Lung cancer risk test trial: study design, participant baseline characteristics, bronchoscopy safety, and establishment of a biospecimen repository.

BACKGROUND: The Lung Cancer Risk Test (LCRT) trial is a prospective cohort study comparing lung cancer incidence among persons with a positive or negative value for the LCRT, a 15 gene test measured in normal bronchial epithelial cells (NBEC). The purpose of this article is to describe the study design, primary endpoint, and safety; baseline characteristics of enrolled individuals; and establishment of a bio-specimen repository. METHODS/DESIGN: Eligible participants were aged 50-90 years, current or former smokers with 20 pack-years or more cigarette smoking history, free of lung cancer, and willing to undergo bronchoscopic brush biopsy for NBEC sample collection. NBEC, peripheral blood samples, baseline CT, and medical and demographic data were collected from each subject. DISCUSSION: Over a two-year span (2010-2012), 403 subjects were enrolled at 12 sites. At baseline 384 subjects remained in study and mean age and smoking history were 62.9 years and 50.4 pack-years respectively, with 34% current smokers. Obstructive lung disease (FEV1/FVC <0.7) was present in 157 (54%). No severe adverse events were associated with bronchoscopic brushing. An NBEC and matched peripheral blood bio-specimen repository was established. The demographic composition of the enrolled group is representative of the population for which the LCRT is intended. Specifically, based on baseline population characteristics we expect lung cancer incidence in this cohort to be representative of the population eligible for low-dose Computed Tomography (LDCT) lung cancer screening. Collection of NBEC by bronchial brush biopsy/bronchoscopy was safe and well-tolerated in this population. These findings support the feasibility of testing LCRT clinical utility in this prospective study. If validated, the LCRT has the potential to significantly narrow the population of individuals requiring annual low-dose helical CT screening for early detection of lung cancer and delay the onset of screening for individuals with results indicating low lung cancer risk. For these individuals, the small risk incurred by undergoing once in a lifetime bronchoscopic sample collection for LCRT may be offset by a reduction in their CT-related risks. The LCRT biospecimen repository will enable additional studies of genetic basis for COPD and/or lung cancer risk. TRIAL REGISTRATION: The LCRT Study, NCT 01130285, was registered with Clinicaltrials.gov on May 24, 2010.

CEBPG regulates ERCC5/XPG expression in human bronchial epithelial cells and this regulation is modified by E2F1/YY1 interactions.

Marked inter-individual variation in lung cancer risk cannot be accounted for solely by cigarette smoke and other environmental exposures. Evidence suggests that variation in bronchial epithelial cell expression of key DNA repair genes plays a role. Variation in these genes correlates with variation in expression of CEBPG and E2F1 transcription factors. Here, we investigated the mechanistic basis for correlation of the DNA repair gene ERCC5 (previously known as XPG) with CEBPG and E2F1. CEBPG expression vector transfected into H23 or H460 cell lines up-regulated endogenous ERCC5 and also luciferase from a reporter construct containing 589 bp of ERCC5 5' regulatory region. A recognition site for CEBPG and a region containing sites for YY1 on the sense strand and E2F1 on the anti-sense strand participated in CEBPG up-regulation of ERCC5. CEBPG, E2F1 and YY1 binding to their respective sites were confirmed by electrophoretic mobility shift assay. Thus, we conclude that CEBPG regulates ERCC5 expression and this regulation is modified by E2F1/YY1 interactions. Several polymorphisms have been identified in these regions and, based on the data presented here, it is reasonable to hypothesize that they may contribute to risk for bronchogenic carcinoma.

Normal bronchial epithelial cell expression of glutathione transferase P1, glutathione transferase M3, and glutathione peroxidase is low in subjects with bronchogenic carcinoma.

Normal bronchial epithelial cells (NBECs) are at risk for damage from inhaled and endogenous oxidative species and from epoxide metabolites of inhaled polycyclic aromatic hydrocarbons. Epidemiological and in vitro data suggest that interindividual variation in this risk may result from variation in NBEC expression of enzymes that inactivate reactive species by conjugating them to glutathione. Quantitative competitive reverse transcription-PCR was used to measure mRNA levels of glutathione transferases (GSTs) and glutathione peroxidases (GSHPxs) in primary NBECs from subjects with or without bronchogenic carcinoma. Mean expression levels (mRNA/10(3) beta-actin mRNA) in NBECs from 23 subjects without bronchogenic carcinoma compared to those from 11 subjects with bronchogenic carcinoma respectively (in parentheses) were: mGST (26.0, 6.11), GSTM3 (0.29, 0.09), combined GSTM1,2,4,5 (0.98, 0.60), GSTT1 (0.84, 0.76), GSTP1 (287, 110), GSHPx (140, 62.1), and GSHPxA (0.43, 0.34). Levels of GSTP1, GSTM3, and GSHPx were significantly (P < 0.05) lower in NBECs from subjects with bronchogenic carcinoma. Further, the gene expression index formed by multiplying the values for mGST x GSTM3 x GSHPx x GSHPxA x GSTP1 had a sensitivity (90%) and specificity (76%) for detecting NBECs from bronchogenic carcinoma subjects that was better than any individual gene. In cultured NBECs derived from eight individuals without bronchogenic carcinoma and incubated under identical conditions such that environmental effects were minimized, the mean level of expression and degree of interindividual variation for each gene evaluated was less than that observed in primary NBECs. Data from these studies support the hypotheses that (a) interindividual variation in risk for bronchogenic carcinoma results in part from interindividual variation in NBEC expression of antioxidant genes; (b) gene expression indices will better identify individuals at risk for bronchogenic carcinoma than individual gene expression values; and (c) both hereditary and environmental exposures contribute to the level of and interindividual variation in gene expression observed in primary NBECs. Many epidemiological studies have been designed to evaluate risk associated with polymorphisms or gene expression levels of putative susceptibility genes based on measurements in surrogate tissues, such as peripheral blood lymphocytes. Based on data presented here, it will be important to include the assessment of NBECs in future studies. Measurement of antioxidant gene expression in NBECs may identify the 5-10% of individuals at risk for bronchogenic carcinoma. Bronchoscopic sampling of NBECs from smokers and ex-smokers then will allow susceptible individuals to be entered into surveillance and/or chemoprevention studies.

Reproducible gene expression measurement among multiple laboratories obtained in a blinded study using standardized RT (StaRT)-PCR.

BACKGROUND: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. METHODS AND RESULTS: In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. CONCLUSION: Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.

Loss of spr1 expression measurable by quantitative RT-PCR in human bronchogenic carcinoma cell lines.

Expression of the small, proline-rich protein (spr1) squamous differentiation marker was measured in five cultured normal and 12 malignant human bronchial epithelial cell (BEC) populations by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Whereas spr1 expression was quantifiable and inducible in all five cultured normal cell populations, in all 12 carcinoma cell lines evaluated it was neither quantifiable nor inducible. Primers spanning the entire spr1 coding sequence amplified full-length PCR product from genomic DNA; therefore, large deletions in the coding region were not responsible for the loss of expression measurable by RT-PCR. This is the first molecular genetic marker reported that distinguishes all normal from all carcinoma cell populations evaluated. Because the spr1 protein is a component of the crosslinked envelope that forms during the squamous differentiation process, we hypothesize that the apparent loss of spr1 gene expression disrupts mechanisms for terminal squamous differentiation in the bronchial epithelium, thereby contributing to malignant transformation.

Effect of acyclovir and interferon on human hematopoietic progenitor cells.

Continuous in vitro exposure of human bone marrow cells to acyclovir (approximately 200 microM) or human leukocyte interferon (approximately 250 U/ml) caused 50% inhibition of granulocyte colony-forming cell differentiation. Colonies expressed in the presence of either agent were reduced both in size and number. Erythroid progenitors were more resistant than granulocyte progenitors to the antiproliferative effects of acyclovir. Progenitor cells of patients recovering from cytotoxic chemotherapy were no more sensitive to the effects of acyclovir or interferon than were cells obtained from patients before chemotherapy.

Localization of tumor suppressor gene candidates by cytogenetic and short tandem repeat analyses in tumorigenic human bronchial epithelial cells.

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.

Expression measurement of many genes simultaneously by quantitative RT-PCR using standardized mixtures of competitive templates.

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.

The gene expression index c-myc x E2F-1/p21 is highly predictive of malignant phenotype in human bronchial epithelial cells.

Recent methodological developments allow expression measurement of many genes simultaneously, thereby revealing patterns of gene expression that can be related to phenotype. We hypothesized that through the use of such methods we could identify patterns of gene expression associated with the malignant phenotype in human bronchial epithelial cells (BEC). To test this hypothesis, a recently developed quantitative reverse transcriptase polymerase chain reaction method was used to assess simultaneously expression of 15 genes mechanistically associated with cell-cycle control (c-myc, E2F-1, p21, rb, PCNA, cyclin D2, cyclin D3, cyclin E, cdc2, CDK2, CDK4, mad, max p21, max p22, and p53) in normal cell cultures from five individuals and in nine different malignant BEC lines. Relative to the mean expression levels in cultured normal cell populations, expression of c-myc, E2F-1, PCNA, cyclin E, and CDK4 messenger RNA (mRNA) were significantly increased and expression of p21 and p53 mRNA were significantly decreased in one or two, but not all three subtypes (squamous, adenocarcinoma and small cell) of carcinoma cell lines evaluated. No single cell-cycle control gene discriminated all three subtypes from normal cell populations. In contrast, the gene expression index c-myc x E2F-1/p21 separated all carcinoma cell lines from all normal cell populations initially evaluated. This malignancy index was validated in an additional three cultured normal BEC and three carcinoma cell lines, as well as three pairs of matched primary normal bronchial epithelial and primary bronchogenic carcinoma samples, and three pairs of matched primary normal lung parenchyma and primary bronchogenic carcinoma tissue. Again, the c-myc x E2F-1/ p21 index successfully discriminated all cultured and primary normal from malignant samples and thereby had a predictive value of 1 (no false positives and no false negatives). We hypothesize that because of functional mutations in cell-cycle regulatory genes (e.g., p53 and/or rb), cells lose the ability to maintain a pattern of gene expression mechanistically associated with normal, division-limited homeostatic equilibrium. Because the c-myc x E2F-1/p21 gene expression index has high specificity for malignant tissue, it will allow confirmation that there is a significant amount of tumor tissue present in small (e.g., fine-needle) biopsy specimens prior to evaluating them for expression of other genes, such as those involved in chemoresistance or radioresistance. In addition, the goal of most gene therapy efforts is to alter levels of gene expression quantitatively. This index and others derived in a similar manner may better define potential gene therapy targets as well as response of targeted genes to therapy.

Measurement of cytochrome P450 2A6 and 2E1 gene expression in primary human bronchial epithelial cells.

Bronchogenic carcinomas arise from bronchial epithelial cells (BECs). Inhalation exposure of BECs to nitrosamines in cigarette smoke is an important exogenous risk factor for malignant transformation of BECs. Thus, an important endogenous risk factor is likely to be the capacity of BECs to metabolize nitrosamines. Among the cytochrome P450 enzymes capable of metabolizing nitrosamines, CYP2A6, CYP2E1 and CYP2B6 are expressed in BECs. In this study, we used quantitative RT-PCR to evaluate expression of CYP2A6 and CYP2E1 in primary human BECs from 12 non-smokers and eight smokers. CYP2A6 was expressed in 20/20 cases and quantifiable in 18/20 cases, with a mean level of 580 mRNA/10(6) beta-actin mRNA. CYP2E1 expression was observed in 9/20 cases, but in all cases it was expressed at levels below our limit of quantification (10 mRNA/10(6) beta-actin mRNA). There was significant (P < 0.05) 20-fold inter-individual variation in expression of CYP2A6. Further, the mean level of CYP2A6 among smokers (260 mRNA/10(6) beta-actin mRNA) was significantly lower than among non-smokers (740 mRNA/10(6) beta-actin mRNA). It is hypothesized that: (i) inter-individual variation in CYP2A6 gene expression may contribute to inter-individual variation in risk for bronchogenic carcinoma; (ii) smoking may reduce the level of expression of CYP2A6 in the BECs of some individuals; and (iii) CYP2A6 is more important than CYP2E1 for metabolic activation of nitrosamines in bronchial epithelial cells.