Prediction of non-muscle invasive bladder cancer outcomes assessed by innovative multimarker prognostic models.

BACKGROUND: We adapted Bayesian statistical learning strategies to the prognosis field to investigate if genome-wide common SNP improve the prediction ability of clinico-pathological prognosticators and applied it to non-muscle invasive bladder cancer (NMIBC) patients. METHODS: Adapted Bayesian sequential threshold models in combination with LASSO were applied to consider the time-to-event and the censoring nature of data. We studied 822 NMIBC patients followed-up >10 years. The study outcomes were time-to-first-recurrence and time-to-progression. The predictive ability of the models including up to 171,304 SNP and/or 6 clinico-pathological prognosticators was evaluated using AUC-ROC and determination coefficient. RESULTS: Clinico-pathological prognosticators explained a larger proportion of the time-to-first-recurrence (3.1 %) and time-to-progression (5.4 %) phenotypic variances than SNPs (1 and 0.01 %, respectively). Adding SNPs to the clinico-pathological-parameters model slightly improved the prediction of time-to-first-recurrence (up to 4 %). The prediction of time-to-progression using both clinico-pathological prognosticators and SNP did not improve. Heritability (h (2)) of both outcomes was <1 % in NMIBC. CONCLUSIONS: We adapted a Bayesian statistical learning method to deal with a large number of parameters in prognostic studies. Common SNPs showed a limited role in predicting NMIBC outcomes yielding a very low heritability for both outcomes. We report for the first time a heritability estimate for a disease outcome. Our method can be extended to other disease models.

Infertility and incident endometrial cancer risk: a pooled analysis from the epidemiology of endometrial cancer consortium (E2C2).

BACKGROUND: Nulliparity is an endometrial cancer risk factor, but whether or not this association is due to infertility is unclear. Although there are many underlying infertility causes, few studies have assessed risk relations by specific causes. METHODS: We conducted a pooled analysis of 8153 cases and 11 713 controls from 2 cohort and 12 case-control studies. All studies provided self-reported infertility and its causes, except for one study that relied on data from national registries. Logistic regression was used to estimate adjusted odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Nulliparous women had an elevated endometrial cancer risk compared with parous women, even after adjusting for infertility (OR=1.76; 95% CI: 1.59-1.94). Women who reported infertility had an increased risk compared with those without infertility concerns, even after adjusting for nulliparity (OR=1.22; 95% CI: 1.13-1.33). Among women who reported infertility, none of the individual infertility causes were substantially related to endometrial cancer. CONCLUSIONS: Based on mainly self-reported infertility data that used study-specific definitions of infertility, nulliparity and infertility appeared to independently contribute to endometrial cancer risk. Understanding residual endometrial cancer risk related to infertility, its causes and its treatments may benefit from large studies involving detailed data on various infertility parameters.

Estimating breast cancer mortality reduction and overdiagnosis due to screening for different strategies in the United Kingdom.

BACKGROUND: The benefits and harms of population-wide mammography screening have been long debated. This study evaluated the impact of screening frequency and age range on breast cancer mortality reduction and overdiagnosis. METHODS: We developed a Markov simulation model for the evaluation of mammography screening in a cohort of British women born in 1935-40. RESULTS: For triennial screening in women aged 47-73, breast cancer mortality reduction and overdiagnosis was 18.1% (95% confidence interval: 17.3%, 19.0%) and 5.6% (5.1%, 6.1%), of all breast cancer deaths and diagnoses, respectively, from age 40 to 85 years. For annual screening in the same age range, estimates for both outcomes increased considerably to 35.0% (34.2%, 35.7%) and 7.6% (7.1%, 8.1%), respectively. For the age extension of triennial screening from 50-70 to 47-73, we estimated 5 (3, 7) incremental breast cancer deaths avoided and 14 (9, 19) incremental cases overdiagnosed per 10 000 women invited for screening. CONCLUSIONS: Estimates of mortality reduction and overdiagnosis were highly dependent on screening frequency, age range, and uptake, which may explain differences between some previous estimates obtained from randomised trials and from service screening.

LINE-1 methylation in leukocyte DNA, interaction with phosphatidylethanolamine N-methyltransferase variants and bladder cancer risk.

BACKGROUND: Aberrant global DNA methylation is shown to increase cancer risk. LINE-1 has been proven a measure of global DNA methylation. The objectives of this study were to assess the association between LINE-1 methylation level and bladder cancer risk and to evaluate effect modification by environmental and genetic factors. METHODS: Bisulphite-treated leukocyte DNA from 952 cases and 892 hospital controls was used to measure LINE-1 methylation level at four CpG sites by pyrosequencing. Logistic regression model was fitted to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). Interactions between LINE-1 methylation levels and environmental and genetic factors were assessed. RESULTS: The risk of bladder cancer followed a nonlinear association with LINE-1 methylation. Compared with subjects in the middle tertile, the adjusted OR for subjects in the lower and the higher tertiles were 1.26 (95% CI 0.99-1.60, P=0.06) and 1.33 (95% CI 1.05-1.69, P=0.02), respectively. This association significantly increased among individuals homozygous for the major allele of five single-nucleotide polymorphisms located in the phosphatidylethanolamine N-methyltransferase gene (corrected P-interaction<0.05). CONCLUSIONS: The findings from this large-scale study suggest that both low and high levels of global DNA methylation are associated with the risk of bladder cancer.

Common genetic variants in the 9p21 region and their associations with multiple tumours.

BACKGROUND: The chromosome 9p21.3 region has been implicated in the pathogenesis of multiple cancers. METHODS: We systematically examined up to 203 tagging SNPs of 22 genes on 9p21.3 (19.9-32.8 Mb) in eight case-control studies: thyroid cancer, endometrial cancer (EC), renal cell carcinoma, colorectal cancer (CRC), colorectal adenoma (CA), oesophageal squamous cell carcinoma (ESCC), gastric cardia adenocarcinoma and osteosarcoma (OS). We used logistic regression to perform single SNP analyses for each study separately, adjusting for study-specific covariates. We combined SNP results across studies by fixed-effect meta-analyses and a newly developed subset-based statistical approach (ASSET). Gene-based P-values were obtained by the minP method using the Adaptive Rank Truncated Product program. We adjusted for multiple comparisons by Bonferroni correction. RESULTS: Rs3731239 in cyclin-dependent kinase inhibitors 2A (CDKN2A) was significantly associated with ESCC (P=7 × 10(-6)). The CDKN2A-ESCC association was further supported by gene-based analyses (Pgene=0.0001). In the meta-analyses by ASSET, four SNPs (rs3731239 in CDKN2A, rs615552 and rs573687 in CDKN2B and rs564398 in CDKN2BAS) showed significant associations with ESCC and EC (P<2.46 × 10(-4)). One SNP in MTAP (methylthioadenosine phosphorylase) (rs7023329) that was previously associated with melanoma and nevi in multiple genome-wide association studies was associated with CRC, CA and OS by ASSET (P=0.007). CONCLUSION: Our data indicate that genetic variants in CDKN2A, and possibly nearby genes, may be associated with ESCC and several other tumours, further highlighting the importance of 9p21.3 genetic variants in carcinogenesis.

PREDICT Plus: development and validation of a prognostic model for early breast cancer that includes HER2.

BACKGROUND: Predict (www.predict.nhs.uk) is an online, breast cancer prognostication and treatment benefit tool. The aim of this study was to incorporate the prognostic effect of HER2 status in a new version (Predict+), and to compare its performance with the original Predict and Adjuvant!. METHODS: The prognostic effect of HER2 status was based on an analysis of data from 10 179 breast cancer patients from 14 studies in the Breast Cancer Association Consortium. The hazard ratio estimates were incorporated into Predict. The validation study was based on 1653 patients with early-stage invasive breast cancer identified from the British Columbia Breast Cancer Outcomes Unit. Predicted overall survival (OS) and breast cancer-specific survival (BCSS) for Predict+, Predict and Adjuvant! were compared with observed outcomes. RESULTS: All three models performed well for both OS and BCSS. Both Predict models provided better BCSS estimates than Adjuvant!. In the subset of patients with HER2-positive tumours, Predict+ performed substantially better than the other two models for both OS and BCSS. CONCLUSION: Predict+ is the first clinical breast cancer prognostication tool that includes tumour HER2 status. Use of the model might lead to more accurate absolute treatment benefit predictions for individual patients.

GSTM1 null and NAT2 slow acetylation genotypes, smoking intensity and bladder cancer risk: results from the New England bladder cancer study and NAT2 meta-analysis.

Associations between bladder cancer risk and NAT2 and GSTM1 polymorphisms have emerged as some of the most consistent findings in the genetic epidemiology of common metabolic polymorphisms and cancer, but their interaction with tobacco use, intensity and duration remain unclear. In a New England population-based case-control study of urothelial carcinoma, we collected mouthwash samples from 1088 of 1171 cases (92.9%) and 1282 of 1418 controls (91.2%) for genotype analysis of GSTM1, GSTT1 and NAT2 polymorphisms. Odds ratios and 95% confidence intervals of bladder cancer among New England Bladder Cancer Study subjects with one or two inactive GSTM1 alleles (i.e. the 'null' genotype) were 1.26 (0.85-1.88) and 1.54 (1.05-2.25), respectively (P-trend = 0.008), compared with those with two active copies. GSTT1 inactive alleles were not associated with risk. NAT2 slow acetylation status was not associated with risk among never (1.04; 0.71-1.51), former (0.95; 0.75-1.20) or current smokers (1.33; 0.91-1.95); however, a relationship emerged when smoking intensity was evaluated. Among slow acetylators who ever smoked at least 40 cigarettes/day, risk was elevated among ever (1.82; 1.14-2.91, P-interaction = 0.07) and current heavy smokers (3.16; 1.22-8.19, P-interaction = 0.03) compared with rapid acetylators in each category; but was not observed at lower intensities. In contrast, the effect of GSTM1-null genotype was not greater among smokers, regardless of intensity. Meta-analysis of the NAT2 associations with bladder cancer showed a highly significant relationship. Findings from this large USA population-based study provided evidence that the NAT2 slow acetylation genotype interacts with tobacco smoking as a function of exposure intensity.

Evaluation of variation in the phosphoinositide-3-kinase catalytic subunit alpha oncogene and breast cancer risk.

BACKGROUND: Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer. METHODS: A single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30 949 cases and 29 788 controls from the Breast Cancer Association Consortium (BCAC). RESULTS: Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95-0.99, P=4.6 × 10(-3)), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96-1.01, P=0.139). CONCLUSION: Common germline variation in PIK3CA does not have a strong influence on the risk of breast cancer.

Prolactin serum levels and breast cancer: relationships with risk factors and tumour characteristics among pre- and postmenopausal women in a population-based case-control study from Poland.

BACKGROUND: Previous prospective studies have found an association between prolactin (PRL) levels and increased risk of breast cancer. Using data from a population-based breast cancer case-control study conducted in two cities in Poland (2000-2003), we examined the association of PRL levels with breast cancer risk factors among controls and with tumour characteristics among the cases. METHODS: We analysed PRL serum levels among 773 controls without breast cancer matched on age and residence to 776 invasive breast cancer cases with available pretreatment serum. Tumours were centrally reviewed and prepared as tissue microarrays for immunohistochemical analysis. Breast cancer risk factors, assessed by interview, were related to serum PRL levels among controls using analysis of variance. Mean serum PRL levels by tumour characteristics are reported. These associations also were evaluated using polytomous logistic regression. RESULTS: Prolactin levels were associated with nulliparity in premenopausal (P=0.05) but not in postmenopausal women. Associations in postmenopausal women included an inverse association with increasing body mass index (P=0.0008) and direct association with use of recent/current hormone therapy (P=0.0006). In case-only analyses, higher PRL levels were more strongly associated with lobular compared with ductal carcinoma among postmenopausal women (P=0.02). Levels were not different by tumour size, grade, node involvement or oestrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2 status. CONCLUSIONS: Our analysis demonstrates that PRL levels are higher among premenopausal nulliparous as compared with parous women. Among postmenopausal women, levels were higher among hormone users and lower among obese women. These results may have value in understanding the mechanisms underlying several breast cancer risk factor associations.

Human epidermal growth factor receptor-2 and estrogen receptor expression, a demonstration project using the residual tissue repository of the Surveillance, Epidemiology, and End Results (SEER) program.

BACKGROUND: In 2001, the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) program established Residual Tissue Repositories (RTR) in the Hawaii, Iowa, and Los Angeles Tumor Registries to collect discarded tissue blocks from pathologic laboratories within their catchment areas. To validate the utility of the RTR for supplementing SEER's central database, we assessed human epidermal growth factor receptor-2 (HER2) and estrogen receptor expression (ER) in a demonstration project. MATERIALS: Using a prepared set of tissue microarrays (TMAs) residing in the Hawaii Tumor Registry (HTR), we performed standard immunohistochemistry. Breast cancers in the TMA were diagnosed in 1995, followed through 2006, and linked to SEER's main database. RESULTS: The TMA included 354 cases, representing 51% of 687 breast cancers in the HTR (1995). The HTR and TMA cases were similar with respect to patient demographics and tumor characteristics. Seventy-six percent (76%, 268 of 354) of TMA cases were HER2+ and/or ER+, i.e., 28 HER2+ER-, 12 HER2+ER+, and 228 HER2-ER+. There were 67 HER2-ER- cases and 19 were unclassified. Age distributions at diagnosis were bimodal with dominant early-onset modes for HER2+ER- tumors and dominant late-onset modes for HER2-ER+ breast cancers. Epidemiologic patterns for concordant HER2+ER+ (double-positive) and HER2-ER- (double-negative) were intermediate to discordant HER2+ER- and HER2-ER+. CONCLUSION: Results showed contrasting incidence patterns for HER2+ (HER2+ER-) and ER+ (HER2-ER+) breast cancers, diagnosed in 1995. Though sample sizes were small, this demonstration project validates the potential utility of the RTR for supplementing the SEER program.

Skewed X chromosome inactivation and early-onset breast cancer.

BACKGROUND: Skewed X chromosome inactivation may be more common in women with epithelial ovarian cancer and early-onset breast cancer. We tested this hypothesis in a group of 235 breast cancer patients and 253 controls (mean age 45.8 years) from a larger population based case control study. METHODS: We measured X chromosome inactivation with the AR gene assay in lymphocyte DNA digested with the methylation specific enzyme HpaII. We judged skewness using an adjusted measure (relative to the undigested sample) with a cut point of 75%, and an unadjusted measure where skewed was defined as > 90% of the signal from one allele in the HpaII digested sample. RESULTS: There were no significant differences in any of the skewing measures between cases and controls. Using the adjusted skewing measure among pre-menopausal subjects under the age of 50, 14% of cases versus 11% of controls were skewed, OR = 1.2, 95% CI 0.6 to 2.3; using the unadjusted measure, OR = 0.9, 95% CI 0.4 to 2.0. CONCLUSIONS: While we cannot rule out a subtle difference of approximately twofold or less, we have failed to find a significant difference in the prevalence of skewed X chromosome inactivation in younger women with breast cancer compared to controls.

Glutathione S-transferase mu and theta polymorphisms and breast cancer susceptibility.

BACKGROUND: The enzymes encoded by the glutathione S-transferase mu 1 (GSTM1) and theta 1 (GSTT1) genes are involved in the metabolism (mainly inactivation, but activation is possible) of a wide range of carcinogens that are ubiquitous in the environment; the enzyme encoded by the GSTT1 gene may also be active in endogenous mutagenic processes. Homozygous deletions of the GSTM1 and GSTT1 genes are commonly found in the population and result in a lack of enzyme activity. This study was undertaken to evaluate the association between GSTM1 and GSTT1 gene polymorphisms and breast cancer risk. METHODS: Our study included 466 women with incident cases of breast cancer occurring from May 1989 through May 1994 and 466 matched control subjects. These individuals were part of a prospective cohort of U.S. women (i.e., the Nurses' Health Study). Odds ratios (ORs) and 95% confidence intervals (CIs) from conditional logistic regression models were used to estimate the association between genetic polymorphisms and breast cancer risk. RESULTS: The GSTM1 and GSTT1 null genotypes were not associated with an increased risk of breast cancer (OR = 1.05 [95% CI = 0.80-1.37] for GSTM1 null; OR = 0. 86 [95% CI = 0.61-1.21] for GSTT1 null). On the contrary, a suggestion of a decreased risk of breast cancer associated with the GSTT1 null genotype was observed among premenopausal women. When considered together, no combination of the GSTM1 and GSTT1 genotypes was associated with an increased risk of breast cancer. The relationship between GSTM1 and GSTT1 gene deletions and breast cancer risk was not substantially modified by cigarette smoking. CONCLUSIONS: Our data provide evidence against a substantially increased risk of breast cancer associated with GSTM1 and/or GSTT1 homozygous gene deletions.

A prospective study of N-acetyltransferase genotype, red meat intake, and risk of colorectal cancer.

Carcinogenic heterocyclic amines are activated by N-acetyltransferase (NAT) enzymes, encoded by NAT1 and NAT2, to genotoxic compounds that can form DNA adducts in the colon epithelium. We have examined the relation of polymorphisms in the genes coding for both enzymes to risk of colorectal cancer and the gene-environment interaction with red meat intake among participants in the prospective Physicians' Health Study. Baseline blood samples from 212 men subsequently diagnosed with colorectal cancer during 13 years of follow-up were genotyped, along with 221 controls. NAT genotypes were analyzed by a PCR-restriction fragment length polymorphism method. Effect modification of the relation of red meat intake and risk of colorectal cancer by NAT genotype was assessed using conditional logistic regression. There was no overall independent association of NAT acetylation genotypes and colorectal cancer risk. The relative risks for the rapid acetylation genotype were 0.93 [95% confidence interval (CI), 0.61-1.42] for NAT1, 0.80 (95% CI, 0.53-1.19) for NAT2, and 0.81 (95% CI, 0.52-1.27) for NAT1/NAT2 combined. We observed a stronger association of red meat intake with cancer risk among NAT rapid acetylators, especially among men 60 years old or older. Among those men who were rapid acetylators for both NAT1 and NAT2, consumption of >1 serving of red meat per day was associated with a relative risk of 5.82 (95% CI, 1.11-30.6) compared with consumption of < or = 0.5 serving per day (P, trend = 0.02). These prospective data, which need to be confirmed in other studies, suggest that polymorphisms in the NAT genes confer differential susceptibility to the effect of red meat consumption on colorectal cancer risk.

Well-done, grilled red meat increases the risk of colorectal adenomas.

Red meat or meat-cooking methods such as frying and doneness level have been associated with an increased risk of colorectal and other cancers. It is unclear whether it is red meat intake or the way it is cooked that is involved in the etiology of colorectal cancer. To address this issue, we developed an extensive food frequency questionnaire module that collects information on meat-cooking techniques as well as the level of doneness for individual meat items and used it in a study of colorectal adenomas, known precursors of colorectal cancer. A case-control study of colorectal adenomas was conducted at the National Naval Medical Center (Bethesda, MD) between April 1994 and September 1996. All cases (n = 146) were diagnosed with colorectal adenomas at sigmoidoscopy or colonoscopy and histologically confirmed. Controls (n = 228) were screened with sigmoidoscopy and found not to have colorectal adenomas. The subjects completed a food frequency questionnaire and answered detailed questions on meat-cooking practices. We used frequency and portion size to estimate grams of meat consumed per day for total meat as well as for meat subgroups defined by cooking methods and doneness levels. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression, adjusted for age, gender, total caloric intake, reason for screening (routine or other), and several established risk factors for colorectal adenomas or cancer, including the use of nonsteroidal anti-inflammatory drugs, physical activity, and pack-years of cigarette smoking. There was an increased risk of 11% per 10 g/day (or 2.5 oz/week) of reported red meat consumption (OR, 1.11; CI, 1.03-1.19). The increased risk was mainly associated with well-done/very well-done red meat, with an excess risk of 29% per 10 g/day (OR, 1.29; CI, 1.08-1.54) versus an excess of 10% per 10 g/day (OR, 1.10; CI, 0.96-1.26) for consumption of rare/medium red meat. High-temperature cooking methods were also associated with increased risk; 26% per 10 g/day (OR, 1.26; CI, 1.06-1.50) of grilled red meat and 15% per 10 g/day (OR, 1.15; CI, 0.97-1.36) of pan-fried red meat consumption. There was an increased risk of colorectal adenomas associated with higher intake of red meat, most of which was due to the subgroup of red meat that was cooked until well done/very well done and/or by high-temperature cooking techniques, such as grilling. These results are consistent with the hypothesis that carcinogenic compounds formed by high-temperature cooking techniques, such as heterocyclic amines and polycyclic aromatic hydrocarbons, may contribute to the risk of developing colorectal tumors.

The use of common genetic polymorphisms to enhance the epidemiologic study of environmental carcinogens.

Overwhelming evidence indicates that environmental exposures, broadly defined, are responsible for most cancer. There is reason to believe, however, that relatively common polymorphisms in a wide spectrum of genes may modify the effect of these exposures. We discuss the rationale for using common polymorphisms to enhance our understanding of how environmental exposures cause cancer and comment on epidemiologic strategies to assess these effects, including study design, genetic and statistical analysis, and sample size requirements. Special attention is given to sources of potential bias in population studies of gene--environment interactions, including exposure and genotype misclassification and population stratification (i.e., confounding by ethnicity). Nevertheless, by merging epidemiologic and molecular approaches in the twenty-first century, there will be enormous opportunities for unraveling the environmental determinants of cancer. In particular, studies of genetically susceptible subgroups may enable the detection of low levels of risk due to certain common exposures that have eluded traditional epidemiologic methods. Further, by identifying susceptibility genes and their pathways of action, it may be possible to identify previously unsuspected carcinogens. Finally, by gaining a more comprehensive understanding of environmental and genetic risk factors, there should emerge new clinical and public health strategies aimed at preventing and controlling cancer.

Factors critical to the design and execution of epidemiologic studies and description of an innovative technology to follow the progression from normal to cancer tissue.

The results obtained from experimental studies of estrogen carcinogenesis need validation in epidemiologic studies. Such studies present additional challenges, however, because variations in human populations are much greater than those in experimental systems and in animal models. Because epidemiologic studies are often used to evaluate modest differences in risk factors, it is essential to minimize sources of errors and to maximize sensitivity, reproducibility, and specificity. In the first part of this chapter, critical factors in designing and executing epidemiologic studies, as well as the influence of sample collection, processing, and storage on data reliability, are discussed. One of the most important requirements is attaining sufficient statistical power to assess small genetic effects and to evaluate interactions between genetic and environmental factors. The second part of this chapter describes innovative technology, namely, Fourier transform-infrared (FT-IR) spectra of DNA that reveal major structural differences at various stages of the progression from normal to cancer tissue. The structural differences become evident from wavenumber-by-wavenumber statistical comparisons of the mean FT-IR spectra of DNA from normal to cancer tissues. This analysis has allowed distinguishing benign tissues from cancer and metastatic tissues in human breast, prostate, and ovarian cancers. This analysis, which requires less than 1 microg of DNA, is predicted to be used for detecting early cancer-related changes at the level of DNA, rather than at the cellular level.

A cross-sectional study of dental caries, intake of confectionery and foods rich in starch and sugars, and salivary counts of Streptococcus mutans in children in Spain.

In this cross-sectional study of 236 schoolchildren living in Manresa, Spain, we evaluated the association between prevalence of dental caries and frequency of consumption of various food groups, including sweetened baked goods and similar foods (rich in starch and sugars) and confectionery (rich in sugars but not starch), using a food-frequency questionnaire. Because Streptococcus mutans is associated with the cariogenicity of carbohydrates, we also evaluated the modification of these associations by salivary counts of this microorganism. Odds ratios (ORs) were used to measure the association between caries and tertiles of consumption. Sex, age, use of fluorides, tooth-brushing frequency, frequency of dental visits, socioeconomic status, and intake of other potentially cariogenic food groups were considered as potential confounders. We did not find a significant association between any of the food groups evaluated and caries prevalence. Failure to detect an association could have been due to the low prevalence of caries in our population (decayed, missing, or filled permanent teeth = 1.3 at age 10.6 y) or to underestimation of the association due to diet misclassification. In this population, the association between consumption of sweetened baked goods and caries appeared to be modified by the numbers of S. mutans [OR = 6.1 (95% CI: 1.6, 23.0) for low compared with high intake in children with moderate-to-high S. mutans counts and OR = 0.3 (95% CI: 0.1, 1.6) for low compared with high intake in children with low S. mutans counts]. These results suggest that a high intake of sweetened baked goods may be a determinant of caries prevalence in children with moderate-to-high salivary counts of S. mutans.

Misclassification in case-control studies of gene-environment interactions: assessment of bias and sample size.

In studies of gene-environment interactions, exposure misclassification can lead to bias in the estimation of an interaction effect and increased sample size. The magnitude of the bias and the consequent increase in sample size for fixed misclassification probabilities are highly dependent on the prevalence of the misclassified factor and on the interaction model. This paper describes a relatively simple approach to assess the impact of misclassification on bias in the estimation of multiplicative or additive interactions and on sample size requirements. Applications of this method illustrate that even small errors in the assessment of environmental or genetic factors can result in biased interaction parameters and substantially increased sample size requirements that can compromise the feasibility of the study. Also, an example is provided where nondifferential misclassification biases an additive interaction parameter away from the null value, even under conditions where a multiplicative interaction parameter will always be biased toward the null value. Efforts to improve the accuracy in measuring both genetic and environmental factors are critical for the valid assessment of gene-environment interactions in case-control studies.

Collection of buccal cell DNA using treated cards.

We devised a simple, noninvasive, cost-efficient technique for collecting buccal cell DNA for molecular epidemiology studies. Subjects (n = 52) brushed their oral mucosa and expectorated the fluid in their mouths, which was applied to "Guthrie" cards pretreated to retard bacterial growth and inhibit nuclease activity (IsoCode, Schleicher and Schuell, Keene, NH). The cards are well-suited for transport and storage because they dry quickly, need no processing, and are compact and lightweight. We stored the samples at room temperature for 5 days to mimic a field situation and then divided them into portions from which DNA was extracted either immediately or after storage for 9 months at room temperature, -20 degrees C, or -70 degrees C. The fresh samples had a median yield of 2.3 microg of human DNA (range, 0.2-53.8 microg), which was adequate for at least 550 PCR reactions. More than 90% of the samples were amplified in all three beta-globin gene fragment assays attempted. DNA extract frozen for 1 week at -20 degrees C also performed well. Stored samples had reduced DNA yields, which achieved statistical significance for room temperature and -70 degrees C, but not -20 degrees C, storage. However, because all of the stored samples tested were successfully amplified, the observed reduction may represent tighter DNA fixation to the card over time rather than loss of genetic material. We conclude that treated cards are an alternative to brushes/swabs and mouth rinses for the collection of buccal cell DNA and offer some advantages over these methods, particularly for large-scale or large-scale or long-term studies involving stored samples and studies in which samples are collected off-site and transported. Future studies that enable direct comparisons of the various buccal cell collection methods are needed.

Vitamin D, calcium, and vitamin D receptor polymorphism in colorectal adenomas.

Experimental studies suggest that vitamin D and calcium protect against cancer by reducing proliferation and inducing differentiation. The effects of vitamin D and calcium may be mediated by the vitamin D receptor (VDR), which is encoded by the VDR gene. The present study investigated whether calcium intake and serum vitamin D, as an integrated measure of intake and endogenous production, were associated with risk of colorectal adenoma, known precursors of invasive colorectal cancer. In addition, the interrelation among vitamin D, calcium, and FokI polymorphism of the VDR gene was investigated. Persons (239) with histologically confirmed colorectal adenomas and 228 control individuals without colorectal adenomas confirmed by sigmoidoscopy were enrolled in this case control study conducted at the National Naval Medical Center, Bethesda, MD. We observed an inverse association of serum 25-OH vitamin D [25-(OH)D] with colorectal adenoma. With each 10 ng/ml increase of serum 25-(OH)D, the risk of colorectal adenoma decreased by 26% (odds ratio 0.74, 95% confidence interval 0.60-0.92). The results provided limited evidence for a weak association between calcium intake and colorectal adenoma (odds ratio 0.97, 95% confidence interval 0.93-1.01 per each 100-mg calcium intake). However, the inverse association of serum 25-(OH)D with colorectal adenoma is suggested to be stronger in subjects with calcium intake above the median (P for multiplicative interaction 0.13). The VDR FokI polymorphism was not significantly associated with colorectal adenoma and did not modify the effect of vitamin D or calcium. In conclusion, the study results suggested a protective effect for vitamin D on colorectal adenoma.