Kristina G. Flores

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.

ATM-Dependent and -Independent Gene Expression Changes in Response to Oxidative Stress, Gamma Irradiation, and UV Irradiation

Abstract Heinloth, A. N., Shackelford, R. E., Innes, C. L., Bennett, L., Li, L., Amin, R. P., Sieber, S. O., Flores, K. G., Bushel, P. R. and Paules, R. S. ATM-Dependent and -Independent Gene Expression Changes in Response to Oxidative Stress, Gamma Irradiation, and UV Irradiation. Radiat. Res. 160, 273–290 (2003). Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by progressive cerebellar degeneration, immunodeficiencies, telangiectasias, sensitivity to ionizing radiation, and high predisposition for malignancies. The ataxia telangiectasia mutated (ATM) gene encodes a protein (ATM) with serine/threonine kinase activity. DNA-double strand breaks are known to increase its kinase activity. While cells from individuals with AT are attenuated in their G1-, S- and G2-phase cell cycle checkpoint functions in response to γ irradiation and oxidative stress, their response to UV irradiation appears to be equivalent to that of wild-type cells. In this study, we investigated changes in gene expression in response to γ irradiation, oxidative stress, and UV irradiation, focusing on the dependence on ATM. Doses for all three treatments were selected that resulted in roughly an equivalent induction of a G1 checkpoint response and inhibition of progression through S phase. To investigate gene expression changes, logarithmically growing wild-type and AT dermal diploid fibroblasts were exposed to either γ radiation (5 Gy), oxidative stress (75 µM t-butyl-hydroperoxide), or UV radiation (7.5 J/m2), and RNA was harvested 6 h after treatment. Gene expression analysis was performed using the NIEHS Human ToxChip 2.0 with approximately 1900 cDNA clones representing known genes and ESTs. All three treatments resulted in distinct patterns of gene expression changes, as shown previously. ATM-dependent and ATM-independent components were detected within these patterns, as were novel indications of involvement of ATM in regulation of transcription factors such as SP1, AP1 and MTF1.

Identification of Distinct and Common Gene Expression Changes After Oxidative Stress and Gamma and Ultraviolet Radiation

The human genome is exposed to many different kinds of DNA‐damaging agents. While most damage is detected and repaired through complex damage recognition and repair machineries, some damage has the potential to escape these mechanisms. Unrepaired DNA damage can give rise to alterations and mutations in the genome in an individual cell, which can result in malignant transformation, especially when critical genes are deregulated. In this study, we investigated gene expression changes in response to oxidative stress, gamma (γ) radiation, and ultraviolet (UV) radiation and their potential implications in cancer development. Doses were selected for each of the three treatments, based on their ability to cause a similar G1 checkpoint induction and slow down in early S‐phase progression, as reflected by a comparable reduction in cyclin E–associated kinase activity of at least 75% in logarithmically growing human dermal diploid fibroblasts. To investigate gene expression changes, logarithmically growing dermal diploid fibroblasts were exposed to either γ radiation (5 Gy), oxidative stress (75 μM of tert‐butyl hydroperoxide (t‐butyl‐OOH)), or UV radiation (UVC) (7.5 J/m2) and RNA was harvested 6 h after treatment. Gene expression was analyzed using the NIEHS Human ToxChip 2.0 with approximately 1901 cDNA clones representing known genes and expressed sequence tags (ESTs). We were able to identify common and distinct responses in dermal diploid fibroblasts to the three different stimuli used. Within our analysis, gene expression profiles in response to γ radiation and oxidative stress appeared to be more similar than profiles expressed after UV radiation. Interestingly, equivalent cyclin E–associated kinase activity reduction with all the three treatments was associated with greater transcriptional changes after UV radiation than after γ radiation and oxidative stress. While samples treated with UV radiation displayed modulations of their mitogen activated protein kinase (MAPK) pathway, γ radiation had its major influence on cell‐cycle progression in S‐phase and mitosis. In addition, cell cultures from different individuals displayed significant differences in their gene expression responses to DNA damage. Published 2003 Wiley‐Liss, Inc.

Expanding Access to BRCA1/2 Genetic Counseling with Telephone Delivery: A Cluster Randomized Trial

BACKGROUND The growing demand for cancer genetic services underscores the need to consider approaches that enhance access and efficiency of genetic counseling. Telephone delivery of cancer genetic services may improve access to these services for individuals experiencing geographic (rural areas) and structural (travel time, transportation, childcare) barriers to access. METHODS This cluster-randomized clinical trial used population-based sampling of women at risk for BRCA1/2 mutations to compare telephone and in-person counseling for: 1) equivalency of testing uptake and 2) noninferiority of changes in psychosocial measures. Women 25 to 74 years of age with personal or family histories of breast or ovarian cancer and who were able to travel to one of 14 outreach clinics were invited to participate. Randomization was by family. Assessments were conducted at baseline one week after pretest and post-test counseling and at six months. Of the 988 women randomly assigned, 901 completed a follow-up assessment. Cluster bootstrap methods were used to estimate the 95% confidence interval (CI) for the difference between test uptake proportions, using a 10% equivalency margin. Differences in psychosocial outcomes for determining noninferiority were estimated using linear models together with one-sided 97.5% bootstrap CIs. RESULTS Uptake of BRCA1/2 testing was lower following telephone (21.8%) than in-person counseling (31.8%, difference = 10.2%, 95% CI = 3.9% to 16.3%; after imputation of missing data: difference = 9.2%, 95% CI = -0.1% to 24.6%). Telephone counseling fulfilled the criteria for noninferiority to in-person counseling for all measures. CONCLUSIONS BRCA1/2 telephone counseling, although leading to lower testing uptake, appears to be safe and as effective as in-person counseling with regard to minimizing adverse psychological reactions, promoting informed decision making, and delivering patient-centered communication for both rural and urban women.

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.

Randomized Noninferiority Trial of Telephone Delivery of BRCA1/2 Genetic Counseling Compared With In-Person Counseling: 1-Year Follow-Up

PURPOSE The ongoing integration of cancer genomic testing into routine clinical care has led to increased demand for cancer genetic services. To meet this demand, there is an urgent need to enhance the accessibility and reach of such services, while ensuring comparable care delivery outcomes. This randomized trial compared 1-year outcomes for telephone genetic counseling with in-person counseling among women at risk of hereditary breast and/or ovarian cancer living in geographically diverse areas. PATIENTS AND METHODS Using population-based sampling, women at increased risk of hereditary breast and/or ovarian cancer were randomly assigned to in-person (n = 495) or telephone genetic counseling (n = 493). One-sided 97.5% CIs were used to estimate the noninferiority effects of telephone counseling on 1-year psychosocial, decision-making, and quality-of-life outcomes. Differences in test-uptake proportions for determining equivalency of a 10% prespecified margin were evaluated by 95% CIs. RESULTS At the 1-year follow-up, telephone counseling was noninferior to in-person counseling for all psychosocial and informed decision-making outcomes: anxiety (difference [d], 0.08; upper bound 97.5% CI, 0.45), cancer-specific distress (d, 0.66; upper bound 97.5% CI, 2.28), perceived personal control (d, -0.01; lower bound 97.5% CI, -0.06), and decisional conflict (d, -0.12; upper bound 97.5% CI, 2.03). Test uptake was lower for telephone counseling (27.9%) than in-person counseling (37.3%), with the difference of 9.4% (95% CI, 2.2% to 16.8%). Uptake was appreciably higher for rural compared with urban dwellers in both counseling arms. CONCLUSION Although telephone counseling led to lower testing uptake, our findings suggest that telephone counseling can be effectively used to increase reach and access without long-term adverse psychosocial consequences. Further work is needed to determine long-term adherence to risk management guidelines and effective strategies to boost utilization of primary and secondary preventive strategies.

Telehealth Personalized Cancer Risk Communication to Motivate Colonoscopy in Relatives of Patients With Colorectal Cancer: The Family CARE Randomized Controlled Trial

PURPOSE The rate of adherence to regular colonoscopy screening in individuals at increased familial risk of colorectal cancer (CRC) is suboptimal, especially among rural and other geographically underserved populations. Remote interventions may overcome geographic and system-level barriers. We compared the efficacy of a telehealth-based personalized risk assessment and communication intervention with a mailed educational brochure for improving colonoscopy screening among at-risk relatives of patients with CRC. METHODS Eligible individuals age 30 to 74 years who were not up-to-date with risk-appropriate screening and were not candidates for genetic testing were recruited after contacting patients with CRC or their next of kin in five states. Enrollees were randomly assigned as family units to either an active, personalized intervention that incorporated evidence-based risk communication and behavior change techniques, or a mailed educational brochure. The primary outcome was medically verified colonoscopy within 9 months of the intervention. RESULTS Of the 481 eligible and randomly assigned at-risk relatives, 79.8% completed the outcome assessments within 9 months; 35.4% of those in the personalized intervention group and 15.7% of those in the comparison group obtained a colonoscopy. In an intent-to-treat analysis, the telehealth group was almost three times as likely to get screened as the low-intensity comparison group (odds ratio, 2.83; 95% CI, 1.87 to 4.28; P < .001). Persons residing in rural areas and those with lower incomes benefitted at the same level as did urban residents. CONCLUSION Remote personalized interventions that consider family history and incorporate evidence-based risk communication and behavior change strategies may promote risk-appropriate screening in close relatives of patients with CRC.

ATM Requirement in Gene Expression Responses to Ionizing Radiation in Human Lymphoblasts and Fibroblasts

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function. (Mol Cancer Res 2006;4(3):197–207)

Native American Ancestry Affects the Risk for Gene Methylation in the Lungs of Hispanic Smokers from New Mexico

RATIONALE Gene promoter methylation detected in sputum predicts lung cancer risk in smokers. Compared with non-Hispanic whites (NHW), Hispanics have a lower age-standardized incidence for lung cancer. OBJECTIVES This study compared the methylation prevalence in sputum of NHWs with Hispanics using the Lovelace Smokers cohort (n = 1998) and evaluated the effect of Native American ancestry (NAA) and diet on biomarkers for lung cancer risk. METHODS Genetic ancestry was estimated using 48 ancestry markers. Diet was assessed by the Harvard University Dietary Assessment questionnaire. Methylation of 12 genes was measured in sputum using methylation-specific polymerase chain reaction. The association between NAA and risk for methylation was assessed using generalized estimating equations. The ethnic difference in the association between pack-years and risk for lung cancer was assessed in the New Mexico lung cancer study. MEASUREMENTS AND MAIN RESULTS Overall Hispanics had a significantly increased risk for methylation across the 12 genes analyzed (odds ratio, 1.18; P = 0.007). However, the risk was reduced by 32% (P = 0.032) in Hispanics with high versus low NAA. In the New Mexico lung cancer study, Hispanic non-small cell lung cancer cases have significantly lower pack-years than NHW counterparts (P = 0.007). Furthermore, compared with NHW smokers, Hispanic smokers had a more rapidly increasing risk for lung cancer as a function of pack-years (P = 0.058). CONCLUSIONS NAA may be an important risk modifier for methylation in Hispanic smokers. Smoking intensity may have a greater impact on risk for lung cancer in Hispanics compared with NHWs.

Interest and Informational Preferences Regarding Genomic Testing for Modest Increases in Colorectal Cancer Risk

Background/Aims: This study explored the interest in genomic testing for modest changes in colorectal cancer risk and preferences for receiving genomic risk communications among individuals with intermediate disease risk due to a family history of colorectal cancer. Methods: Surveys were conducted on 272 men and women at intermediate risk for colorectal cancer enrolled in a randomized trial comparing a remote personalized risk communication intervention (TeleCARE) aimed at promoting colonoscopy to a generic print control condition. Guided by Leventhals Common Sense Model of Self-Regulation, we examined demographic and psychosocial factors possibly associated with interest in SNP testing. Descriptive statistics and logistic regression models were used to identify factors associated with interest in SNP testing and preferences for receiving genomic risk communications. Results: Three-fourths of participants expressed interest in SNP testing for colorectal cancer risk. Testing interest did not markedly change across behavior modifier scenarios. Participants preferred to receive genomic risk communications from a variety of sources: printed materials (69.5%), oncologists (54.8%), primary-care physicians (58.4%), and the web (58.1%). Overall, persons who were unmarried (p = 0.029), younger (p = 0.003) and with greater cancer-related fear (p = 0.019) were more likely to express interest in predictive genomic testing for colorectal cancer risk. In a stratified analysis, cancer-related fear was associated with the interest in predictive genomic testing in the intervention group (p = 0.017), but not the control group. Conclusions: Individuals with intermediate familial risk for colorectal cancer are highly interested in genomic testing for modest increases in disease risk, specifically unmarried persons, younger age groups and those with greater cancer fear.