James C. Willey

Evaluation of DNA microarray results with quantitative gene expression platforms

We have evaluated the performance characteristics of three quantitative gene expression technologies and correlated their expression measurements to those of five commercial microarray platforms, based on the MicroArray Quality Control (MAQC) data set. The limit of detection, assay range, precision, accuracy and fold-change correlations were assessed for 997 TaqMan Gene Expression Assays, 205 Standardized RT (Sta)RT-PCR assays and 244 QuantiGene assays. TaqMan is a registered trademark of Roche Molecular Systems, Inc. We observed high correlation between quantitative gene expression values and microarray platform results and found few discordant measurements among all platforms. The main cause of variability was differences in probe sequence and thus target location. A second source of variability was the limited and variable sensitivity of the different microarray platforms for detecting weakly expressed genes, which affected interplatform and intersite reproducibility of differentially expressed genes. From this analysis, we conclude that the MAQC microarray data set has been validated by alternative quantitative gene expression platforms thus supporting the use of microarray platforms for the quantitative characterization of gene expression.

Using RNA sample titrations to assess microarray platform performance and normalization techniques

We have assessed the utility of RNA titration samples for evaluating microarray platform performance and the impact of different normalization methods on the results obtained. As part of the MicroArray Quality Control project, we investigated the performance of five commercial microarray platforms using two independent RNA samples and two titration mixtures of these samples. Focusing on 12,091 genes common across all platforms, we determined the ability of each platform to detect the correct titration response across the samples. Global deviations from the response predicted by the titration ratios were observed. These differences could be explained by variations in relative amounts of messenger RNA as a fraction of total RNA between the two independent samples. Overall, both the qualitative and quantitative correspondence across platforms was high. In summary, titration samples may be regarded as a valuable tool, not only for assessing microarray platform performance and different analysis methods, but also for determining some underlying biological features of the samples.

ABCC5, ERCC2, XPA and XRCC1 transcript abundance levels correlate with cisplatin chemoresistance in non-small cell lung cancer cell lines

BackgroundAlthough 40–50% of non-small cell lung cancer (NSCLC) tumors respond to cisplatin chemotherapy, there currently is no way to prospectively identify potential responders. The purpose of this study was to determine whether transcript abundance (TA) levels of twelve selected DNA repair or multi-drug resistance genes (LIG1, ERCC2, ERCC3, DDIT3, ABCC1, ABCC4, ABCC5, ABCC10, GTF2H2, XPA, XPC and XRCC1) were associated with cisplatin chemoresistance and could therefore contribute to the development of a predictive marker. Standardized RT (StaRT)-PCR, was employed to assess these genes in a set of NSCLC cell lines with a previously published range of sensitivity to cisplatin. Data were obtained in the form of target gene molecules relative to 106 β-actin (ACTB) molecules. To cancel the effect of ACTB variation among the different cell lines individual gene expression values were incorporated into ratios of one gene to another. Each two-gene ratio was compared as a single variable to chemoresistance for each of eight NSCLC cell lines using multiple regression. In an effort to validate these results, six additional lines then were evaluated.ResultsFollowing validation, single variable models best correlated with chemoresistance (p < 0.001), were ERCC2/XPC, ABCC5/GTF2H2, ERCC2/GTF2H2, XPA/XPC and XRCC1/XPC. All single variable models were examined hierarchically to achieve two variable models. The two variable model with the highest correlation was (ABCC5/GTF2H2, ERCC2/GTF2H2) with an R2 value of 0.96 (p < 0.001).ConclusionThese results provide markers suitable for assessment of small fine needle aspirate biopsies in an effort to prospectively identify cisplatin resistant tumors.

MRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of β-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR × FPGH)/(RFC × FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.

Variation in transcriptional regulation of cyclin dependent kinase inhibitor p21waf1/cip1 among human bronchogenic carcinomas

BackgroundCell proliferation control depends in part on the carefully ordered regulation of transcription factors. The p53 homolog p73, contributes to this control by directly upregulating the cyclin dependent kinase inhibitor, p21waf1/cip1. E2F1, an inducer of cell proliferation, directly upregulates p73 and in some systems upregulates p21 directly. Because of its central role in controlling cell proliferation, upregulation of p21 has been explored as a modality for treating bronchogenic carcinoma (BC). Improved understanding of p21 transcriptional regulation will facilitate identification of BC tissues that are responsive to p21-directed therapies. Toward this goal, we investigated the role that E2F1 and p73 each play in the transcriptional regulation of p21.ResultsAmong BC samples (N = 21) p21 transcript abundance (TA) levels varied over two orders of magnitude with values ranging from 400 to 120,000 (in units of molecules/106 molecules β-actin). The p21 values in many BC were high compared to those observed in normal bronchial epithelial cells (BEC) (N = 18). Among all BC samples, there was no correlation between E2F1 and p21 TA but there was positive correlation between E2F1 and p73α (p < 0.001) TA. Among BC cell lines with inactivated p53 and wild type p73 (N = 7) there was positive correlation between p73α and p21 TA (p < 0.05). Additionally, in a BC cell line in which both p53 and p73 were inactivated (H1155), E2F1 TA level was high (50,000), but p21 TA level was low (470). Transiently expressed exogenous p73α in the BC cell line Calu-1, was associated with a significant (p < 0.05) 90% increase in p21 TA and a 20% reduction in E2F1 TA. siRNA mediated reduction of p73 TA in the N417 BC cell line was associated with a significant reduction in p21 TA level (p < 0.01).Conclusionp21 TA levels vary considerably among BC patients which may be attributable to 1) genetic alterations in Rb and p53 and 2) variation in TA levels of upstream transcription factors E2F1 and p73. Here we provide evidence that p73 upregulates p21 TA in BC tissues and upregulated p21 TA may result from E2F1 upregulation of p73 but not from E2F1 directly.

Stable low-level expression of p21WAF1/CIP1 in A549 human bronchogenic carcinoma cell line-derived clones down-regulates E2F1 mRNA and restores cell proliferation control

BackgroundDeregulated cell cycle progression and loss of proliferation control are key properties of malignant cells. In previous studies, an interactive transcript abundance index (ITAI) comprising three cell cycle control genes, [MYC × E2F1]/p21 accurately distinguished normal from malignant bronchial epithelial cells (BEC), using a cut-off threshold of 7,000. This cut-off is represented by a line with a slope of 7,000 on a bivariate plot of p21 versus [MYC × E2F1], with malignant BEC above the line and normal BEC below the line. This study was an effort to better quantify, at the transcript abundance level, the difference between normal and malignant BEC. The hypothesis was tested that experimental elevation of p21 in a malignant BEC line would decrease the value of the [MYC × E2F1]/p21 ITAI to a level below this line, resulting in loss of immortality and limited cell population doubling capacity. In order to test the hypothesis, a p21 expression vector was transfected into the A549 human bronchogenic carcinoma cell line, which has low constitutive p21 TA expression relative to normal BEC.ResultsFollowing transfection of p21, four A549/p21 clones with stable two-fold up-regulated p21 expression were isolated and expanded. For each clone, the increase in p21 transcript abundance (TA) was associated with increased total p21 protein level, more than 5-fold reduction in E2F1 TA, and 10-fold reduction in the [MYC × E2F1]/p21 ITAI to a value below the cut-off threshold. These changes in regulation of cell cycle control genes were associated with restoration of cell proliferation control. Specifically, each transfectant was capable of only 15 population doublings compared with unlimited population doublings for parental A549. This change was associated with an approximate 2-fold increase in population doubling time to 38.4 hours (from 22.3 hrs), resumption of contact-inhibition, and reduced dividing cell fraction as measured by flow cytometric DNA analysis.ConclusionThese results, likely due to increased p21-mediated down-regulation of E2F1 TA at the G1/S phase transition, are consistent with our hypothesis. Specifically, they provide experimental confirmation that a line with slope of 7,000 on the p21 versus [MYC × E2F1] bivariate plot quantifies the difference between normal and malignant BEC at the level of transcript abundance.

Effect of cigarette smoking on major histological types of lung cancer in men

This study examined the effect of cigarette smoking parameters such as intensity, duration, age at initiation, and quitting on the development of different histological types of lung cancer in men. We used data from a case-control study conducted in Philadelphia between 1985 and 1987. Cases included 482 men with histologically confirmed lung cancer diagnosed in 15 selected hospitals in Philadelphia. Controls were selected from a neighborhood survey of men in Philadelphia conducted concurrently to the case recruitment. Most aspects of smoking were associated with all the major histological types of lung cancer. Number of cigarettes smoked per day was the strongest predictor of risk of developing lung cancer. Early age at initiation of smoking significantly increased the risk of small cell carcinoma (odds ratio = 3.0; 95% CI, 1.1-8.4). Quitting smoking reduced the risk of squamous cell and adenocarcinoma; however, it did not affect the risk of small cell lung cancer. The findings of this study suggest the need for greater emphasis on smoking prevention programs, especially in adolescents.

Developing a sustainable process to provide quality control materials for genetic testing

Purpose: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community.Methods: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps.Results: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step.Conclusions: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

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.

Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR

BackgroundProbe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as β-actin (ACTB).ResultsThe NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired students t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns.ConclusionStaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further, it correlates well with Taqman real time PCR in terms of quantitative and discriminatory ability. This label-free, inexpensive technique may provide the ability to generate prognostically important molecular signatures unique to individual tumors and may enable identification of novel therapeutic targets.