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In previous studies, we reported that key antioxidant and DNA repair genes are regulated differently in normal bronchial epithelial cells of lung cancer cases compared with non–lung cancer controls. In an effort to develop a biomarker for lung cancer risk, we evaluated the transcript expressions of 14 antioxidant, DNA repair, and transcription factor genes in normal bronchial epithelial cells (HUGO names CAT, CEBPG, E2F1, ERCC4, ERCC5, GPX1, GPX3, GSTM3, GSTP1, GSTT1, GSTZ1, MGST1, SOD1, and XRCC1). A test comprising these 14 genes accurately identified the lung cancer cases in two case-control studies. The receiver operating characteristic–area under the curve was 0.82 (95% confidence intervals, 0.68–0.91) for the first case-control set (25 lung cancer cases and 24 controls), and 0.87 (95% confidence intervals, 0.73–0.96) for the second set (18 cases and 22 controls). For each gene included in the test, the key difference between cases and controls was altered distribution of transcript expression among cancer cases compared with controls, with more lung cancer cases expressing at both extremes among all genes (Kolmorogov-Smirnov test, D = 0.0795; P = 0.041). A novel statistical approach was used to identify the lower and upper boundaries of transcript expression that optimally classifies cases and controls for each gene. Based on the data presented here, there is an increased prevalence of lung cancer diagnosis among individuals that express a threshold number of key antioxidant, DNA repair, and transcription factor genes at either very high or very low levels in the normal airway epithelium. [Cancer Res 2009;69(22):8629–35]

Supplementary Table 1 from Pattern of Antioxidant and DNA Repair Gene Expression in Normal Airway Epithelium Associated with Lung Cancer Diagnosis

SUMMARYThe 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 enables more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. This advancement was enabled by designing a synthetic internal standard spike-in 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.

Summary 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.

Background: Detection of rare variants in traditional and liquid biopsies is becoming increasingly important for cancer diagnosis, monitoring and treatment. Next Generation Sequencing (NGS) has the capacity to detect known and novel variants in small biological samples with a high level of sensitivity. However, the limit of detection is affected by sample DNA concentration and integrity, technical error and number of unique molecules captured and sequenced (library complexity). Unique molecular identifiers (UMl) and random fragment end analysis commonly are used to measure complexity yet both are reported to have systematic biases leading to a non-random read distribution when measured, altering interpretation of results and potentially skewing data. Here we describe the use of a spike in complexity calibration ladder comprising synthetic DNA internal standard competitors (IS) as an orthogonal measure of library complexity. Methods: An Endogenous Complexity Calibration Ladder (ECCL) comprising multiple unique synthetic IS at different concentrations was created. All ECCL IS share homology with each other and endogenous human SCGB1A1 sequence but each contain nucleotide changes at different positions along the sequence string so that they can be distinguished. The ECCL was mixed with several IS targeting different TP53 exons in a fixed proportion. The ECCL/TP53 IS mixture is designed to be used in either amplicon or hybrid-capture library preparation. Here, amplicon libraries were generated. Molecule numbers and ratios between IS were determined using deep sequencing of multiple replicates. The ECCL/TP53 IS mix was spiked into a commercial human gDNA prior to NGS library preparation. Serial dilutions (1.5-, 3-, 6-, 12- and 24-fold) and intentional addition of inhibitors were conducted to stress and test the system. The ECCL/TP53 IS mixture also was spiked into gDNA from 60 primary airway epithelial cell (AEC) specimens prior to library preparation and NGS to measure TP53 variant fraction while controlling for complexity. Results: Observed complexity measured using the ECCL was close to expected for each serially diluted mixture of gDNA and ECCL/TP53 IS. The precision of complexity measurement for less dilute and less inhibited samples was limited by absence of finer titration of the least concentrated IS in ECCL. Use of ECCL enabled measurement of inter-sample variation in complexity among the 60 AEC specimens. Conclusions: The ECCL controls for both sample and library specific variation in complexity, and enables better estimation of the lower limit of detection for variant allele fraction (VAF). Additionally, it is compatible with the use of target specific IS which allow for improved measurement of technical sequencing error. The variation in complexity observed in primary AEC gDNA samples demonstrates the need to account for this when assessing rare variant allele fraction. Citation Format: Erin L. Crawford, Tian Chen, Daniel J. Craig, James C. Willey. Use of a synthetic spike-in ladder to measure NGS library complexity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2286.

Background: Lung cancer is the leading cause of cancer-related death in men and women in the United States. Based on evidence from controlled trials in the United States and Europe that low dose CT (LDCT) screening significantly reduces lung cancer mortality, the United States Preventative Services Tasks Force (USPSTF) recommends LDCT screening for individuals with high demographic risk based on age and smoking history. However, a large fraction of lung cancers occur in individuals who do not meet LDCT screening threshold criteria. Thus, there is a need for biomarkers that supplement demographic factors to more accurately detect those at highest lung cancer risk and include additional individuals who will benefit from LDCT screening. An initial study from this lab demonstrated that TP53 mutations with 0.05-1.0% variant allele fraction (VAF) were significantly more prevalent (p Methods: AEC specimens were prospectively collected through the National Cancer Institute Early Detection Research Network (EDRN) program from 60 subjects at high demographic risk for lung cancer by bronchoscopic brush biopsy at Vanderbilt University Medical Center. Genomic (g)DNA was extracted from each AEC specimen and stored at -80 degrees Celsius. Subjects matched for age and smoking history who subsequently did or did not develop lung cancer were identified and gDNA specimens, blinded with respect to cancer status, were shipped to the University of Toledo. gDNA specimens were quantified and 50,000 gDNA copies were included in each NGS library preparation. Synthetic DNA internal standards (IS) were prepared for multiple lung cancer driver mutations within the TP53 gene and mixed with each AEC gDNA specimen prior to competitive multiplex PCR NGS library preparation. By controlling for technical error, this approach enables reliable detection of mutations with VAF as low as 5 x 10-4 (0.05%) (Craig et al, BMC Cancer, 2019). Results: Following library preparation and sequencing on Illumina Novaseq, there was sufficient library complexity to detect TP53 VAF 90% of AEC specimens. The data are currently in pipeline analysis. After analysis is complete, VCF files will be sent to Vanderbilt for unblinding and validation of the biomarker. Conclusions: Based on preliminary data, we expect that sufficient AEC gDNA is available from most subjects enrolled in the EDRN program for TP53 low VAF biomarker analysis, and the previously reported method (Craig et al, BMC Cancer, 2019) is reliable and feasible. The current study should provide additional information regarding validity of the biomarker. Citation Format: Daniel J. Craig, Erin L. Crawford, Pierre P. Massion, Thomas Morrison, James C. Willey. Low frequency TP53 mutations in airway epithelial cells serve as lung cancer risk biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2525.

BackgroundStandardized 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.MethodsSynthetic 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.ResultsTP53 mutations with 0.05–1.0% VAF were more prevalent (p ConclusionsApplication 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.

The burden of cardiovascular disease and death in chronic kidney disease (CKD) outpaces that of the other diseases and is not adequately described by traditional risk factors alone. Diminished activity of paraoxonase (PON)-1 is associated with increased oxidant stress, a common feature underlying the pathogenesis of CKD. We aimed to assess the prognostic value of circulating PON-1 protein and PON lactonase activity on adverse clinical outcomes across various stages and etiologies of CKD. Circulating PON-1 protein levels and PON lactonase activity were measured simultaneously in patients with CKD as well as a cohort of apparently healthy non-CKD subjects. Both circulating PON-1 protein levels and PON lactonase activity were significantly lower in CKD patients compared to the non-CKD subjects. Similarly, across all stages of CKD, circulating PON-1 protein and PON lactonase activity were significantly lower in patients with CKD compared to the non-CKD controls. Circulating PON lactonase activity, but not protein levels, predicted future adverse clinical outcomes, even after adjustment for traditional risk factors. The combination of lower circulating protein levels and higher activity within the CKD subjects were associated with the best survival outcomes. These findings demonstrate that diminished circulating PON lactonase activity, but not protein levels, predicts higher risk of future adverse clinical outcomes in patients with CKD.

Background: Lung cancer is the leading cause of cancer-related death in men and women, and cigarette smoking is the most significant preventable risk factor. Early detection through annual low dose CT screening reduces mortality but has low positive predictive value and specificity, in part due to low lung cancer incidence ( Methods: Synthetic DNA internal standards (IS) were prepared for each of 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 enabled reliable detection of mutations with VAF as low as 5 x 10-4 (0.05%). This method was then applied in a retrospective case-control study. Specifically, AEC specimens were 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 Conclusions: Measurement of very low frequency 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. Citation Format: Daniel J. Craig, Thomas Morrison, Sadik Khuder, Erin L. Crawford, Leihong Wu, Joshua Xu, Thomas M. Blomquist, James C. Willey. TP53, PIK3CA, and BRAF somatic mutations in airway epithelial field of injury associated with lung cancer risk [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4609.

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.

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

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

Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Leprdb/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Leprdb/J mice were gavaged with 50 &mu, g/kg, 100 &mu, g/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15&ndash, 17 mice/group). Early mortality was observed in both the 50 &mu, g/kg (1/17, 6%), and 100 &mu, g/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.

Background: Identification of actionable mutations in circulating tumor DNA (ctDNA) enables gene-targeted therapy of solid tumors based on a simple blood test. NGS methods that attach a unique molecular identifier (UMI) to each DNA molecule reduce variant allele fraction (VAF) limit of detection (LOD) to 0.5% was >95%, but reproducibility at 0.1-0.5% VAF was about 70%. Given limited value of UMI NGS when size of ctDNA specimen restricted VAF LOD to 0.5%, we tested the hypothesis that synthetic internal standard (IS) spike-in molecules provide reliable alternative quality control while eliminating UMI-imposed sequencing burden. Methods: We synthesized IS for 61 actionable mutations, cloned them into pUC vectors, confirmed each IS to be wild-type sequence, linearized, quantified abundance and combined at a 1:1 genome copy mixture. An aliquot of IS mixture was added to the ctDNA reference material at 1:1 genome copy prior to library prep, followed by Illumina HiSeq sequencing, separation of target from IS reads using custom splitter, then pipeline analysis on Qiagen CLC Genomics Workbench. Results: Analysis of each target relative to IS reliably identified base-substitution sequencing errors at each measured actionable mutation site and determined that VAF for each was 200,000 reads per target in each sample, with analysis relative to IS spike-ins, 20,000 reads was sufficient (10,000 reads for the specimen and 10,000 reads for the spike-in). Because the IS spike-in molecules were validated to be wild-type, any variants were assumed to be due to technical error. Thus, a z-score comparing sequence count for a) specimen wild-type, b) specimen mutant, c) IS wild-type, and d) IS mutant enabled calculation of confidence level regarding each biological variant call. Conclusion: Preliminary data indicate that IS spike-in mixtures enable reliable analysis of ctDNA mutation fraction to LOD of 0.5% without need for UMI. Disclaimer: The views expressed here are those of the authors only and do not necessarily express the views/policies of the FDA. Citation Format: Daniel J. Craig, Erin L. Crawford, Joshua Xu, Thomas M. Blomquist, Leihong Wu, Thomas Morrison, James C. Willey. Novel method for NGS analysis of actionable mutations in circulating tumor DNA specimens: improved quality control and 20-fold lower sequencing required [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 432.

Targeted next generation sequencing (NGS) panel testing is now a standard of care for management of advanced stage cancer. At present, most targeted NGS oncology testing occurs with formalin fixed paraffin embedded (FFPE) tissue. This is due to the established utility and prevalence of FFPE in routine histologic classification, grading, staging, and ancillary testing. In addition, FFPE materials are an optimally suited medium for determining adequacy of the specimen prior to downstream NGS targeted panel testing. For these reasons, as well as a growing trend of minimally invasive procedures, clinical specimens are increasingly prioritized for FFPE to meet the bulk of standard oncology care. Currently, formalin fixation time guidelines do exist for specific immunohistochemical and in-situ hybridization oncology tests. However, specific FFPE processing guidelines have yet to be established that would optimize targeted NGS panel testing performance. In this study, our aim was to develop contrived FFPE materials using the FDA-led Sequencing Quality Control Phase II (SEQC2) consortium working group 2 (WG2) cell line materials. The overarching goal was to use these FFPE materials to identify an upper limit of formalin time associated with minimal impact on somatic variant calling performance in targeted NGS oncology panels. The SEQC2 - WG2 normal lymphoblast cell line was selected as a well-characterized test material to undergo systematic alteration in formalin fixation time (1, 2, 6 and 24 hours) prior to “routine” tissue processing and embedding (FFPE). These FFPE cell-block materials, derived from a single sample source (with both extracted FFPE DNA as well as shaved sections), were distributed to multiple labs running various targeted NGS amplicon and hybridization oncology panels. The expected outcome of these studies is to provide general guidance to the community on how formalin time in FFPE specimens impacts targeted NGS oncology panel testing performance. In addition, we hope to identify genomic regions (internal control regions) that serve as markers for the full range of formalin time FFPE-effect. This information will assist in improving the lower limit of detection for variant calling ( Citation Format: Thomas M. Blomquist, Erin Crawford, James Willey, Joshua Xu, Onco-panel Working Group of the Sequencing Quality Control Phase 2 (SEQC2) Consortium. Impact of formalin time on targeted NGS performance in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3539.

Background: Inter-individual variation in risk for lung cancer is based on variation in both exposure to environmental factors and genetic predisposition. As such, it is reasonable to hypothesize that prevalence of mutations in the airway epithelium will vary based on the combined effects of these lung cancer risk factors. Genes most commonly mutated in non-small cell lung cancer were recently reported from The Cancer Genome Atlas (TCGA) project. The purpose of this study was to use targeted deep sequencing to assess the prevalence of these mutations in bronchial epithelial cells (BEC) of individuals at high demographic risk for lung cancer. Methods: We enrolled 19 individuals with varying demographic risk for lung cancer, who were undergoing standard-of-care bronchoscopy into an IRB-approved research study in which we collected 1-5 million BEC via bronchoscopic brush biopsy and extracted genomic DNA. 10 subjects were smokers with lung cancer, 6 subjects were smokers without lung cancer, and 3 subjects were non-smokers without lung cancer. For targeted deep sequencing, we prepared synthetic spike-in competitive internal standards for each of 12 gene loci commonly mutated in lung cancer based on TCGA results and combined them into an IS mixture (ISM). DNA aliquots from each patient were quantified and mixed with equivalent genome equivalents of ISM prior to library preparation to control for technical artifacts associated with library preparation and NGS platform-specific error. The DNA input varied depending on sample quantity; there were 5 samples with 50,000 genome copies loaded, 12 samples with 100,000 genome copies loaded, and 2 samples with 1 million genome copies loaded. This approach enabled reliable measurement of 95% confidence limits of variant allele frequency (VAF), controlled for technical sequencing error, and allowed confident call of variants at a VAF of 0.1% and lower. Libraries for specimens from each of the 19 subjects were first sequenced using the Illumina MiSeq platform to ensure equal coverage across targets and samples. Deeper sequencing was then obtained using the Illumina NextSeq platform. Results: Preliminary results showed that smokers harbored mutations up to 1.6% in gene loci associated with non-small cell lung cancer according to TCGA, while none were identified in non-smoking controls. Conclusion: Initial results suggest that airway epithelial cells in smokers acquire somatic mutations that gradually lead to malignant conversion. Deep sequencing replicate experiments are underway to expand these results. Citation Format: Daniel J. Craig, Thomas M. Blomquist, Erin L. Crawford, James C. Willey. Targeted deep sequencing of driver mutations in airway epithelial cells from smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3537.

Inter-individual variation in CCAAT/enhancer binding protein gamma (CEBPG) transcript expression in normal human bronchial epithelial cells (NBEC) is associated with predisposition to lung cancer. We hypothesize that this inter-individual variation is in part explained by cis-acting genetic variation in CEBPG. To test this hypothesis we measured transcript expression derived from each parental copy of CEBPG (ie, allele-specific expression; ASE). There was a significant 2.9-fold higher cell cycle-specific variation in ASE of CEBPG rs2772 A compared to C allele ( P < 0.001). In 20% of NBEC samples, CEBPG rs2772 A allele was expressed on average 2.10 fold greater than rs2772 C allele. These data support the hypothesis that genetic variation in linkage disequilibrium with rs2772 influences regulation of CEBPG transcript expression through a trans-effect downstream of RNA polymerase II transcription and confirm that cis-acting genetic variation contributes to inter-individual variation in CEBPG transcript expression in NBEC, which is associated with variation in lung cancer risk.

Reliable breakpoint cluster region (BCR)--Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 10(3) GUSB values were significantly reduced (P = 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 10(3) GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection.

Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world's most important finfish diseases, killing80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR(®) green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR(®) green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0×10(0)-1.2×10(5) VHSv/10(6)actb1 molecules in wild caught fishes and 1.0×10(0)-8.4×10(5) molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean=1.9×10(4)) than those without (1.1×10(3)) (p0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv.

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.

Background: Genome-wide association studies (GWAS) and candidate gene studies suggest that telomere maintenance genes participate in etiology of lung cancer, idiopathic pulmonary fibrosis, and COPD. Telomeres are nucleoprotein structures located at the end of chromosomes that are maintained by telomerase encoded by hTERT. Telomerase is active in cancers, has the ability to aid in rapid cell division, and is dysfunctional in chronic conditions induced by inflammation as such as diabetes, renal failure, and COPD. hTERT is a known downstream target of β-catenin and WNT signaling. However, little is known regarding expression levels of these genes in normal bronchial epithelial cells (NBEC). The purpose of this study is to investigate inter-individual variation in regulation of β-catenin and WNT in NBEC, and if present, identify variants in regulatory region of human telomerase reverse transcriptase (hTERT) that could be used as candidate biomarkers for lung cancer risk. Methods: NBEC specimens from subjects with COPD (n=5) or without COPD (n=9) were obtained by bronchoscopy brush under IRB approved protocol. β-catenin, and WNT4 were measured by competitive multiplex-RT-PCR. Results: Both β-catenin and WNT4 displayed significant inter-individual variation as measured by Intra-Class Correlation (ICC) analysis (β-catenin ICC = 0.47, 95% Confidence Interval (0.2, 0.55), p < 0.05; WNT4 ICC = 0.60, 95% Confidence Interval (0.35, 0.85), p < 0.05). In patients with COPD, WNT4 expression was significantly higher in current smokers compared to former smokers (p Citation Format: Rose Zolondek, Daniel J. Craig, Erin L. Crawford, James C. Willey. Inter-individual variation in hTERT regulation pathway genes in normal bronchial epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1488.

The human genome is exposed to a variety of exogenous and endogenous assaults capable of inducing DNA damage and contributing to diseases like cancer. Multiple DNA repair mechanisms are tasked with identifying and repairing these aberrant nucleotides, but damage is occasionally missed giving rise to somatic mutations. As we age, the relative abundance of somatic mutations increases, which may increase our risk for developing cancer. In addition, inter-individual variation in somatic mutation prevalence suggests an inter-individual variation in (a) inherited DNA repair capacity and/or (b) exposure to environmental mutagens. Therefore, somatic mutation prevalence may serve as an end-point biomarker for cancer risk, as it provides critical information regarding both. While Next Generation Sequencing (NGS) has provided unparalleled access to the human genome and revolutionized the field of cancer genomics, its implementation into the clinical setting has been limited by technical errors introduced during library preparation and on the sequencing platform. This makes identification of rare variants below 10-4 mutations per nucleotide through NGS a challenge, and the use of somatic mutations to characterize cancer risk unfeasible. To address this issue, unique molecular indexes (UMI) were assigned to each genomic copy, enabling reliable identification of rare somatic mutations versus technical errors. We hypothesize that measurement of somatic mutation prevalence in normal bronchial epithelial cells (NBEC) will serve as an end-point biomarker for lung cancer risk by identifying both (a) inherited sub-optimal DNA repair/protection and (b) exposure to inhaled environmental mutagens including components of cigarette smoke, radon, and/or occupational hazards. In our initial pilot study, we used genomic DNA derived from an A549 cell line to optimize an ERCC5 gene-specific, dual-indexed PCR method for UMI assignment, followed by sequencing on the Illumina MiSeq platform. In parallel analyses, UMI correction using 25,000 genomic copies provided over 10-fold greater sensitivity in identifying somatic mutations compared to no UMI correction. Based on this pilot, we obtained three clinical NBEC specimens via cytology brush biopsy of grossly normal (non-cancerous) airway from (a) heavy smoker with lung cancer, (b) heavy smoker without lung cancer, and (c) non-smoking control. UMIs were assigned to 50,000 genomic copies at 16 loci, increasing our theoretical limit of detection to approximately 1 in 4x107 nucleotides. Results thus far support further application of this approach in studies to assess NBEC somatic mutation prevalence as a biomarker for cancer risk. Citation Format: Daniel J. Craig, Mazzin Elsamaloty, Thomas M. Blomquist, Erin L. Crawford, James C. Willey. Using rare variants to characterize lung cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2222.

Background: The potential for next generation sequencing (NGS) analysis in personalized diagnosis and treatment of cancer is advancing rapidly. Measurement of mutation fraction less than 5% may be clinically relevant in analysis of circulating tumor DNA and/or small cytologic samples. However, due to uncertainty introduced by variation in sample quality, the mutation fraction lower limit of detection (LOD) reported by clinical laboratories does not typically extend beyond this 5% threshold. Even with a more conservative LOD criterion, (e.g., 20% mutation fraction), poor harmony may result from inter-laboratory variation in skills to assess sample quality as well as specimen characteristics that are not evident even to expert clinical pathologists. Thus, a critical unmet need is methods that harmonize reporting of mutation fraction measured by NGS by accurately estimating specimen-specific confidence limits for each measurement. Methods: We designed a mixture of spike-in competitive internal standards for tumor somatic mutations in 32 currently actionable genes that enable quality-controlled measurement by targeted NGS using either PCR-amplicon or hybrid-capture enrichment. We also developed a data analysis pipeline suitable to separate target and internal standard sequencing reads prior to analysis of target copy number and sequencing errors. Through participation in the FDA-led SEquencing Quality Control Project Phase 2 (SEQC2), where a set of highly characterized cell line mixtures was developed to assess cross-site reproducibility and mutation detection accuracy of multiple targeted sequencing platforms, we aim to comprehensively evaluate the utility of the spike-in mixture with various targeted NGS panels to measure 95% confidence limits around each somatic mutation fraction measurement. Results: In pilot studies, combining the mixture of internal standards with specimens prior to NGS library preparation enabled reliable measurement of 95% confidence limits around each somatic mutation measurement. Further, using the internal standards to control for specimen adequacy enabled reliable measurement of actionable mutation fraction at as low as 0.01% in suitable specimens. Conclusion: We expect that the full cross-site SEQC2 study will provide further support for the role of competitive spike-in mixtures to provide reliable 95% confidence limits around each measurement of somatic mutation fraction in a clinical specimen. Disclaimer: The views expressed here are those of the authors only and do not necessarily express the views/policies of the FDA. Citation Format: James C. Willey, Thomas M. Blomquist, Erin L. Crawford, Joshua Xu, Leihong Wu, Thomas Morrison. Inter-laboratory harmonization of next generation sequencing somatic mutation assays for cancer response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1623.

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

In previous studies, we reported that key antioxidant and DNA repair genes are regulated differently in normal bronchial epithelial cells of lung cancer cases compared with non–lung cancer controls. In an effort to develop a biomarker for lung cancer risk, we evaluated the transcript expressions of 14 antioxidant, DNA repair, and transcription factor genes in normal bronchial epithelial cells (HUGO names CAT, CEBPG, E2F1, ERCC4, ERCC5, GPX1, GPX3, GSTM3, GSTP1, GSTT1, GSTZ1, MGST1, SOD1, and XRCC1). A test comprising these 14 genes accurately identified the lung cancer cases in two case-control studies. The receiver operating characteristic–area under the curve was 0.82 (95% confidence intervals, 0.68–0.91) for the first case-control set (25 lung cancer cases and 24 controls), and 0.87 (95% confidence intervals, 0.73–0.96) for the second set (18 cases and 22 controls). For each gene included in the test, the key difference between cases and controls was altered distribution of transcript expression among cancer cases compared with controls, with more lung cancer cases expressing at both extremes among all genes (Kolmorogov-Smirnov test, D = 0.0795; P = 0.041). A novel statistical approach was used to identify the lower and upper boundaries of transcript expression that optimally classifies cases and controls for each gene. Based on the data presented here, there is an increased prevalence of lung cancer diagnosis among individuals that express a threshold number of key antioxidant, DNA repair, and transcription factor genes at either very high or very low levels in the normal airway epithelium. [Cancer Res 2009;69(22):8629–35]

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.

bisulfite converted and purified from 3.5ml plasma samples utilizing a commercially available kit. In a casecontrol design 149 specimens from lung cancer patients and age-matched healthy individuals were evaluated. The diagnostic performance was compared to the results from a previously conducted training study on 151 specimens. Data and Results: For the training study including 59 LC subjects the performance of the marker panel reached very high sensitivity (78–95%) at reasonable rates of false positives (5–36%). The area under the receiver operating characteristic (ROC) curve was AUC 1⁄4 0.91. This clinical performance was confirmed in a similar sized marker validation study of an independent set of 149 patients. A total of 49 LC cases and 100 age-matched clinical controls were tested. At 95% specificity level sensitivity was 67%. Sensitivity of 95% was observed at a specificity level of 62%. Sensitivitywas evenly high acrossmajor histological types and stage groups. The AUC 1⁄4 0.94 observed in the testing studywas not statistically different from the result of the training set (p-value > 0.05). Conclusions: The combined analysis of the two DNA methylation markers SHOX2 and PTGER4 can be used to detect lung cancer in plasma samples with very high sensitivity at reasonable false positive rate. These findings were derived in a training study and confirmed in this marker validation study. The marker panel is being used as the basis for the development of a blood-based lung cancer diagnostic tool with very high sensitivity. This new diagnostic tool may ultimately show clinical utility in combination with current imaging techniques by reducing the false positive rate of such methods and has the potential for broader applications in definition of the at-risk screening population.

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 60 CA cases and 60 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: The best classifier comprised 11 features including age and 10 transcript abundance assays representing nine genes; specifically, CDKN1A, E2F1, ERCC5, GPX1, GSTP1, KEAP1, TP63, XPA, and XRCC1. After cross-validation, the Receiver Operator Characteristic area under the curve was 0.94 (95% CI: 0.92-0.96) and overall classification accuracy was 88.3% (95% CI 82.4%-94.1%). Using a threshold set at 95% sensitivity, specificity was > 70%. Conclusions: The LCRT biomarker reported here displayed high specificity 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. Citation Format: Jiyoun Yeo, Erin Crawford, Xiaolu Zhang, Sadik Khuder, Tian Chen, Albert Levin, Thomas Blomquist, James Willey. A lung cancer risk classifier comprising genome maintenance genes measured in normal bronchial epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4737. doi:10.1158/1538-7445.AM2017-4737

Background Reverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed. Methods We developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples. Results Reagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R2>0.99), signal-to-analyte response (slope 1.0±0.05), lower detection threshold (

Radon exposure is associated with increased risk for bronchogenic carcinoma. Mutagenesis analyses have revealed that radon induces mostly multi-locus chromosome deletions. Based on these findings, it was hypothesized that deletion analysis of multiple radon-induced malignant transformants would reveal common mutations in chromosomal regions containing tumor suppressor genes responsible for malignant transformation. This hypothesis was supported by a previous study in which tumorigenic derivatives of the human papillomavirus 18-immortalized human bronchial epithelial cell line BEP2D were established following irradiation with 30 cGy of high linear energy transfer radon-simulated alpha-particles. Herein, we describe the analyses of 10 additional tumorigenic derivative cell lines resulting from the irradiation of five additional independent BEP2D populations. The new transformants have common cytogenetic changes, including the loss of chromosome (ch)Y, one of three copies of ch8, one of two copies of ch11p15-pter and one of three copies of ch14. These changes are the same as those reported previously. Analysis of PCR-amplified short tandem repeats of informative loci confirmed the loss of heterozygosity (LOH) at 12 loci spanning the length of ch8 in cell lines from four of the total of eight irradiation treatments to date and the loss of chY in all cell lines (8 of 8). LOH analysis with a total of 17 informative loci confirmed loss on ch14 in transformants from seven of eight irradiation treatments and indicated a 0.5-1.7 cM region of common involvement centered around locus D14S306. No LOH was detected at any of the informative loci on ch11. The overall results support our stated hypothesis. Further studies are currently in progress to determine whether the ch8 and ch14 regions contain genes with tumor suppressor function in bronchial epithelial cells.

Progress toward complete sequencing of all human genes through the Human Genome Project has already resulted in a need for methods that allow quantitative expression measurement of multiple genes simultaneously. It is increasingly recognized that relative measurement of multiple genes will provide more mechanistic information regarding cell pathophysiology than measurement of individual genes one by one or by methods that do not allow direct intergene comparison. In this study, previously described quantitative reverse transcription-polymerase chain reaction methods were modified in an effort to provide a rapid, simple method for this purpose. Internal standard competitive templates (CTs) were prepared for each gene and were combined in a single solution containing CTs for more than 40 genes at defined concentrations relative to one another. Any subsequent dilution of the CT mixture did not alter the relationship of one CT to another. Because the same CT standard solution or a dilution of it was used in all experiments, data obtained from different experiments were easily compared. The use of multiple CT mixtures with different housekeeping gene to target gene ratios provided a linear dynamic range spanning the range of expression of all genes thus far evaluated. CT stock solutions were used to simultaneously quantify the expression of 25 genes relative to beta-actin and glyceraldehyde-3-phosphate dehydrogenase in normal and malignant bronchial epithelial cells. Because the CT concentrations were known, data in the form of both absolute messenger RNA (mRNA) copy number and mRNA relative to housekeeping gene mRNA were obtained. The methods and reagents described will allow rapid, quantitative measurement of multiple genes simultaneously, using inexpensive and widely available equipment. Furthermore, the CT standard solution may be distributed to other investigators for interlaboratory standardization of experimental conditions.

Orthologous introns have identical positions relative to the coding sequence in orthologous genes of different species. By analyzing the complete genomes of five plants we generated a database of 40,512 orthologous intron groups of dicotyledonous plants, 28,519 orthologous intron groups of angiosperms, and 15,726 of land plants (moss and angiosperms). Multiple sequence alignments of each orthologous intron group were obtained using the Mafft algorithm. The number of conserved regions in plant introns appeared to be hundreds of times fewer than that in mammals or vertebrates. Approximately three quarters of conserved intronic regions among angiosperms and dicots, in particular, correspond to alternatively-spliced exonic sequences. We registered only a handful of conserved intronic ncRNAs of flowering plants. However, the most evolutionarily conserved intronic region, which is ubiquitous for all plants examined in this study, including moss, possessed multiple structural features of tRNAs, which caused us to classify it as a putative tRNA-like ncRNA. Intronic sequences encoding tRNA-like structures are not unique to plants. Bioinformatics examination of the presence of tRNA inside introns revealed an unusually long-term association of four glycine tRNAs inside the Vac14 gene of fish, amniotes, and mammals.

Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.

The number of one of the grants from the National Cancer Institute is incorrect. The Funding Statement should read: "Significant portions of this study were paid for with funding provided by National Institutes of Health, National Cancer Institute (RC2-{"type":"entrez-nucleotide","attrs":{"text":"CA148572","term_id":"35049744","term_text":"CA148572"}}CA148572 and IMAT R21-CA138397) and National Heart Lung and Blood Institute (RO1-{"type":"entrez-nucleotide","attrs":{"text":"HL108016","term_id":"1051681137","term_text":"HL108016"}}HL108016); and the University of Toledo Medical Center George Isaac Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Background: Antioxidant (AO), DNA repair (DNAR), and cell cycle control (CCC) genes play a role in protecting normal bronchial epithelial cells (NBEC) from damage, and sub-optimal function is associated with risk for COPD and lung cancer. CEBP transcription factors regulate key AO and DNA repair genes in NBEC. In this study, we investigated the association between CEBP transcription factor function and regulation of these gene pathways in NBEC of cancer (CA) and non-cancer (NC) COPD subjects. Methods: NBEC samples were obtained by bronchoscopy from 66 CA cases and 60 matched NC controls, and 30 NC COPD and 30 NC non-COPD controls defined by spirometry (FEV1 Citation Format: Jiyoun Yeo, Erin Crawford, Xiaolu Zhang, Albert Levin, James Willey. Altered regulation of CEBP transcription factor family in normal bronchial epithelial cells of subjects with lung cancer or COPD. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1983.