TP53 mutation prevalence in normal airway epithelium as a biomarker for lung cancer risk.

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.

The 18-month efficacy of an Intensive LifeStyle Modification Program (ILSM) to reduce type 2 diabetes risk among rural women: a cluster randomized controlled trial.

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.

Microcystin-LR aerosol induces inflammatory responses in healthy human primary airway epithelium.

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.

Cross-ancestry genome-wide meta-analysis of 61,047 cases and 947,237 controls identifies new susceptibility loci contributing to lung cancer.

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.

Deep oncopanel sequencing reveals within block position-dependent quality degradation in FFPE processed samples.

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.

The 6-Month Efficacy of an Intensive Lifestyle Modification Program on Type 2 Diabetes Risk Among Rural Women with Prior Gestational Diabetes Mellitus: a Cluster Randomized Controlled Trial.

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.

Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions.

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.

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

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.

Diagnosed and Undiagnosed COVID-19 in US Emergency Department Health Care Personnel: A Cross-sectional Analysis.

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.

Advancing NGS quality control to enable measurement of actionable mutations in circulating tumor DNA.

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.

Platelet-T cell aggregates in lung cancer patients: Implications for thrombosis.

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.

Technical advance in targeted NGS analysis enables identification of lung cancer risk-associated low frequency TP53, PIK3CA, and BRAF mutations in airway epithelial cells.

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.

BIRC5 is a target for molecular imaging and detection of human pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer mortality with a dismal overall survival rate and an urgent need for detection of minute tumors. Current diagnostic modalities have high sensitivity and specificity for larger tumors, but not for minute PDAC. In this study, we test the feasibility of a precision diagnostic platform for detecting and localizing minute human PDAC in mice. This platform includes: 1) defining BIRC5 as an early PDAC-upregulated gene and utilizing an enhanced BIRC5 super-promoter to drive expression of dual Gaussia luciferase (GLuc) and sr39 thymidine kinase (sr39TK) reporter genes exponentially and specifically in PDAC; 2) utilizing a genetically-engineered AAV2RGD to ensure targeted delivery of GLuc and sr39TK specifically to PDAC; 3) using serologic GLuc and sr39TK microPET/CT imaging to detect and localize minute human PDAC in mice. The study demonstrates feasibility of a precision diagnostic platform using an integrated technology through a multiple-stage amplification strategy of dual reporter genes to enhance the specificity and sensitivity of detection and localization of minute PDAC tumors and currently undetectable disease.

Epidemiology of lower extremity peripheral artery disease in veterans.

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.

RNAseq analysis of bronchial epithelial cells to identify COPD-associated genes and SNPs.

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.

A novel synthetic human insulin super promoter for targeting PDX-1-expressing pancreatic cancer.

Our previous studies have shown that a rat insulin promoter II fragment (RIP) was used to effectively target pancreatic adenocarcinoma (PDAC) and insulinoma that over-express pancreatic and duodenal homeobox-1 (PDX-1). To enhance the activity and specificity of the human insulin promoter, we engineered a synthetic human insulin super-promoter (SHIP). Reporter assay demonstrated that SHIP1 was the most powerful promoter among all of the SHIPs and had far greater activity than the endogenous human insulin promoters and RIP in PDAC expressing PDX-1. Over-expression, knockdown and competitive inhibition of PDX-1 expression assay proved that PDX-1 is a critical transcript factor to regulate the activity of SHIP1. SHIP1-driven viral thymidine kinase followed by ganciclovir (SHIP1-TK/GCV) resulted in cytotoxicity to PDAC cells in vitro. Systemic delivery of SHIP1-TK/GCV in PDAC xenograft mice significantly suppressed PANC-1 tumor growth in vivo greater than RIP-TK/GCV and CMV-TK/GCV controls (p < .05). These preclinical data suggest that SHIP1 is a powerful novel promoter that can be used to target human PDAC expressing PDX-1 in clinical trials. Furthermore, this novel strategy of engineering synthetic super-promoters could be used for other cancer targets.

A lung cancer risk classifier comprising genome maintenance genes measured in normal bronchial epithelial cells.

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.

Haplotype and diplotype analyses of variation in ERCC5 transcription cis-regulation in normal bronchial epithelial cells.

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.

Control for stochastic sampling variation and qualitative sequencing error in next generation sequencing.

BACKGROUND: Clinical implementation of Next-Generation Sequencing (NGS) is challenged by poor control for stochastic sampling, library preparation biases and qualitative sequencing error. To address these challenges we developed and tested two hypotheses. METHODS: Hypothesis 1: Analytical variation in quantification is predicted by stochastic sampling effects at input of a) amplifiable nucleic acid target molecules into the library preparation, b) amplicons from library into sequencer, or c) both. We derived equations using Monte Carlo simulation to predict assay coefficient of variation (CV) based on these three working models and tested them against NGS data from specimens with well characterized molecule inputs and sequence counts prepared using competitive multiplex-PCR amplicon-based NGS library preparation method comprising synthetic internal standards (IS). Hypothesis 2: Frequencies of technically-derived qualitative sequencing errors (i.e., base substitution, insertion and deletion) observed at each base position in each target native template (NT) are concordant with those observed in respective competitive synthetic IS present in the same reaction. We measured error frequencies at each base position within amplicons from each of 30 target NT, then tested whether they correspond to those within the 30 respective IS. RESULTS: For hypothesis 1, the Monte Carlo model derived from both sampling events best predicted CV and explained 74% of observed assay variance. For hypothesis 2, observed frequency and type of sequence variation at each base position within each IS was concordant with that observed in respective NTs (R2 = 0.93). CONCLUSION: In targeted NGS, synthetic competitive IS control for stochastic sampling at input of both target into library preparation and of target library product into sequencer, and control for qualitative errors generated during library preparation and sequencing. These controls enable accurate clinical diagnostic reporting of confidence limits and limit of detection for copy number measurement, and of frequency for each actionable mutation.

Availability of consumer prices for bunion surgery.

BACKGROUND: Today, insurance insulates most patients from the true costs of the health care services they consume. Economists believe that the absence of price signals incentivizes patients to pursue more extensive care than they would otherwise. Reformers propose restoring price consciousness to patients as a way to tame the soaring costs of American health care. To test this idea, we decided to gauge the availability and variability of price quotes for a common elective surgery-bunion repair. METHODS: Orthopedic clinics were sorted by state and randomly selected from an online directory maintained by the American Orthopaedic Foot and Ankle Society. Each selected clinic was contacted up to 3 times in an attempt to get a full, bundled price quote using a standardized patient script. If this was unavailable, an isolated quote for the physician fee alone was solicited. RESULTS: Of the 141 clinics contacted, 56 (39.7%) could provide a physician price estimate and 12 (8.5%) could give a complete bundled estimate, including hospital fees. The overall mean bundled price quoted was $18 332, while the overall mean physician fee quoted was $2487. There was no statistically significant difference in the mean price quoted by academic and private clinics, nor was regional variation observed. CONCLUSION: We found low price availability for elective bunion procedures. CLINICAL RELEVANCE: However, the wide variation observed in the prices that were quoted suggests that a very determined patient may be able to spend substantially less on an elective surgery if they were willing to select a provider carefully.