RESUMEN
BACKGROUND: There is a need for biomarkers that improve accuracy compared with current demographic risk indices to detect individuals at the highest lung cancer risk. Improved risk determination will enable more effective lung cancer screening and better stratification of lung nodules into high or low-risk category. We previously reported discovery of a biomarker for lung cancer risk characterized by increased prevalence of TP53 somatic mutations in airway epithelial cells (AEC). Here we present results from a validation study in an independent retrospective case-control cohort. METHODS: Targeted next generation sequencing was used to identify mutations within three TP53 exons spanning 193 base pairs in AEC genomic DNA. RESULTS: TP53 mutation prevalence was associated with cancer status (P < 0.001). The lung cancer detection receiver operator characteristic (ROC) area under the curve (AUC) for the TP53 biomarker was 0.845 (95% confidence limits 0.749-0.942). In contrast, TP53 mutation prevalence was not significantly associated with age or smoking pack-years. The combination of TP53 mutation prevalence with PLCOM2012 risk score had an ROC AUC of 0.916 (0.846-0.986) and this was significantly higher than that for either factor alone (P < 0.03). CONCLUSIONS: These results support the validity of the TP53 mutation prevalence biomarker and justify taking additional steps to assess this biomarker in AEC specimens from a prospective cohort and in matched nasal brushing specimens as a potential non-invasive surrogate specimen.
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Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/genética , Detección Precoz del Cáncer , Estudios Prospectivos , Estudios Retrospectivos , Epitelio , Biomarcadores , Pulmón , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Many lifestyle interventions have demonstrated efficacy up to one-year follow-up, yet maintaining improvements at longer-term follow-up is a well-recognized worldwide challenge, especially in underserved areas. The purpose of this study is to compare the 18-month efficacy of an Intensive LifeStyle Modification Program to usual care in reducing the risk for type 2 diabetes (T2D) among women with a history of gestational diabetes mellitus (GDM). METHODS: We conducted a two-arm, cluster randomized controlled trial among women with a history of GDM in China. A total of 16 towns (clusters) in two distinct rural areas in south-central China were randomly selected (8 towns per area) and assigned (1:1) to the intervention (Intensive LifeStyle Modification Program) or control (usual care) group with stratification in the two rural areas. The strategies for maintaining intervention effects were used (including setting recursive goals and providing a supportive environment, etc.) under the guidance of social cognitive theory. The primary outcome was a change in T2D risk; secondary outcomes included glycemic, weight-related, behavioral, and psychological variables. All outcomes were collected at baseline, 6, and 18 months. All participants entered the intention-to-treat analysis. Data were analyzed via generalized estimation equation models (accounting for clusters) at the individual level, with subgroup analysis included in the model. RESULTS: The sample included 320 women from 16 clusters (20 women per cluster). At 18 months, the intervention group demonstrated a significant improvement in T2D risk score, fasting blood glucose, body mass index (BMI), waist circumference, intention to eat low glycemic index food, perceived stress, quality of life in psychological and environmental domains, and social support over time (p < 0.05) based on the intention-to-treat analysis set. Subgroup analysis showed a significant interaction effect on T2D risk score in subgroups of different BMI, waist circumference, and blood glucose (p < 0.05). CONCLUSIONS: Over 18 months, the Intensive LifeStyle Modification Program reduced T2D risk among rural women with a history of GDM in China. Women who were overweight, had high abdominal adiposity, or had blood glucose intolerance benefited more from this intervention. This program serves as a potential diabetes prevention model for women with a history of GDM in low-resource settings worldwide. TRIAL REGISTRATION: Registered on Chinese Clinical Trial Registry (ChiCTR1800015023) on 1st March 2018, http://www.chictr.org.cn/showproj.aspx?proj=25569.
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Diabetes Mellitus Tipo 2 , Diabetes Gestacional , Embarazo , Humanos , Femenino , Diabetes Mellitus Tipo 2/prevención & control , Glucemia , Calidad de Vida , Diabetes Gestacional/prevención & control , Estilo de VidaRESUMEN
This study aimed to evaluate the efficacy of an intensive lifestyle modification program tailored to rural Chinese women with prior gestational diabetes mellitus compared with usual care. In a cluster randomized controlled trial, 16 towns (clusters) in two distinct rural areas in China were randomly selected (8 towns per district); and 320 women with prior gestational diabetes mellitus were recruited from these towns. With stratification for the two study districts, eight towns (160 women) were randomly assigned to the intervention group of a tailored intensive lifestyle modification program and 8 towns (160 women) to the control group. Process measures were collected on attendance, engagement, fidelity, and satisfaction. Primary efficacy outcomes included glycemic and weight-related outcomes, while secondary efficacy outcomes were behavioral outcomes and type 2 diabetes risk score, which were collected at baseline, 3-month, and 6-month follow-up. Generalized estimation equations were used to analyze the data. High attendance (72% of sessions), engagement (67% of interactive activities and group discussions), fidelity (98%), and satisfaction (92%) with the tailored intensive lifestyle modification program were achieved. There were significant reductions in fasting blood glucose, oral glucose tolerance test 2 h, waist circumference, and type 2 diabetes risk score of participants in the intervention group compared to the control group (p < .05). There was no significant intervention effect on body mass index or behavioral outcomes (p > .05). In this study, we demonstrate the successful efficacy of an Intensive Lifestyle Modification Program in reducing type 2 diabetes risk among younger women with prior gestational diabetes mellitus. This tailored program delivered by local healthcare providers is a promising approach for diabetes prevention in rural China, reducing health disparities in rural communities about diabetes prevention. Registered in the Chinese Clinical Trial Registry (ChiCTR2000037956) on 3rd Jan 2018.
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Diabetes Mellitus Tipo 2 , Diabetes Gestacional , Glucemia , Diabetes Mellitus Tipo 2/prevención & control , Diabetes Gestacional/prevención & control , Femenino , Humanos , Estilo de Vida , Embarazo , Población RuralRESUMEN
STUDY OBJECTIVE: We determine the percentage of diagnosed and undiagnosed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among a sample of US emergency department (ED) health care personnel before July 2020. METHODS: This was a cross-sectional analysis of ED health care personnel in 20 geographically diverse university-affiliated EDs from May 13, to July 8, 2020, including case counts of prior laboratory-confirmed coronavirus disease 2019 (COVID-19) diagnoses among all ED health care personnel, and then point-in-time serology (with confirmatory testing) and reverse transcriptase-polymerase chain reaction testing in a sample of volunteers without a previous COVID-19 diagnosis. Health care staff were categorized as clinical (physicians, advanced practice providers, and nurses) and nonclinical (clerks, social workers, and case managers). Previously undiagnosed infection was based on positive SARS-CoV-2 serology or reverse transcriptase-polymerase chain reaction result among health care personnel without prior diagnosis. RESULTS: Diagnosed COVID-19 occurred in 2.8% of health care personnel (193/6,788), and the prevalence was similar for nonclinical and clinical staff (3.8% versus 2.7%; odds ratio 1.5; 95% confidence interval 0.7 to 3.2). Among 1,606 health care personnel without previously diagnosed COVID-19, 29 (1.8%) had evidence of current or past SARS-CoV-2 infection. Most (62%; 18/29) who were seropositive did not think they had been infected, 76% (19/25) recalled COVID-19-compatible symptoms, and 89% (17/19) continued to work while symptomatic. Accounting for both diagnosed and undiagnosed infections, 4.6% (95% confidence interval 2.8% to 7.5%) of ED health care personnel were estimated to have been infected with SARS-CoV-2, with 38% of those infections undiagnosed. CONCLUSION: In late spring and early summer 2020, the estimated prevalence of severe acute respiratory syndrome coronavirus 2 infection was 4.6%, and greater than one third of infections were undiagnosed. Undiagnosed SARS-CoV-2 infection may pose substantial risk for transmission to other staff and patients.
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COVID-19/epidemiología , Servicio de Urgencia en Hospital/estadística & datos numéricos , Personal de Salud/estadística & datos numéricos , SARS-CoV-2 , Adulto , COVID-19/diagnóstico , Estudios Transversales , Femenino , Hospitales Universitarios/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Estados Unidos/epidemiologíaRESUMEN
BACKGROUND: Standardized Nucleic Acid Quantification for SEQuencing (SNAQ-SEQ) is a novel method that utilizes synthetic DNA internal standards spiked into each sample prior to next generation sequencing (NGS) library preparation. This method was applied to analysis of normal appearing airway epithelial cells (AEC) obtained by bronchoscopy in an effort to define a somatic mutation field effect associated with lung cancer risk. There is a need for biomarkers that reliably detect those at highest lung cancer risk, thereby enabling more effective screening by annual low dose CT. The purpose of this study was to test the hypothesis that lung cancer risk is characterized by increased prevalence of low variant allele frequency (VAF) somatic mutations in lung cancer driver genes in AEC. METHODS: Synthetic DNA internal standards (IS) were prepared for 11 lung cancer driver genes and mixed with each AEC genomic (g) DNA specimen prior to competitive multiplex PCR amplicon NGS library preparation. A custom Perl script was developed to separate IS reads and respective specimen gDNA reads from each target into separate files for parallel variant frequency analysis. This approach identified nucleotide-specific sequencing error and enabled reliable detection of specimen mutations with VAF as low as 5 × 10- 4 (0.05%). This method was applied in a retrospective case-control study of AEC specimens collected by bronchoscopic brush biopsy from the normal airways of 19 subjects, including eleven lung cancer cases and eight non-cancer controls, and the association of lung cancer risk with AEC driver gene mutations was tested. RESULTS: TP53 mutations with 0.05-1.0% VAF were more prevalent (p < 0.05) and also enriched for tobacco smoke and age-associated mutation signatures in normal AEC from lung cancer cases compared to non-cancer controls matched for smoking and age. Further, PIK3CA and BRAF mutations in this VAF range were identified in AEC from cases but not controls. CONCLUSIONS: Application of SNAQ-SEQ to measure mutations in the 0.05-1.0% VAF range enabled identification of an AEC somatic mutation field of injury associated with lung cancer risk. A biomarker comprising TP53, PIK3CA, and BRAF somatic mutations may better stratify individuals for optimal lung cancer screening and prevention outcomes.
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Fosfatidilinositol 3-Quinasa Clase I/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Pulmonares/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Bronquios/metabolismo , Bronquios/patología , Estudios de Casos y Controles , Detección Precoz del Cáncer , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Pronóstico , Estudios RetrospectivosRESUMEN
OBJECTIVE: The objective of this study was to describe the epidemiology, clinical features, outcomes, and predictors of mortality in veterans with peripheral artery disease (PAD). METHODS: We used national data from the Veterans Health Administration from fiscal years 2009 to 2011 to identify patients with a new diagnosis of PAD. Within this cohort, we describe characteristics of the patients, use of recommended medications, and clinical outcomes during a 3-year follow-up (fiscal year 2014). We used Cox proportional hazards regression to examine predictors of mortality and adverse limb outcomes (amputation and hospitalization for critical limb ischemia [CLI]) during follow-up. RESULTS: A total of 175,865 patients with a new diagnosis of PAD were included. The mean age was 69.9 years; 97.8% were male, and 67.7% were white. Nearly 77% of patients had hypertension, 46.5% had diabetes, 23% had chronic obstructive pulmonary disease, and 12.9% had renal failure. A prescription for statins was filled by 60.8%, and 34.9% received high-intensity statins within 90 days of PAD diagnosis. At 1 year, 2.6% underwent revascularization, 1.3% developed CLI, and 1.1% underwent amputation. During a median follow-up of 3.8 years, a total of 28.6% patients died (6.7% at 1 year), and 3.7% developed a limb outcome (2.0% at 1 year). Predictors of mortality included advanced age, comorbidities, and CLI or amputation at presentation. In contrast, prescription with statins was associated with lower mortality. Similar findings were present with regard to predictors of adverse limb outcomes, except that older age was associated with a lower risk of amputation or CLI. CONCLUSIONS: We found that veterans with PAD have a high prevalence of comorbid conditions and have a significant risk of mortality and limb loss. A substantial proportion of veterans with PAD are not prescribed recommended medications, especially statin therapy. Our data highlight important opportunities for improving care of veterans with PAD.
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Isquemia/epidemiología , Extremidad Inferior/irrigación sanguínea , Enfermedad Arterial Periférica/epidemiología , Salud de los Veteranos , Factores de Edad , Anciano , Anciano de 80 o más Años , Amputación Quirúrgica , Comorbilidad , Enfermedad Crítica , Bases de Datos Factuales , Femenino , Adhesión a Directriz , Hospitalización , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Incidencia , Isquemia/diagnóstico , Isquemia/mortalidad , Isquemia/terapia , Masculino , Persona de Mediana Edad , Enfermedad Arterial Periférica/diagnóstico , Enfermedad Arterial Periférica/mortalidad , Enfermedad Arterial Periférica/terapia , Guías de Práctica Clínica como Asunto , Pautas de la Práctica en Medicina , Prevalencia , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Estados Unidos/epidemiología , United States Department of Veterans Affairs , Salud de los Veteranos/normasRESUMEN
BACKGROUND: There is a need for more powerful methods to identify low-effect SNPs that contribute to hereditary COPD pathogenesis. We hypothesized that SNPs contributing to COPD risk through cis-regulatory effects are enriched in genes comprised by bronchial epithelial cell (BEC) expression patterns associated with COPD. METHODS: To test this hypothesis, normal BEC specimens were obtained by bronchoscopy from 60 subjects: 30 subjects with COPD defined by spirometry (FEV1/FVC < 0.7, FEV1% < 80%), and 30 non-COPD controls. Targeted next generation sequencing was used to measure total and allele-specific expression of 35 genes in genome maintenance (GM) genes pathways linked to COPD pathogenesis, including seven TP53 and CEBP transcription factor family members. Shrinkage linear discriminant analysis (SLDA) was used to identify COPD-classification models. COPD GWAS were queried for putative cis-regulatory SNPs in the targeted genes. RESULTS: On a network basis, TP53 and CEBP transcription factor pathway gene pair network connections, including key DNA repair gene ERCC5, were significantly different in COPD subjects (e.g., Wilcoxon rank sum test for closeness, p-value = 5.0E-11). ERCC5 SNP rs4150275 association with chronic bronchitis was identified in a set of Lung Health Study (LHS) COPD GWAS SNPs restricted to those in putative regulatory regions within the targeted genes, and this association was validated in the COPDgene non-hispanic white (NHW) GWAS. ERCC5 SNP rs4150275 is linked (D' = 1) to ERCC5 SNP rs17655 which displayed differential allelic expression (DAE) in BEC and is an expression quantitative trait locus (eQTL) in lung tissue (p = 3.2E-7). SNPs in linkage (D' = 1) with rs17655 were predicted to alter miRNA binding (rs873601). A classifier model that comprised gene features CAT, CEBPG, GPX1, KEAP1, TP73, and XPA had pooled 10-fold cross-validation receiver operator characteristic area under the curve of 75.4% (95% CI: 66.3%-89.3%). The prevalence of DAE was higher than expected (p = 0.0023) in the classifier genes. CONCLUSIONS: GM genes comprised by COPD-associated BEC expression patterns were enriched for SNPs with cis-regulatory function, including a putative cis-rSNP in ERCC5 that was associated with COPD risk. These findings support additional total and allele-specific expression analysis of gene pathways with high prior likelihood for involvement in COPD pathogenesis.
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Bronquios/patología , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Células Epiteliales/metabolismo , Proteínas Nucleares/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Factores de Transcripción/genética , Alelos , Estudios de Casos y Controles , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Enfermedad Pulmonar Obstructiva Crónica/patología , Sitios de Carácter Cuantitativo , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: Annual low dose CT (LDCT) screening of individuals at high demographic risk reduces lung cancer mortality by more than 20%. However, subjects selected for screening based on demographic criteria typically have less than a 10% lifetime risk for lung cancer. Thus, there is need for a biomarker that better stratifies subjects for LDCT screening. Toward this goal, we previously reported a lung cancer risk test (LCRT) biomarker comprising 14 genome-maintenance (GM) pathway genes measured in normal bronchial epithelial cells (NBEC) that accurately classified cancer (CA) from non-cancer (NC) subjects. The primary goal of the studies reported here was to optimize the LCRT biomarker for high specificity and ease of clinical implementation. METHODS: Targeted competitive multiplex PCR amplicon libraries were prepared for next generation sequencing (NGS) analysis of transcript abundance at 68 sites among 33 GM target genes in NBEC specimens collected from a retrospective cohort of 120 subjects, including 61 CA cases and 59 NC controls. Genes were selected for analysis based on contribution to the previously reported LCRT biomarker and/or prior evidence for association with lung cancer risk. Linear discriminant analysis was used to identify the most accurate classifier suitable to stratify subjects for screening. RESULTS: After cross-validation, a model comprising expression values from 12 genes (CDKN1A, E2F1, ERCC1, ERCC4, ERCC5, GPX1, GSTP1, KEAP1, RB1, TP53, TP63, and XRCC1) and demographic factors age, gender, and pack-years smoking, had Receiver Operator Characteristic area under the curve (ROC AUC) of 0.975 (95% CI: 0.96-0.99). The overall classification accuracy was 93% (95% CI 88%-98%) with sensitivity 93.1%, specificity 92.9%, positive predictive value 93.1% and negative predictive value 93%. The ROC AUC for this classifier was significantly better (p < 0.0001) than the best model comprising demographic features alone. CONCLUSIONS: The LCRT biomarker reported here displayed high accuracy and ease of implementation on a high throughput, quality-controlled targeted NGS platform. As such, it is optimized for clinical validation in specimens from the ongoing LCRT blinded prospective cohort study. Following validation, the biomarker is expected to have clinical utility by better stratifying subjects for annual lung cancer screening compared to current demographic criteria alone.
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Biomarcadores de Tumor/análisis , Bronquios/citología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Antioxidantes/análisis , Antioxidantes/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biopsia , Bronquios/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/epidemiología , Reacción en Cadena de la Polimerasa , Curva ROC , Reproducibilidad de los Resultados , Estudios Retrospectivos , Medición de Riesgo , Tomografía Computarizada EspiralRESUMEN
Excision repair cross-complementation group 5 (ERCC5) gene plays an important role in nucleotide excision repair, and dysregulation of ERCC5 is associated with increased lung cancer risk. Haplotype and diplotype analyses were conducted in normal bronchial epithelial cells (NBEC) to better understand mechanisms responsible for interindividual variation in transcript abundance regulation of ERCC5 We determined genotypes at putative ERCC5 cis-regulatory SNPs (cis-rSNP) rs751402 and rs2296147, and marker SNPs rs1047768 and rs17655. ERCC5 allele-specific transcript abundance was assessed by a recently developed targeted sequencing method. Syntenic relationships among alleles at rs751402, rs2296147, and rs1047768 were assessed by allele-specific PCR followed by Sanger sequencing. We then assessed association of ERCC5 allele-specific expression at rs1047768 with haplotype and diplotype structure at cis-rSNPs rs751402 and rs2296147. Genotype analysis revealed significantly (P < 0.005) higher interindividual variation in allelic ratios in cDNA samples relative to matched gDNA samples at both rs1047768 and rs17655. By diplotype analysis, mean expression was higher at the rs1047768 alleles syntenic with rs2296147 T allele compared with rs2296147 C allele. Furthermore, mean expression was lower at rs17655 C allele, which is syntenic with G allele at a linked SNP rs873601 (D' = 0.95). These data support the conclusions that in NBEC, T allele at SNP rs2296147 upregulates ERCC5, variation at rs751402 does not alter ERCC5 regulation, and that C allele at SNP rs17655 downregulates ERCC5 Variation in ERCC5 transcript abundance associated with allelic variation at these SNPs could result in variation in NER function in NBEC and lung cancer risk.
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Bronquios/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Células Epiteliales/metabolismo , Haplotipos/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple/genética , Factores de Transcripción/genética , Anciano , Alelos , Estudios de Casos y Controles , Femenino , Regulación de la Expresión Génica/genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Regulación hacia Arriba/genéticaRESUMEN
A 23-year-old male presented from a nursing home with hypotension, tachycardia, diaphoresis and electrocardiographic evidence of right ventricular strain that was confirmed by echocardiography. His differential diagnosis included sepsis and pulmonary embolism. A high-resolution computed tomography scan demonstrated no pulmonary emboli but did demonstrate multiple bilateral pulmonary nodules. Upon questioning he admitted to injecting a long-acting narcotic that had been manually macerated, dissolved in saline, and injected through an indwelling intravenous line. Lung biopsy findings were consistent with cellulose-induced perivascular granulomatosis. Cellulose granulomatosis can be seen in patients who inject medications designed for oral use and should be considered in patients who present with acute pulmonary hypertension.
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Granuloma de Cuerpo Extraño/diagnóstico , Narcóticos/efectos adversos , Embolia Pulmonar/diagnóstico , Detección de Abuso de Sustancias , Trastornos Relacionados con Sustancias/diagnóstico , Enfermedad Aguda , Celulosa/análisis , Diagnóstico Diferencial , Glucocorticoides/uso terapéutico , Granuloma de Cuerpo Extraño/diagnóstico por imagen , Granuloma de Cuerpo Extraño/tratamiento farmacológico , Humanos , Inyecciones Intravenosas , Enfermedades Pulmonares/diagnóstico por imagen , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/patología , Masculino , Pacientes Ambulatorios , Embolia Pulmonar/diagnóstico por imagen , Embolia Pulmonar/tratamiento farmacológico , Sepsis/diagnóstico , Sepsis/diagnóstico por imagen , Trastornos Relacionados con Sustancias/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
Harmful algal blooms plague bodies of freshwater globally. These blooms are often composed of outgrowths of cyanobacteria capable of producing the heptapeptide Microcystin-LR (MC-LR) which is a well-known hepatotoxin. Recently, MC-LR has been detected in aerosols generated from lake water. However, the risk for human health effects due to MC-LR inhalation exposure have not been extensively investigated. In this study, we exposed a fully differentiated 3D human airway epithelium derived from 14 healthy donors to MC-LR-containing aerosol once a day for 3 days. Concentrations of MC-LR ranged from 100 pM to 1 µM. Although there were little to no detrimental alterations in measures of the airway epithelial function (i.e. cell survival, tissue integrity, mucociliary clearance, or cilia beating frequency), a distinct shift in the transcriptional activity was found. Genes related to inflammation were found to be upregulated such as C-C motif chemokine 5 (CCL5; log2FC = 0.57, p = 0.03) and C-C chemokine receptor type 7 (CCR7; log2FC = 0.84, p = 0.03). Functionally, conditioned media from MC-LR exposed airway epithelium was also found to have significant chemo-attractive properties for primary human neutrophils. Additionally, increases were found in the concentration of secreted chemokine proteins in the conditioned media such as CCL1 (log2FC = 5.07, p = 0.0001) and CCL5 (log2FC = 1.02, p = 0.046). These results suggest that MC-LR exposure to the human airway epithelium is capable of inducing an inflammatory response that may potentiate acute or chronic disease.
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Microcistinas , Agua , Aerosoles/toxicidad , Medios de Cultivo Condicionados , Epitelio , Humanos , Toxinas Marinas , Microcistinas/toxicidad , Receptores CCR7RESUMEN
BACKGROUND: Clinical laboratories routinely use formalin-fixed paraffin-embedded (FFPE) tissue or cell block cytology samples in oncology panel sequencing to identify mutations that can predict patient response to targeted therapy. To understand the technical error due to FFPE processing, a robustly characterized diploid cell line was used to create FFPE samples with four different pre-tissue processing formalin fixation times. A total of 96 FFPE sections were then distributed to different laboratories for targeted sequencing analysis by four oncopanels, and variants resulting from technical error were identified. RESULTS: Tissue sections that fail more frequently show low cellularity, lower than recommended library preparation DNA input, or target sequencing depth. Importantly, sections from block surfaces are more likely to show FFPE-specific errors, akin to "edge effects" seen in histology, while the inner samples display no quality degradation related to fixation time. CONCLUSIONS: To assure reliable results, we recommend avoiding the block surface portion and restricting mutation detection to genomic regions of high confidence.
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Formaldehído , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Adhesión en Parafina , Análisis de Secuencia de ADN , Fijación del TejidoRESUMEN
To identify new susceptibility loci to lung cancer among diverse populations, we performed cross-ancestry genome-wide association studies in European, East Asian and African populations and discovered five loci that have not been previously reported. We replicated 26 signals and identified 10 new lead associations from previously reported loci. Rare-variant associations tended to be specific to populations, but even common-variant associations influencing smoking behavior, such as those with CHRNA5 and CYP2A6, showed population specificity. Fine-mapping and expression quantitative trait locus colocalization nominated several candidate variants and susceptibility genes such as IRF4 and FUBP1. DNA damage assays of prioritized genes in lung fibroblasts indicated that a subset of these genes, including the pleiotropic gene IRF4, potentially exert effects by promoting endogenous DNA damage.
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Estudio de Asociación del Genoma Completo , Neoplasias Pulmonares , Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Pulmonares/genética , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Proteínas de Unión al ARN/genéticaRESUMEN
The primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next-generation sequencing (NGS) method that will enable more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. To accomplish this, a synthetic internal standard spike-in was designed for each actionable mutation target, suitable for use in NGS following hybrid capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position in each sample. True-positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.
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ADN Tumoral Circulante , Humanos , ADN Tumoral Circulante/genética , Mutación/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Medicina de Precisión/métodos , Control de CalidadRESUMEN
BACKGROUND: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. RESULTS: All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. CONCLUSION: This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.
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Biomarcadores de Tumor , Pruebas Genéticas/métodos , Genómica/métodos , Neoplasias/genética , Oncogenes , Variaciones en el Número de Copia de ADN , Pruebas Genéticas/normas , Genómica/normas , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Mutación , Neoplasias/diagnóstico , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.
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ADN Tumoral Circulante/genética , Oncología Médica , Neoplasias/genética , Medicina de Precisión , Análisis de Secuencia de ADN/normas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Límite de Detección , Guías de Práctica Clínica como Asunto , Reproducibilidad de los ResultadosRESUMEN
ERCC5 (XPG) is a key component of the nucleotide excision DNA repair pathway. In two recent case-control studies, we determined that ERCC5 transcript expression pattern in grossly normal human bronchial epithelial cells (NBEC) was different in individuals diagnosed with lung cancer compared with non-lung cancer controls. In this study, we tested the hypothesis that variation at cis-acting sites contributed to observed variation in ERCC5 transcript expression in NBEC. Allele-specific expression (ASE) was measured at transcribed polymorphic site rs1047768 in exon 2 of ERCC5 in NBEC complementary DNA (cDNA) of 22 individuals using allele-specific competitive polymerase chain reaction. ASE at rs1047768 was then assessed for association with allelotype at polymorphic sites rs751402 (E2F1 and YY1 recognition and response site) and rs2296147 (putative P53 recognition site) in the proximal promoter and 5' untranslated region, respectively, of ERCC5. Interindividual variation in recombination between rs751402, rs2296147 and rs1047768 in poly-heterozygotes was controlled for by allele-specific sequencing. Measured rs1047768 T:C allelic ratio was (i) significantly higher in NBEC cDNA compared with genomic DNA controls (P < 0.001) among samples heterozygous at both rs751402 and rs2296147; (ii) less high (P = 0.02) for samples homozygous at rs751402 but heterozygous at rs2296147 and (iii) not significantly different (P = 0.18) for doubly homozygous individuals. Here, we demonstrate that rs751402 A allele and rs2296147 T allele are associated with higher ASE of ERCC5 T allele transcript at rs1047768 in NBEC.
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Bronquios/metabolismo , Proteínas de Unión al ADN/genética , Factor de Transcripción E2F1/fisiología , Endonucleasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/fisiología , Factor de Transcripción YY1/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Células Epiteliales/metabolismo , Femenino , Variación Genética , Genotipo , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Mensajero/análisisRESUMEN
Platelet-leukocyte aggregates (PLAs) are associated with increased thrombosis risk. The influence of PLA formation is especially important for cancer patients, since thrombosis accounts for approximately 10% of cancer-associated deaths. Our objective was to characterize and quantify PLAs in whole blood samples from lung cancer patients compared to healthy volunteers with the intent to analyze PLA formation in the context of lung cancer-associated thrombosis. Consenting lung cancer patients (57) and healthy volunteers (56) were enrolled at the Dana Cancer Center at the University of Toledo Health Science Campus. Peripheral blood samples were analyzed by flow cytometry. Patient medical history was reviewed through electronic medical records. Most importantly, we found lung cancer patients to have higher percentages of platelet-T cell aggregates (PTCAs) than healthy volunteers among both CD4+ T lymphocyte and CD8+ T lymphocyte populations. Our findings demonstrate that characterization of PTCAs may have clinical utility in differentiating lung cancer patients from healthy volunteers and stratifying lung cancer patients by history of thrombosis.
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Plaquetas/patología , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/complicaciones , Linfocitos T/patología , Trombosis/sangre , Trombosis/etiología , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Estudios de Casos y Controles , Agregación Celular , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Adulto JovenRESUMEN
We have evaluated the performance characteristics of three quantitative gene expression technologies and correlated their expression measurements to those of five commercial microarray platforms, based on the MicroArray Quality Control (MAQC) data set. The limit of detection, assay range, precision, accuracy and fold-change correlations were assessed for 997 TaqMan Gene Expression Assays, 205 Standardized RT (Sta)RT-PCR assays and 244 QuantiGene assays. TaqMan is a registered trademark of Roche Molecular Systems, Inc. We observed high correlation between quantitative gene expression values and microarray platform results and found few discordant measurements among all platforms. The main cause of variability was differences in probe sequence and thus target location. A second source of variability was the limited and variable sensitivity of the different microarray platforms for detecting weakly expressed genes, which affected interplatform and intersite reproducibility of differentially expressed genes. From this analysis, we conclude that the MAQC microarray data set has been validated by alternative quantitative gene expression platforms thus supporting the use of microarray platforms for the quantitative characterization of gene expression.
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Perfilación de la Expresión Génica/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Garantía de la Calidad de Atención de Salud/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We have assessed the utility of RNA titration samples for evaluating microarray platform performance and the impact of different normalization methods on the results obtained. As part of the MicroArray Quality Control project, we investigated the performance of five commercial microarray platforms using two independent RNA samples and two titration mixtures of these samples. Focusing on 12,091 genes common across all platforms, we determined the ability of each platform to detect the correct titration response across the samples. Global deviations from the response predicted by the titration ratios were observed. These differences could be explained by variations in relative amounts of messenger RNA as a fraction of total RNA between the two independent samples. Overall, both the qualitative and quantitative correspondence across platforms was high. In summary, titration samples may be regarded as a valuable tool, not only for assessing microarray platform performance and different analysis methods, but also for determining some underlying biological features of the samples.