an order of magnitude even though this mutation does not change the global fold of the RNA. These results demonstrate that formation of a single stacking interaction at a C2'-endo nucleotide comprises the rate-determining step for folding an entire 154 nucleotide RNA. C2'-endo nucleotides exhibit slow local dynamics in structures spanning isolated helices to complex tertiary interactions. Because the motif is both simple and ubiquitous, C2'-endo nucleotides may function as molecular timers in many RNA folding and ligand recognition reactions.
Asunto(s)
Conformación de Ácido Nucleico , ARN Bacteriano/química , Acilación , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Secuencia de Bases , Indicadores y Reactivos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Catalítico/química , ARN Catalítico/genética , ARN Catalítico/metabolismo , Ribonucleasa P/química , Ribonucleasa P/genética , Ribonucleasa P/metabolismo , Eliminación de SecuenciaRESUMEN
Hydroxyl-selective electrophiles, including N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7), are broadly useful for RNA structure analysis because they react preferentially with the ribose 2'-OH group at conformationally unconstrained or flexible nucleotides. Each nucleotide in an RNA has the potential to form an adduct with these reagents to yield a comprehensive, nucleotide-resolution, view of RNA structure. However, it is possible that factors other than local structure modulate reactivity. To evaluate the influence of base identity on the intrinsic reactivity of each nucleotide, we analyze NMIA and 1M7 reactivity using four distinct RNAs, under both native and denaturing conditions. We show that guanosine and adenosine residues have identical intrinsic 2'-hydroxyl reactivities at pH 8.0 and are 1.4 and 1.7 times more reactive than uridine and cytidine, respectively. These subtle, but statistically significant, differences do not impact the ability of selective 2'-hydroxyl acylation analyzed by primer extension-based (SHAPE) methods to establish an RNA secondary structure or monitor RNA folding in solution because base-specific influences are much smaller than the reactivity differences between paired and unpaired nucleotides.
Asunto(s)
Anhídridos/química , Radical Hidroxilo/química , ARN/química , Ribosa/química , ortoaminobenzoatos/química , Acilación , VIH-1/genética , Conformación de Ácido Nucleico , ARN/genética , ARN/metabolismo , ARN Ribosómico/genética , Ribonucleasa P/genéticaRESUMEN
By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 104 cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling. For complete information on the use and execution of this protocol, please refer to Mair et al. (2020).
Asunto(s)
Perfilación de la Expresión Génica/métodos , Proteínas/análisis , Análisis de la Célula Individual/métodos , Anticuerpos/inmunología , Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas/genética , Proteínas/inmunología , Proteómica/métodos , Análisis de Secuencia de ARN/métodos , Transcriptoma/genéticaRESUMEN
An important unmet experimental objective is to analyze local RNA structure in a way that is strictly governed by solvent accessibility. Essentially all chemical probes currently used to evaluate RNA (and DNA) structure via formation of stable covalent adducts employ carbon-based electrophiles, which undergo nucleophilic attack from limited spatial orientations and via highly polar transition states. Reaction by these classical electrophiles is therefore gated by both solvent accessibility and additional electrostatic factors. In contrast, silicon electrophiles react via their d-orbitals and consequently can undergo nucleophilic attack from many spatial orientations. In this work, we explore the use of silanes to react indiscriminately with RNA such that the primary factor governing reactivity is solvent accessibility. We show that N,N-(dimethylamino)dimethylchlorosilane (DMAS-Cl) reacts at the guanosine N2 position to yield a near-perfect measure (r >or= 0.82) of solvent accessibility in an RNA with a complex tertiary structure. This silane-based chemistry represents a direct and quantitative approach for probing solvent accessibility at the base pairing face of guanosine in RNA.
Asunto(s)
Guanosina/química , ARN/química , Silanos/química , Solventes/química , Aldehídos/química , Antiportadores/genética , Aptámeros de Nucleótidos/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Butanonas , Nucleótidos de Desoxiguanina/química , Inosina/química , Magnesio/química , Modelos Químicos , Estructura Molecular , Conformación de Ácido Nucleico , Secuencias Reguladoras de Ácido Ribonucleico/genéticaRESUMEN
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry yields quantitative RNA secondary and tertiary structure information at single nucleotide resolution. SHAPE takes advantage of the discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl group is modulated by local nucleotide flexibility in the RNA backbone. Flexible nucleotides are reactive toward hydroxyl-selective electrophiles, whereas constrained nucleotides are unreactive. Initial versions of SHAPE chemistry, which employ isatoic anhydride derivatives that react on the minute time scale, are emerging as the ideal technology for monitoring equilibrium structures of RNA in a wide variety of biological environments. Here, we extend SHAPE chemistry to a benzoyl cyanide scaffold to make possible facile time-resolved kinetic studies of RNA in approximately 1 s snapshots. We then use SHAPE chemistry to follow the time-dependent folding of an RNase P specificity domain RNA. Tertiary interactions form in two distinct steps with local tertiary contacts forming an order of magnitude faster than long-range interactions. Rate-determining tertiary folding requires minutes despite that no non-native interactions must be disrupted to form the native structure. Instead, overall folding is limited by simultaneous formation of interactions approximately 55 A distant in the RNA. Time-resolved SHAPE holds broad potential for understanding structural biogenesis and the conformational interconversions essential to the functions of complex RNA molecules at single nucleotide resolution.
Asunto(s)
ARN/química , Hidrólisis , Datos de Secuencia Molecular , Estructura Molecular , ARN/metabolismo , Ribonucleasa P/metabolismo , Especificidad por Sustrato , Factores de TiempoRESUMEN
RNA molecules undergo local conformational dynamics on timescales spanning picoseconds to minutes. Slower local motions have the greater potential to govern RNA folding, ligand recognition, and ribonucleoprotein assembly reactions but are difficult to detect in large RNAs with complex structures. RNA SHAPE chemistry employs acylation of the ribose 2'-hydroxyl position to measure local nucleotide flexibility in RNA and is well-characterized by a mechanism in which each nucleotide samples unreactive (closed) and reactive (open) states. We monitor RNA conformational dynamics over distinct time domains by varying the electrophilicity of the acylating reagent. Select C2'-endo nucleotides are nonreactive toward fast reagents but reactive toward slower SHAPE reagents in both model RNAs and in a large RNA with a tertiary fold. We conclude, first, that the C2'-endo conformation by itself does not govern SHAPE reactivity. However, some C2'-endo nucleotides undergo extraordinarily slow conformational changes, on the order of 10(-4) s(-1). Due to their distinctive local dynamics, C2'-endo nucleotides have the potential to function as rate-determining molecular switches and are likely to play central, currently unexplored, roles in RNA folding and function.
Asunto(s)
Conformación de Ácido Nucleico , Nucleótidos/química , ARN/química , Acilación , Secuencia de Bases , Hidrólisis , Cinética , Datos de Secuencia Molecular , TermodinámicaRESUMEN
Purpose: To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility.Experimental Design: Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples.Results: Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%-100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non-small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker.Conclusions: High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery. Clin Cancer Res; 24(15); 3539-49. ©2018 AACR.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , ADN Tumoral Circulante/genética , Genómica , Neoplasias/genética , Biomarcadores de Tumor , Ácidos Nucleicos Libres de Células/sangre , ADN Tumoral Circulante/sangre , Femenino , Genotipo , Técnicas de Genotipaje , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mutación , Neoplasias/sangre , Neoplasias/patologíaRESUMEN
Purpose: Cell-free DNA (cfDNA) sequencing provides a noninvasive method for obtaining actionable genomic information to guide personalized cancer treatment, but the presence of multiple alterations in circulation related to treatment and tumor heterogeneity complicate the interpretation of the observed variants.Experimental Design: We describe the somatic mutation landscape of 70 cancer genes from cfDNA deep-sequencing analysis of 21,807 patients with treated, late-stage cancers across >50 cancer types. To facilitate interpretation of the genomic complexity of circulating tumor DNA in advanced, treated cancer patients, we developed methods to identify cfDNA copy-number driver alterations and cfDNA clonality.Results: Patterns and prevalence of cfDNA alterations in major driver genes for non-small cell lung, breast, and colorectal cancer largely recapitulated those from tumor tissue sequencing compendia (The Cancer Genome Atlas and COSMIC; r = 0.90-0.99), with the principal differences in alteration prevalence being due to patient treatment. This highly sensitive cfDNA sequencing assay revealed numerous subclonal tumor-derived alterations, expected as a result of clonal evolution, but leading to an apparent departure from mutual exclusivity in treatment-naïve tumors. Upon applying novel cfDNA clonality and copy-number driver identification methods, robust mutual exclusivity was observed among predicted truncal driver cfDNA alterations (FDR = 5 × 10-7 for EGFR and ERBB2), in effect distinguishing tumor-initiating alterations from secondary alterations. Treatment-associated resistance, including both novel alterations and parallel evolution, was common in the cfDNA cohort and was enriched in patients with targetable driver alterations (>18.6% patients).Conclusions: Together, these retrospective analyses of a large cfDNA sequencing data set reveal subclonal structures and emerging resistance in advanced solid tumors. Clin Cancer Res; 24(15); 3528-38. ©2018 AACR.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , ADN Tumoral Circulante/genética , Evolución Clonal/genética , Neoplasias/genética , Biomarcadores de Tumor/genética , Ácidos Nucleicos Libres de Células/sangre , ADN Tumoral Circulante/sangre , Variaciones en el Número de Copia de ADN/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Femenino , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mutación , Neoplasias/sangre , Neoplasias/patologíaRESUMEN
Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.
Asunto(s)
ADN de Neoplasias/biosíntesis , ADN de Neoplasias/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/sangre , Neoplasias/genética , Femenino , Humanos , MasculinoRESUMEN
Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.
Asunto(s)
Análisis Mutacional de ADN/métodos , ADN de Neoplasias/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Neoplasias/genética , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cetuximab/administración & dosificación , Fosfatidilinositol 3-Quinasa Clase I , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/química , Femenino , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/genética , Estudios Prospectivos , Proteínas Proto-Oncogénicas p21(ras)/genética , Reproducibilidad de los ResultadosRESUMEN
Knowledge of RNA structure is critical to understanding both the important functional roles of RNA in biology and the engineering of RNA to control biological systems. This article contains a protocol for selective 2'-hydroxyl acylation analyzed by primer extension and sequencing (SHAPE-Seq) that, through a combination of structure-dependent chemical probing and next-generation sequencing technologies, achieves structural characterization of hundreds of RNAs in a single experiment. This protocol is applicable in a variety of conditions, and represents an important tool for understanding RNA biology. The protocol includes methods for the design and synthesis of RNA mixtures for study, and the construction and analysis of structure-dependent sequencing libraries that reveal structural information of the RNAs in the mixtures. The methods are generally applicable to studying RNA structure and interactions in vitro in a variety of conditions, and allows for the rapid characterization of RNA structures in a high-throughput manner. Curr. Protoc. Chem. Biol. 4:275-297 © 2012 by John Wiley & Sons, Inc.
RESUMEN
Translation of hepatitis C viral proteins requires an internal ribosome entry site (IRES) located in the 5' untranslated region of the viral mRNA. The core domain of the hepatitis C virus (HCV) IRES contains a four-way helical junction that is integrated within a predicted pseudoknot. This domain is required for positioning the mRNA start codon correctly on the 40S ribosomal subunit during translation initiation. Here, we present the crystal structure of this RNA, revealing a complex double-pseudoknot fold that establishes the alignment of two helical elements on either side of the four-helix junction. The conformation of this core domain constrains the open reading frame's orientation for positioning on the 40S ribosomal subunit. This structure, representing the last major domain of HCV-like IRESs to be determined at near-atomic resolution, provides the basis for a comprehensive cryoelectron microscopy-guided model of the intact HCV IRES and its interaction with 40S ribosomal subunits.
Asunto(s)
Codón Iniciador/química , Hepacivirus/química , ARN Mensajero/química , ARN Viral/química , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Secuencia de Bases , Clonación Molecular , Biología Computacional , Microscopía por Crioelectrón , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Desplegamiento Proteico , Ribosomas/química , Transcripción Genética , Proteínas Virales/químicaRESUMEN
RNA selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry exploits the discovery that conformationally dynamic nucleotides preferentially adopt configurations that facilitate reaction between the 2'-OH group and a hydroxyl-selective electrophile, such as benzoyl cyanide (BzCN), to form a 2'-O-adduct. BzCN is ideally suited for quantitative, time-resolved analysis of RNA folding and ribonucleoprotein (RNP) assembly mechanisms because this reagent both reacts with flexible RNA nucleotides and also undergoes auto-inactivating hydrolysis with a half-life of 0.25 s at 37 degrees C. RNA folding is initiated by addition of Mg(2+) or protein, or other change in solution conditions, and nucleotide resolution structural images are obtained by adding aliquots of the evolving reaction to BzCN and then 'waiting' for 1 second. Sites of the 2'-O-adduct formation are subsequently scored as stops to primer extension using reverse transcriptase. This time-resolved SHAPE protocol makes it possible to obtain 1-second structural snapshots in time-resolved kinetic studies for RNAs of arbitrary length and complexity in a straightforward and concise experiment.