Matt J. Barnett

Lung cancer susceptibility locus at 5p15.33

We carried out a genome-wide association study of lung cancer (3,259 cases and 4,159 controls), followed by replication in 2,899 cases and 5,573 controls. Two uncorrelated disease markers at 5p15.33, rs402710 and rs2736100 were detected by the genome-wide data (P = 2 × 10−7 and P = 4 × 10−6) and replicated by the independent study series (P = 7 × 10−5 and P = 0.016). The susceptibility region contains two genes, TERT and CLPTM1L, suggesting that one or both may have a role in lung cancer etiology.

A Mouse to Human Search for Plasma Proteome Changes Associated with Pancreatic Tumor Development

Background The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer. Methods and Findings Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer. Conclusions Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection.

Assessing Lead Time of Selected Ovarian Cancer Biomarkers: A Nested Case–Control Study

Background CA125, human epididymis protein 4 (HE4), mesothelin, B7-H4, decoy receptor 3 (DcR3), and spondin-2 have been identified as potential ovarian cancer biomarkers. Except for CA125, their behavior in the prediagnostic period has not been evaluated. Methods Immunoassays were used to determine concentrations of CA125, HE4, mesothelin, B7-H4, DcR3, and spondin-2 proteins in prediagnostic serum specimens (1–11 samples per participant) that were contributed 0–18 years before ovarian cancer diagnosis from 34 patients with ovarian cancer (15 with advanced-stage serous carcinoma) and during a comparable time interval before the reference date from 70 matched control subjects who were participating in the Carotene and Retinol Efficacy Trial. Lowess curves were fit to biomarker levels in cancer patients and control subjects separately to summarize mean levels over time. Receiver operating characteristic curves were plotted, and area-under-the curve (AUC) statistics were computed to summarize the discrimination ability of these biomarkers by time before diagnosis. Results Smoothed mean concentrations of CA125, HE4, and mesothelin (but not of B7-H4, DcR3, and spondin-2) began to increase (visually) in cancer patients relative to control subjects approximately 3 years before diagnosis but reached detectable elevations only within the final year before diagnosis. In descriptive receiver operating characteristic analyses, the discriminatory power of these biomarkers was limited (AUC statistics range = 0.56–0.75) but showed increasing accuracy with time approaching diagnosis (eg, AUC statistics for CA125 were 0.57, 0.68, and 0.74 for ≥4, 2–4, and <2 years before diagnosis, respectively). Conclusion Serum concentrations of CA125, HE4, and mesothelin may provide evidence of ovarian cancer 3 years before clinical diagnosis, but the likely lead time associated with these markers appears to be less than 1 year.

Occurrence of Autoantibodies to Annexin I, 14-3-3 Theta and LAMR1 in Prediagnostic Lung Cancer Sera

PURPOSE We have implemented a high throughput platform for quantitative analysis of serum autoantibodies, which we have applied to lung cancer for discovery of novel antigens and for validation in prediagnostic sera of autoantibodies to antigens previously defined based on analysis of sera collected at the time of diagnosis. MATERIALS AND METHODS Proteins from human lung adenocarcinoma cell line A549 lysates were subjected to extensive fractionation. The resulting 1,824 fractions were spotted in duplicate on nitrocellulose-coated slides. The microarrays produced were used in a blinded validation study to determine whether annexin I, PGP9.5, and 14-3-3 theta antigens previously found to be targets of autoantibodies in newly diagnosed patients with lung cancer are associated with autoantibodies in sera collected at the presymptomatic stage and to determine whether additional antigens may be identified in prediagnostic sera. Individual sera collected from 85 patients within 1 year before a diagnosis of lung cancer and 85 matched controls from the Carotene and Retinol Efficacy Trial (CARET) cohort were hybridized to individual microarrays. RESULTS We present evidence for the occurrence in lung cancer sera of autoantibodies to annexin I, 14-3-3 theta, and a novel lung cancer antigen, LAMR1, which precede onset of symptoms and diagnosis. CONCLUSION Our findings suggest potential utility of an approach to diagnosis of lung cancer before onset of symptoms that includes screening for autoantibodies to defined antigens.

Polymorphisms in Oxidative Stress–Related Genes Are Not Associated with Prostate Cancer Risk in Heavy Smokers

Oxidative stress, associated with aging and inflammation, is likely to play a role in the etiology of prostate cancer. We evaluated potential associations between gene variants that result in reduced neutralization of reactive oxygen species (ROS; MnSOD Ala-16Val, CAT −262 C>T, and GPX1 Pro200Leu) and prostate cancer risk among 724 men with incident prostate cancer who participated in the Carotene and Retinol Efficacy Trial (CARET) cohort, a randomized trial for the prevention of lung cancer among men with a history of smoking and/or asbestos exposure. Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated by logistic regression. Nested case-control analyses included study participants with available DNA (n = 533 cases and 1,470 controls), matched for race, age, and length of follow-time. Overall, there were no associations between genotypes of MnSOD, CAT, and GPX1 and prostate cancer risk, although among men diagnosed before age 65, CAT TT genotype was associated with increased risk (OR, 2.0; 95% CI, 0.97-3.95). Further analyses stratified by factors related to environmental oxidative stress exposures did not modify associations. When calculating the number of risk alleles of MnSOD, CAT, and GPX1 hypothetically related to reduced protection against ROS, there was a nonsignificant relationship between prostate cancer and carriage of five or more risk alleles, in comparison to men with less than five risk alleles (OR, 2.0; 95% CI, 0.90-4.42). In conclusion, it does not seem that variants in MnSOD, CAT, or GPX1 have an influence on prostate cancer risk in this cohort of men who were smokers or exposed to asbestos, although it is possible that cumulative defects in protection from oxidative stress may result in increased risk of the disease. (Cancer Epidemiol Biomarkers Prev 2007;16(6):1115–20)

Iron intake, oxidative stress-related genes (MnSOD and MPO) and prostate cancer risk in CARET cohort

Iron overload may increase prostate cancer risk through stimulation of oxidative stress, and endogenous pro- and antioxidant capabilities, i.e. manganese superoxide dismutase (MnSOD) and myeloperoxidase (MPO), may modify these associations. We investigated this hypothesis in the Carotene and Retinol Efficacy Trial cohort in a nested case-control study. Although there was no association between iron intake and risk overall, there was a suggestion of increased risk of clinically aggressive prostate cancer with higher iron intake [odds ratio (OR) = 1.4, 95% confidence interval (CI) = 0.9-2.0]. Associations were most notable for men with aggressive prostate cancer who were below the median consumption of total fruits and vegetables (OR = 1.8, 95% CI = 1.1-3.2). Associations between MPO -463 G to A genotype (rs2333227) and prostate cancer risk were only noted among men with aggressive cancer, with more than a 2-fold risk reduction among men with AA genotypes (OR = 0.4, 95% CI = 0.2-1.0); MnSOD was not associated with risk overall, but the MnSOD T to C (Val-9Ala, rs4880) polymorphism modified associations between risk of clinically aggressive prostate cancer and dietary iron intake (P for interaction = 0.02). Among aggressive cancer cases with the TT genotype, higher iron intake level was associated with >2-fold increase in risk (OR = 2.3, 95% CI = 1.0-4.9), whereas there was no association among men with CC genotypes (OR = 0.9, 95% CI = 0.4-2.3). Although interactions were not significant, there were similar patterns for MPO genotype, iron intake and risk. These findings suggest that higher iron intake may be associated with risk of clinically aggressive prostate cancer, and that endogenous antioxidant capabilities may modify these associations.

Identification of 14-3-3θ as an antigen that induces a humoral response in lung cancer

We have implemented a strategy to identify tumor antigens that induce a humoral immune response in lung cancer based on the analysis of tumor cell proteins. Chromatographically fractionated protein extracts from three lung cancer cell lines were subjected to Western blotting and hybridization with individual sera to determine serum antibody binding. Two sets of sera were initially investigated. One set consisted of sera from 19 newly diagnosed subjects with lung adenocarcinoma and 19 matched controls. A second independent set consisted of sera from 26 newly diagnosed subjects with lung adenocarcinoma and 24 controls matched for age, gender, and smoking history. One protein that exhibited significant reactivity with both sets of cancer sera ( P = 0.0008) was confidently identified by mass spectrometry as 14-3-3𝛉. Remarkably, significant autoantibody reactivity against 14-3-3𝛉 was also observed in an analysis of a third set consisting of 18 prediagnostic lung cancer sera collected as part of the Beta-Carotene and Retinol Efficacy Trial cohort study, relative to 19 matched controls ( P = 0.0042). A receiver operating characteristic curve constructed with a panel of three proteins consisting of 14-3-3𝛉 identified in this study, plus annexin 1 and protein gene product 9.5 proteins previously identified as associated with autoantibodies in lung cancer, gave a sensitivity of 55% at 95% specificity (area under the curve, 0.838) in discriminating lung cancer at the preclinical stage from matched controls. [Cancer Res 2007;67(24):12000–6]

Association between imatinib transporters and metabolizing enzymes genotype and response in newly diagnosed chronic myeloid leukemia patients receiving imatinib therapy.

Imatinib has so far been the first-choice treatment in chronic myeloid leukemia with excellent results. However, only a proportion of patients achieve major molecular response – hence the need to find biological predictors of outcome to select the optimal therapeutic strategy now that more potent inhibitors are available. We investigated a panel of 20 polymorphisms in seven genes, potentially associated with the pharmacogenetics of imatinib, in a subset of 189 patients with newly diagnosed chronic myeloid leukemia enrolled in the TOPS trial. The analysis included polymorphisms in the transporters hOCT1, MDR1, ABCG2, OCTN1, and OATP1A2, and in the metabolizing genes CYP3A4 and CYP3A5. In the overall population, the OCTN1 C allele (rs1050152), a simple combination of polymorphisms in the hOCT1 gene and another combination in the genes involved in imatinib uptake were significantly associated with major molecular response. The combination of polymorphisms in imatinib uptake was also significantly associated with complete molecular response. Analyses restricted to Caucasians highlighted the significant association of MDR1 CC (rs60023214) genotype with complete molecular response. We demonstrate the usefulness of a pharmacogenetic approach for stratifying patients with chronic myeloid leukemia according to their likelihood of achieving a major or complete molecular response to imatinib. This represents an attractive opportunity for therapy optimization, worth testing in clinical trials.

Dietary Supplement Use and Prostate Cancer Risk in the Carotene and Retinol Efficacy Trial

We investigated dietary supplement use and prostate cancer risk in the Carotene and Retinol Efficacy Trial (CARET). CARET was a randomized, double-blinded, placebo-controlled trial testing a daily dose of 30 mg β-carotene + 25,000 IU retinyl palmitate for lung cancer prevention (1985-1996; active follow-up occurred through 2005). Secondary outcomes, including prostate cancer, were also assessed. Participants were queried about dietary supplements, health history, family history of cancer, smoking, and lifestyle habits. Cox proportional hazards regression estimated multivariate-adjusted relative risk [and 95% confidence intervals (95% CI)] of prostate cancer for dietary supplement users and nonusers with or without the high-dose CARET vitamins during the intervention and postintervention phases. After an average of 11 years of follow-up, 890 prostate cancer cases were reported. Neither the CARET nor other supplements were associated with total prostate cancer risk. For aggressive prostate cancer, men in the CARET intervention arm who used additional supplements had a relative risk for aggressive prostate cancer (Gleason ≥7 or stage III/IV) of 1.52 (95% CI, 1.03-2.24; P < 0.05), relative to all others. These associations disappeared in the postintervention period (0.75; 95% CI, 0.51-1.09). Conversely, there was no association of CARET + other supplements with nonaggressive disease, relative to all others. There was no effect modification by smoking or time on CARET intervention in any analyses. CARET only included smokers, so findings reported here may not apply to nonsmokers. Our results are consistent with other studies suggesting that dietary supplements may influence prostate cancer risk.(Cancer Epidemiol Biomarkers Prev 2009;18(8):2202–6)

Diet and exercise habits of patients with diabetes, dyslipidemia, cardiovascular disease or hypertension

Objective: The objective of this study was to determine whether free-living individuals diagnosed with diabetes, dyslipidemia, cardiovascular disease or hypertension follow standard dietary recommendations for treatment of these diet-modifiable disorders. Methods: Data are from 1,782 adult men and women who completed an annual clinic visit as part of a large study of diet and health. Usual dietary intake over the previous month was assessed with a self-administered food frequency questionnaire. Trained staff obtained a detailed medical history and information on health and exercise habits, measured height and weight, and collected a fasting blood specimen to measure total serum cholesterol, triglycerides and carotenoids. Multivariate linear regression was used to test associations of diet-modifiable chronic diseases with diet and exercise habits. Results: 42% of the study sample reported at least one diet-modifiable disease or risk factor for disease. These individuals had higher total serum cholesterol (p < 0.001) and triglycerides (p < 0.001) compared to those without these conditions. Diabetics consumed a greater percent of energy from fat (p < 0.01), and men with hypertension consumed a greater percent energy from saturated fat (p < 0.05) compared to those without these conditions. There were few other differences in dietary intake between diseased and healthy individuals, and on average, all participants had diets that were not consistent with recommended guidelines for prevention or treatment of these diet-modifiable disorders. Forty-six percent of all participants were overweight or obese, and BMI was significantly higher among participants with at least one diet-modifiable disorder (p < 0.001). Healthy and diseased participants exercised about 17 minutes per day, and compared to non-diabetics, persons with diabetes exercised with 25% less intensity (p < 0.05). Conclusion: Participants in this sample with diet-modifiable disorders reported that they are motivated to eat less fat, but most are still overweight or obese, consume a diet high in fat and low in fruits and vegetables and engage in very little physical exercise. New strategies are needed to help patients adopt and maintain healthful dietary practices that will reduce their risk.