Thomas M. Blomquist

Pattern of antioxidant and DNA repair gene expression in normal airway epithelium associated with lung cancer diagnosis

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.

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.

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.

A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

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 (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples. Conclusions This quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.

CEBPG Exhibits Allele-Specific Expression in Human Bronchial Epithelial Cells.

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.

Abstract 2890: ERCC5 variant rs2296147 T-allele creates a predicted TP53 binding site and up-regulates transcript abundance in normal bronchial epithelial cells, while rs17655 C-allele is linked to miRNA binding site variant and down regulates

Background: Excision repair cross-complementation group 5 (ERCC5) gene plays an important role in nucleotide excision repair (NER) and dysregulation of ERCC5 is associated with increased lung cancer risk. This study was conducted to characterize cis-acting genetic variants responsible for inter-individual variation in ERCC5 transcript regulation in normal bronchial epithelial cells (NBEC). Methods: We determined genotypes at putative ERCC5 cis-regulatory single nucleotide polymorphic sites (SNP) rs751402 and rs2296147, and marker SNPs rs1047768 and rs17655. Using a recently developed targeted sequencing method, ERCC5 allele-specific transcript abundance was assessed in NBEC RNA from 55 individuals heterozygous for rs1047768 and 21 subjects heterozygous for rs17655. Syntenic relationships among alleles at rs751402, rs2296147 and rs1047768 were assessed by allele-specific PCR followed by Sanger sequencing. We assessed association of NBEC ERCC5 allele-specific expression at rs1047768 with haplotype and diplotype structure at putative ERCC5 promoter cis-regulatory SNPs rs751402 and rs2296147. Results: Genotype analysis revealed higher inter-individual variation in allelic ratios in cDNA samples relative to matched gDNA samples at both rs1047768 and rs17655 (p Conclusions: These data support the conclusion that T allele at SNP rs2296147 creates a TP53 binding site and up-regulates ERCC5 while C allele at SNP rs873601 creates a miRNA binding site and down-regulates ERCC5. Variation in ERCC5 transcript abundance associated with allelic variation at these SNPs is likely associated with variation in NER function in NBEC and lung cancer risk. Citation Format: Xiaolu Zhang, Erin L. Crawford, Thomas M. Blomquist, Sadik A. Khuder, Jiyoun Yeo, Albert M. Levin, James C. Willey. ERCC5 variant rs2296147 T-allele creates a predicted TP53 binding site and up-regulates transcript abundance in normal bronchial epithelial cells, while rs17655 C-allele is linked to miRNA binding site variant and down regulates. [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 2890.

Quality Control Methods for Optimal BCR-ABL1 Clinical Testing in Human Whole Blood Samples

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.

Flexibility of BIV TAR-Tat: Models of peptide binding

Abstract A new approach in determining local residue flexibility from base-amino acid contact frequencies is applied to the twelve million lattice chains modeling BIV Tat peptide binding to TAR RNA fragment. Many of the resulting key features in flexibility correspond to RMSD calculations derived from a set of five NMR derived structures (X. Ye, R. A. Kumar, and D. J. Patel, Protein Data Bank: Database of three-dimensional structures determined from NMR (1996)) and binding studies of mutants (L. Chen and A. D. Frankel, Proc. Natl. Acad. Sci. USA 92, 5077–5081 (1995)). The lattice and RMSD calculations facilitate the identification of peptide hinge regions that can best utilize the introduction of Gly or other flexible residues. This approach for identifying potential sites amenable to substitution of more flexible residues to enhance peptide binding to RNA targets could be a useful design tool.

Abstract 4892: Methods for accurate reporting of confidence intervals in clinical applications of next generation sequencing (NGS)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA BACKGROUND: A challenge to clinical implementation of Next-Generation Sequencing (NGS) is lack of appropriate quality control including control for: a) adequate sample loading, b) variation in target amplification efficiency, and c) variation in loading of prepared NGS specimen onto sequencing platform. Polymerase chain reaction steps used in preparation for NGS can yield a large number of sequencing reads from a small number of starting nucleic acid molecules (e.g. small and/or degraded samples from FNA specimens or FFPE tissue), resulting in large stochastic sampling variation. At present, methods to quantify the analyzable fraction of target nucleic acid are not available or are not suitable for small specimens. As a result, current practice is to rely on sequencing coverage data that may provide false assurance of adequate specimen sampling during molecular analysis, and this has potential to negatively impact patient care. We hypothesize that coefficient of variation (CV) for amplicon-based NGS assay measurements is largely predicted by Poisson (i.e. stochastic) sampling effects for a nucleic acid target at two key points: 1) input molarity (i.e. number of intact molecules) and 2) sequencing coverage (i.e. read counts). METHODS: To test this hypothesis we developed three working models using Monte Carlo simulation and derived equations to predict expected CV based on sequence read count and/or intact molecules mesaured for a given nucleic acid target. These expectation models were tested against empirically derived data from cross-mixtures of two cell lines (H23 and H520) known to be homozygous for opposite alleles at four polymorphic sites (rs769217, rs1042522, rs735482 and rs2298881). Cell lines were mixed to produce limiting inputs of one allele relative to the other, then prepared for NGS such that a broad range of combinations of limiting allelic molecule inputs and/or sequence read counts were observed (46 sets of allelic measurement at all 4 loci). Intra-assay measurement of intact and amplifiable molecules was accomplished using recently described competitive multiplex-PCR amplicon-based NGS specimen preparation (Blomquist, et. al. 2013). RESULTS: Observed CV for measurement at varying input copies and sequencing read counts were compared to expected CV. Actual measured CV was on average 13- and 1.5-fold higher than expected CV based on sequencing reads or molecule input measurements alone, respectively. For the model derived from both sequencing reads and molecule input measurements, expected CV was very close to measured (average [measured CV/expected CV] = 1.01) and explained 74% of observed assay variance. CONCLUSIONS: NGS-based diagnostic tests that do not take into account both input concentration of intact target nucleic acid material and associated sequencing read coverage may not provide accurate reporting of confidence intervals in specimens with limited or degraded material. Citation Format: Erin L. Crawford, Thomas Blomquist, James C. Willey. Methods for accurate reporting of confidence intervals in clinical applications of next generation sequencing (NGS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4892. doi:10.1158/1538-7445.AM2015-4892

Abstract 3401: Inter-individual variation in MUC5B allele specific expression in normal bronchial epithelial cells and relationship to lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Familial interstitial pneumonia and idiopathic pulmonary fibrosis are associated with increased risk of lung cancer. Seibold et al (NEJM, 2011) recently identified a common variant in the putative promoter of MUC5B (rs35705950) associated with level of MUC5B expression in lung tissue and also with development of these diseases. The goal of this study was to determine whether the MUC5B rs35705950 variant is associated with a) MUC5B allele-specific expression (ASE) or total expression in normal bronchial epithelial cells (NBEC) or b) lung cancer risk. Through funding in part from RC2 CA148572 and HL108016 we collected NBEC samples from over 500 subjects with lung cancer or at risk for lung cancer. Methods: This was a pilot study of 85 subjects (26 cancer cases and 59 non-cancer controls). RNA was extracted from normal bronchial airway brush NBEC specimens of 85 subjects and reverse transcribed. Using next generation sequencing (NGS), allele-specific expression (ASE) was measured as allelic imbalance in each cDNA at three marker SNPs in MUC5B coding region (rs2075859, rs2943510, and rs4963059) selected for common (>0.05) minor allele frequency, using matched peripheral blood cell gDNA as a control. Specifically, each cDNA and matched gDNA sample was subjected to targeted competitive template multiplex PCR amplicon library generation followed by NGS (Blomquist et al, PLOS one, 2013) on Illumina Hiseq platform. This NGS method controlled for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. The genotype at putative cis-regulatory SNP rs35705950 was assessed in gDNA from 95 subjects (31 cancer cases and 64 non-cancer controls) including those assessed for ASE using a TaqMan® SNP assay. Results: There was significant (p 3 log) but not associated with lung cancer. Conclusions: These results support previous observations that there is significant inter-individual variation in cis-regulation of MUC5B in NBEC and suggest that this variation also may contribute to inherited lung cancer risk. Lack of association between rs35705950 genotype and MUC5B ASE may have been due to role of other cis-regulatory factors, and/or environmentally associated transfactors that contributed to large inter-individual variation in total MUC5B expression. Citation Format: Xiaolu Zhang, Jiyoun Yeo, Erin L. Crawford, Thomas M. Blomquist, James C. Willey. Inter-individual variation in MUC5B allele specific expression in normal bronchial epithelial cells and relationship to lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3401. doi:10.1158/1538-7445.AM2014-3401