Richard E. Crowell

The XRCC1 399 glutamine allele is a risk factor for adenocarcinoma of the lung

Defects in the repair and maintenance of DNA increase risk for cancer. X-ray cross-complementing group 1 protein (XRCC1) is involved with the repair of DNA single-strand breaks. A nucleotide substitution of guanine to adenine leading to a non-conservative amino acid change was identified in the XRCC1 gene at codon 399 (Arg/Gln). This change is associated with higher levels of aflatoxin B1-adducts and glycophorin A somatic mutations. A case-control study was conducted to test the hypothesis that the 399Gln allele is positively associated with risk for adenocarcinoma of the lung. XRCC1 genotypes were assessed at codon 399 in 172 cases of lung adenocarcinoma and 143 cancer-free controls. Two ethnic populations were represented, non-Hispanic White and Hispanic. The distribution of XRCC1 genotypes differed between cases and controls. Among cases, 47.7% were Arg/Arg, 35.5% were Arg/Gln, and 16.9% were Gln/Gln. Among controls, XRCC1 allele frequencies were 45.5% for Arg/Arg, 44.8% for Arg/Gln, and 9.8% for Gln/Gln. Logistic regression analysis was used to assess the association between lung adenocarcinoma and the G/G genotype relative to the A/A or A/G genotypes. In non-Hispanic White participants, the lung cancer risk associated with the G/G genotype increased significantly after adjustment for age (OR=2.81; 95% CI, 1.2-7.9; P=0.03) and increased further after adjustment for smoking (OR=3.25; 95% CI, 1.2-10.7; P=0.03). Among all groups, a significant association was found between the G/G homozygote and lung cancer (OR=2.45; 95% CI, 1.1-5.8; P=0.03) after adjustment for age, ethnicity, and smoking. This study links a functional polymorphism in the critical repair gene XRCC1 to risk for adenocarcinoma of the lung.

Gene Promoter Methylation in Plasma and Sputum Increases with Lung Cancer Risk

Purpose: Lung cancer is the leading cause of cancer mortality in the United States, due in part to the lack of a validated and effective screening approach for early detection. The prevalence for methylation of seven and three genes was examined in DNA from sputum and plasma, respectively, from women at different risk for lung cancer. Experimental Design: Lung cancer survivors (n = 56), clinically cancer-free smokers (n = 121), and never smokers (n = 74) comprised the study population. Plasma was collected from all three groups, whereas sputum was collected from lung cancer survivors and smokers. Results: Methylation was detected in plasma DNA from 10 of 74 women who never smoked. Prevalence for methylation of the p16 gene in plasma was highest in lung cancer survivors. Lung cancer survivors showed a significant increase in the odds of having at least one or more genes methylated in plasma (odds ratio, 3.6; 95% confidence interval, 1.9-9.1) than never smokers. The prevalence for methylation of the O6-methylguanine-DNA methyltransferase, ras effector homologue 1, death associated protein kinase, and PAX5α genes in sputum was significantly higher in lung cancer survivors compared with smokers. Lung cancer survivors had 6.2-fold greater odds (95% confidence interval, 2.1-18.5) for methylation of three or more genes in sputum compared with smokers. Methylation was more commonly detected in sputum than plasma for O6-methylguanine-DNA methyltransferase and ras effector homologue 1, but not p16, in lung cancer survivors. Conclusion: Concomitant methylation of multiple gene promoters in sputum is strongly associated with lung cancer risk.

Cardiopulmonary manifestations of hantavirus pulmonary syndrome.

OBJECTIVE To describe the clinical characteristics of a group of patients infected with the newly recognized hantavirus in the Southwestern United States. DESIGN Case series. SETTING Tertiary referral center. PATIENTS All patients with confirmed hantavirus infection admitted to the University of New Mexico Hospital between May 1, 1993 and January 1, 1994. INTERVENTIONS Records of patients with hantavirus infection were reviewed to collect all pertinent clinical data. MEASUREMENTS AND MAIN RESULTS Pulmonary disease in these patients was characterized by hypoxemia covering a wide range of severity. The cause of hypoxemia was an increased permeability (noncardiac) pulmonary edema which could be differentiated from hydrostatic (cardiac) pulmonary edema by its association with low pulmonary artery occlusion pressures and increased protein content of edema fluid. Hemodynamic measurements in severe cases showed a shock state characterized by a low cardiac index (range 1.6 to 3.0 L/min/min2), a low stroke volume index (range 10.5 to 29 mL/m2), and high systemic vascular resistance index (range 1,653 to 2,997 dyne.sec/cm5.m2). Progression to death was associated with worsening cardiac dysfunction unresponsive to treatment and causing oxygen debt and lactic acidosis. CONCLUSIONS The two major life-threatening pathophysiologic changes in Hantavirus Pulmonary Syndrome are increased permeability pulmonary edema, and an atypical form of septic shock caused by myocardial depression and hypovolemia.

Multivitamins, Folate, and Green Vegetables Protect against Gene Promoter Methylation in the Aerodigestive Tract of Smokers

One promising approach for early detection of lung cancer is by monitoring gene promoter hypermethylation events in sputum. Epidemiologic studies suggest that dietary fruits and vegetables and the micronutrients they contain may reduce risk of lung cancer. In this study, we evaluated whether diet and multivitamin use influenced the prevalence of gene promoter methylation in cells exfoliated from the aerodigestive tract of current and former smokers. Members (N = 1,101) of the Lovelace Smokers Cohort completed the Harvard Food Frequency Questionnaire and provided a sputum sample that was assessed for promoter methylation of eight genes commonly silenced in lung cancer and associated with risk for this disease. Methylation status was categorized as low (fewer than two genes methylated) or high (two or more genes methylated). Logistic regression models were used to identify associations between methylation status and 21 dietary variables hypothesized to affect the acquisition of gene methylation. Significant protection against methylation was observed for leafy green vegetables [odds ratio (OR) = 0.83 per 12 monthly servings; 95% confidence interval (95% CI), 0.74-0.93] and folate (OR, 0.84 per 750 microg/d; 95% CI, 0.72-0.99). Protection against gene methylation was also seen with current use of multivitamins (OR, 0.57; 95% CI, 0.40-0.83). This is the first cohort-based study to identify dietary factors associated with reduced promoter methylation in cells exfoliated from the airway epithelium of smokers. Novel interventions to prevent lung cancer should be developed based on the ability of diet and dietary supplements to affect reprogramming of the epigenome.

Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation.

Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could affect strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break (DSB) repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of DSB repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as three or more of eight genes methylated in sputum, compared with smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-PKc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with seven or more risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ataxia-telangiectasia mutated was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify DSB DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, which is, in turn, associated with elevated risk for lung cancer.

The A/G Allele of Rs16906252 Predicts for MGMT Methylation and Is Selectively Silenced in Premalignant Lesions from Smokers and in Lung Adenocarcinomas

Purpose: To address the association between sequence variants within the MGMT (O6-methylguanine-DNA methyltransferase) promoter–enhancer region and methylation of MGMT in premalignant lesions from smokers and lung adenocarcinomas, their biological effects on gene regulation, and targeting MGMT for therapy. Experimental Design: Single nucleotide polymorphisms (SNP) identified through sequencing a 1.9 kb fragment 5′ of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1,731 sputum samples from smokers. The effect of promoter haplotypes on MGMT expression was tested using a luciferase reporter assay and cDNA expression analysis along with allele-specific sequencing for methylation. The response of MGMT methylated lung cancer cell lines to the alkylating agent temozolomide (TMZ) was assessed. Results: The A allele of rs16906252 and the haplotype containing this SNP were strongly associated with increased risk for MGMT methylation in adenocarcinomas (ORs ≥ 94). This association was observed to a lesser extent in sputum samples in both smoker cohorts. The A allele was selectively methylated in primary lung tumors and cell lines heterozygous for rs16906252. With the most common haplotype as the reference, a 20 to 41% reduction in promoter activity was seen for the haplotype carrying the A allele that correlated with lower MGMT expression. The sensitivity of lung cancer cell lines to TMZ was strongly correlated with levels of MGMT methylation and expression. Conclusions: These studies provide strong evidence that the A allele of a MGMT promoter–enhancer SNP is a key determinant for MGMT methylation in lung carcinogenesis. Moreover, TMZ treatment may benefit a subset of lung cancer patients methylated for MGMT. Clin Cancer Res; 17(7); 2014–23. ©2011 AACR.

Nonsteroidal antiinflammatory agents inhibit upregulation of CD11b, CD11c, and CD35 in neutrophils stimulated by formyl-methionine-leucine-phenylalanine

Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit PMN aggregation, chemotaxis, degranulation, and superoxide anion production stimulated by synthetic formyl peptides. Many of these functions are dependent upon receptors for the complement components C3b and iC3b (CD35 and CD11b, respectively), or the adherence molecules CD11b and CD11c. Using flow cytometry and specific monoclonal antibodies, we studied the effects of the NSAID piroxicam and indomethacin on the upregulation of these cell surface proteins on PMNs stimulated with FMLP, C5a, or ionomycin. Incubation of PMNs at 37 °C increased the expression of all three of these surface proteins. A further increase was induced by stimulation with FMLP, C5a, or ionomycin. Both piroxicam and indomethacin inhibited FMLP-induced upregulation of CD11b, CD1 le, and CD35, but neither drug affected the upregulation of these surface molecules induced by C5a or ionomycin. Furthermore, piroxicam had no effect on 37°C-induced upregulation of any of the surface proteins, while indomethacin showed no effect on 37°C-induced CD11b upregulation but suppressed CD11c and CD35 upregulation. Inhibition of surface protein upregulation by FMLP was not due to inhibition of FMLP binding to PMNs. We conclude that piroxicam and indomethacin inhibit FMLP receptor-mediated upregulation of CD 11b, CD11c, and CD35 in PMNs, but have no effect on the upregulation of these molecules by ionomycin or C5a. These data suggest that piroxicam and indomethacin interfere with postreceptor signaling events specific to PMN stimulation by FMLP.

Attitudes and Beliefs of Primary Care Providers in New Mexico About Lung Cancer Screening Using Low-Dose Computed Tomography.

Introduction On the basis of results from the National Lung Screening Trial (NLST), national guidelines now recommend using low-dose computed tomography (LDCT) to screen high-risk smokers for lung cancer. Our study objective was to characterize the knowledge, attitudes, and beliefs of primary care providers about implementing LDCT screening. Methods We conducted semistructured interviews with primary care providers practicing in New Mexico clinics for underserved minority populations. The interviews, conducted from February through September 2014, focused on providers’ tobacco cessation efforts, lung cancer screening practices, perceptions of NLST and screening guidelines, and attitudes about informed decision making for cancer screening. Investigators iteratively reviewed transcripts to create a coding structure. Results We reached thematic saturation after interviewing 10 providers practicing in 6 urban and 4 rural settings; 8 practiced at federally qualified health centers. All 10 providers promoted smoking cessation, some screened with chest x-rays, and none screened with LDCT. Not all were aware of NLST results or current guideline recommendations. Providers viewed study results skeptically, particularly that 95% of abnormal LDCT results were false positives, the need to screen 320 patients to prevent 1 lung cancer death, and the small proportion of minority participants. Providers were uncertain whether New Mexico had the necessary infrastructure to support high-quality screening, and worried about access barriers and financial burdens for rural, underinsured populations. Providers noted the complexity of discussing benefits and harms of screening and surveillance with their patient population. Conclusion Providers have several concerns about the feasibility and appropriateness of implementing LDCT screening. Effective lung cancer screening programs will need to educate providers and patients to support informed decision making and to ensure that high-quality screening can be efficiently delivered in community practice.

Lung biopsy: Is it necessary?

OBJECTIVE Lung biopsy is associated with substantial mortality rates. We reviewed our experience with this operation, primarily in patients with immunocompetence, to determine whether the results justify the continued performance of this procedure. METHODS We conducted a retrospective review of all diagnostic lung biopsies performed at 3 university-affiliated hospitals between July 1, 1992, and December 31, 1998. RESULTS There were 75 patients: 25 patients were treated electively, 17 were treated on an urgent basis, 27 patients on an emergency basis, and the urgency was unclear in 6 patients. Significant beneficial therapeutic changes were made in 15 of 25 elective procedures (60%), in 16 of 17 urgent procedures (94%), and in 11 of 27 emergency procedures (41%; P =.001). Significant beneficial therapeutic changes consisted of immunosuppression in 13 of 15 (87%) patients treated on an elective basis, in 9 of 16 (56%) treated on an urgent basis, and in 9 of 11 (82%) treated on an emergency basis in whom therapy was altered (P =.14). Operative death was 0 of 25 for elective operations (0%), 3 of 17 for urgent operations (18%), and 14 of 26 for emergency operations (54%). Multivariable analysis of operative death showed urgency to be the only significant predictor of death (P =.002). CONCLUSIONS In patients with immunocompetence, elective and urgent lung biopsies have acceptable operative mortality rates and frequently result in important beneficial therapeutic changes. Consequently biopsies are appropriate in these patients. Emergency biopsies are associated with high operative mortality rates and rarely result in a therapeutic change other than immunosuppression. These patients should not undergo lung biopsy if they are in stable condition and should be treated empirically with immunosuppression without operation if their condition is deteriorating.

Haplotypes of DNMT1 and DNMT3B are associated with mutagen sensitivity induced by benzo[a]pyrene diol epoxide among smokers

The mutagen sensitivity assay is an in vitro measure of DNA repair capacity used to evaluate intrinsic susceptibility for cancer. The high heritability of mutagen sensitivity to different mutagens validates the use of this phenotype to predict cancer susceptibility. However, genetic determinants of mutagen sensitivity have not been fully characterized. Recently, several studies found that three major cytosine DNA methyltransferases (DNMTs), especially DNMT1, have a direct role in the DNA damage response, independent of their methyltransferase activity. This study evaluated the hypothesis that sequence variants in DNMT1, DNMT3A and DNMT3B are associated with mutagen sensitivity induced by the tobacco carcinogen benzo[a]pyrene diol epoxide (BPDE) in 278 cancer-free smokers. Single-nucleotide polymorphisms (n = 134) dispersed over the entire gene and regulatory regions of these DNMTs were genotyped by the Illumina Golden Gate Assay. DNA sequence variation in the DNMT1 and DNMT3B loci was globally associated with breaks per cell (P < 0.04 for both). No global association between DNMT3A and breaks per cell was seen (P = 0.09). Two haplotypes in block1 of DNMT1 (H284) and 3B (H70) were associated with 16 and 24% increase in breaks per cell, respectively. Subjects with three or four adverse haplotypes of both DNMT1 and 3B had a 50% elevation in mean level of breaks per cell compared with persons without adverse alleles (P = 0.004). The association between sequence variants of DNMT1 and 3B and mutagen sensitivity induced by BPDE supports the involvement of these DNMTs in protecting the cell from DNA damage.