Obesity and inflammation markers in relation to leukocyte telomere length in a cross-sectional study of persons with Barrett's esophagus.

BACKGROUND: Telomere shortening is associated with increasing age, male gender and lifestyle factors such as obesity and smoking. Inflammation has also been implicated in cellular senescence and may promote telomere shortening in chronic conditions such as obesity and diabetes. However, little is known about the relationship between markers of obesity and inflammation, and leukocyte telomere length (LTL). METHODS: LTL was measured using quantitative polymerase chain reaction in peripheral leukocytes from 295 individuals diagnosed with Barrett's esophagus (BE) between 1995 and 2009. Data on lifestyle variables including obesity and smoking were collected at in-person interviews. Biomarkers of obesity (leptin, adiponectin), diabetes (glucose, insulin), inflammation (C-reactive protein, Interleukin-6, surface tumor necrosis factor receptor (sTNFR) I & II) and oxidative stress (F2-isoprostanes) were measured in stored blood samples. We examined associations between these covariates and LTL in a cross-sectional analysis using linear and logistic regression models, adjusting for possible confounders. RESULTS: LTL was significantly associated with age (r = -0.30, p < 0.001), gender (r = 0.14 for females, p = 0.01) and inversely associated with cigarette pack-years (r = -0.11, p = 0.04). Odds of having short LTL were significantly higher for participants in the highest tertile for sTNF-RI (Odds ratio adjusted for age, gender, smoking, and obesity = 2.19; 95 % CI 1.00-4.85, p-trend = 0.02). LTL was not significantly associated with any other lifestyle factors, including smoking or obesity, or other inflammation-, obesity-/diabetes-related biomarkers measured. CONCLUSIONS: Increasing age, male gender, smoking history, and sTNF-RI levels were associated with short LTL among persons with BE but no correlations were observed between LTL and other inflammatory markers or measures of obesity. Larger longitudinal studies are necessary in order to further establish the potential relationships between obesity, inflammation markers and LTL.

High Goblet Cell Count Is Inversely Associated with Ploidy Abnormalities and Risk of Adenocarcinoma in Barrett's Esophagus.

PURPOSE: Goblet cells may represent a potentially successful adaptive response to acid and bile by producing a thick mucous barrier that protects against cancer development in Barrett's esophagus (BE). The aim of this study was to determine the relationship between goblet cells (GC) and risk of progression to adenocarcinoma, and DNA content flow cytometric abnormalities, in BE patients. EXPERIMENTAL DESIGN: Baseline mucosal biopsies (N=2988) from 213 patients, 32 of whom developed cancer during the follow up period, enrolled in a prospective dynamic cohort of BE patients were scored in a blinded fashion, for the total number (#) of GC, mean # of GC/crypt (GC density), # of crypts with ≥ 1 GC, and the proportion of crypts with ≥1 GC, in both dysplastic and non-dysplastic epithelium separately. The relationship between these four GC parameters and DNA content flow cytometric abnormalities and adenocarcinoma outcome was compared, after adjustment for age, gender, and BE segment length. RESULTS: High GC parameters were inversely associated with DNA content flow cytometric abnormalities, such as aneuploidy, ploidy >2.7N, and an elevated 4N fraction > 6%, and with risk of adenocarcinoma. However, a Kaplan-Meier analysis showed that the total # of GC and the total # crypts with ≥1 GC were the only significant GC parameters (p<0.001 and 0.003, respectively). CONCLUSIONS: The results of this study show, for the first time, an inverse relationship between high GC counts and flow cytometric abnormalities and risk of adenocarcinoma in BE. Further studies are needed to determine if GC depleted foci within esophageal columnar mucosa are more prone to neoplastic progression or whether loss of GC occurs secondary to underlying genetic abnormalities.

Assessment of Esophageal Adenocarcinoma Risk Using Somatic Chromosome Alterations in Longitudinal Samples in Barrett's Esophagus.

Cancers detected at a late stage are often refractory to treatments and ultimately lethal. Early detection can significantly increase survival probability, but attempts to reduce mortality by early detection have frequently increased overdiagnosis of indolent conditions that do not progress over a lifetime. Study designs that incorporate biomarker trajectories in time and space are needed to distinguish patients who progress to an early cancer from those who follow an indolent course. Esophageal adenocarcinoma is characterized by evolution of punctuated and catastrophic somatic chromosomal alterations and high levels of overall mutations but few recurrently mutated genes aside from TP53. Endoscopic surveillance of Barrett's esophagus for early cancer detection provides an opportunity for assessment of alterations for cancer risk in patients who progress to esophageal adenocarcinoma compared with nonprogressors. We investigated 1,272 longitudinally collected esophageal biopsies in a 248 Barrett's patient case-cohort study with 20,425 person-months of follow-up, including 79 who progressed to early-stage esophageal adenocarcinoma. Cancer progression risk was assessed for total chromosomal alterations, diversity, and chromosomal region-specific alterations measured with single-nucleotide polymorphism arrays in biopsies obtained over esophageal space and time. A model using 29 chromosomal features was developed for cancer risk prediction (area under receiver operator curve, 0.94). The model prediction performance was robust in two independent esophageal adenocarcinoma sets and outperformed TP53 mutation, flow cytometric DNA content, and histopathologic diagnosis of dysplasia. This study offers a strategy to reduce overdiagnosis in Barrett's esophagus and improve early detection of esophageal adenocarcinoma and potentially other cancers characterized by punctuated and catastrophic chromosomal evolution.

Bacterial Composition of the Human Upper Gastrointestinal Tract Microbiome Is Dynamic and Associated with Genomic Instability in a Barrett's Esophagus Cohort.

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) has increased nearly five-fold over the last four decades in the United States. Barrett's esophagus, the replacement of the normal squamous epithelial lining with a mucus-secreting columnar epithelium, is the only known precursor to EAC. Like other parts of the gastrointestinal (GI) tract, the esophagus hosts a variety of bacteria and comparisons among published studies suggest bacterial communities in the stomach and esophagus differ. Chronic infection with Helicobacter pylori in the stomach has been inversely associated with development of EAC, but the mechanisms underlying this association remain unclear. METHODOLOGY: The bacterial composition in the upper GI tract was characterized in a subset of participants (n=12) of the Seattle Barrett's Esophagus Research cohort using broad-range 16S PCR and pyrosequencing of biopsy and brush samples collected from squamous esophagus, Barrett's esophagus, stomach corpus and stomach antrum. Three of the individuals were sampled at two separate time points. Prevalence of H. pylori infection and subsequent development of aneuploidy (n=339) and EAC (n=433) was examined in a larger subset of this cohort. RESULTS/SIGNIFICANCE: Within individuals, bacterial communities of the stomach and esophagus showed overlapping community membership. Despite closer proximity, the stomach antrum and corpus communities were less similar than the antrum and esophageal samples. Re-sampling of study participants revealed similar upper GI community membership in two of three cases. In this Barrett's esophagus cohort, Streptococcus and Prevotella species dominate the upper GI and the ratio of these two species is associated with waist-to-hip ratio and hiatal hernia length, two known EAC risk factors in Barrett's esophagus. H. pylori-positive individuals had a significantly decreased incidence of aneuploidy and a non-significant trend toward lower incidence of EAC.

Integrative post-genome-wide association analysis of CDKN2A and TP53 SNPs and risk of esophageal adenocarcinoma.

Incidence of esophageal adenocarcinoma (EA) in Western countries has increased markedly in recent decades. Although several risk factors have been identified for EA and its precursor, Barrett's esophagus (BE), including reflux, Caucasian race, male gender, obesity, and smoking, less is known about the role of inherited genetic variation. Frequent somatic mutations in the tumor suppressor genes CDKN2A and TP53 were recently reported in EA tumors, while somatic alterations at 9p (CDKN2A) and 17p (TP53) have been implicated as predictors of progression from BE to EA. Motivated by these findings, we used data from a genome-wide association study of 2515 EA cases and 3207 controls to analyze 37 germline single nucleotide polymorphisms at the CDKN2A and TP53 loci. Three CDKN2A polymorphisms were nominally associated (P < 0.05) with reduced risk of EA: rs2518720 C>T [intronic, odds ratio 0.90, P = 0.0121, q = 0.3059], rs3088440 G>A (3'UTR, odds ratio 0.84, P = 0.0186, q = 0.3059), and rs4074785 C>T (intronic, odds ratio 0.85, P = 0.0248, q = 0.3059). None of the TP53 single nucleotide polymorphisms reached nominal significance. Two of the CDKN2A variants identified were also associated with reduced risk of progression from BE to EA, when assessed in a prospective cohort of 408 BE patients: rs2518720 (hazard ratio 0.57, P = 0.0095, q = 0.0285) and rs3088440 (hazard ratio 0.34, P = 0.0368, q = 0.0552). In vitro functional studies of rs3088440, a single nucleotide polymorphism located in the seed sequence of a predicted miR-663b binding site, suggested a mechanism whereby the G>A substitution may attenuate miR-663b-mediated repression of the CDKN2A transcript. This study provides the first evidence that germline variation at the CDKN2A locus may influence EA susceptibility.

Inflammation and oxidative stress markers and esophageal adenocarcinoma incidence in a Barrett's esophagus cohort.

BACKGROUND: Persons with Barrett's esophagus experience increased risk of esophageal adenocarcinoma. Prediagnostic inflammation markers predict several cancers, but their role in predicting esophageal adenocarcinoma is unknown. METHODS: We investigated whether biomarkers of inflammation [C-reactive protein (CRP), interleukin-6 (IL6), soluble tumor necrosis factor (sTNF) receptors I and II], and of oxidative stress (F2-isoprostanes) predicted progression to esophageal adenocarcinoma in a prospective cohort of 397 patients with Barrett's esophagus, 45 of whom developed esophageal adenocarcinoma. Biomarkers were measured in stored plasma samples from two time points during follow-up, the mean of which served as the primary predictor. Adjusted hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression. RESULTS: CRP level above the median was associated with an 80% increased risk of esophageal adenocarcinoma. The HR and 95% CI adjusted for age, gender, and further adjusted for waist-hip ratio and smoking were 1.98 (1.05-3.73) and 1.77 (0.93-3.37), respectively, with Ptrend for continuous CRP = 0.04. Persons with IL6 levels above the median also had almost 2-fold increased risk [HR and 95% CI adjusted for age and gender, and further adjusted for waist-hip ratio and smoking were 1.95 (1.03-3.72) and 1.79 (0.93-3.43), respectively, but no evidence of a trend was observed]. Concentrations of TNF receptors and F2-isoprostanes were not associated with esophageal adenocarcinoma risk. CONCLUSIONS: Further research is needed to evaluate the role of inflammation and associated markers in esophageal adenocarcinoma development in persons with Barrett's esophagus. IMPACT: This prospective study suggests that inflammation markers, particularly CRP and IL6, may help identify persons at higher risk of progression to esophageal adenocarcinoma.

Intraindividual variability over time in plasma biomarkers of inflammation and effects of long-term storage.

PURPOSE: Systemic measures of chronic inflammation, often based on a single blood draw, are frequently used to study the associations between inflammation and chronic diseases such as cancer. However, more information is needed on the measurement error in these markers due to laboratory error, within-person variation over time, and long-term storage. METHODS: We investigated the intraindividual variability of inflammation markers C-reactive protein (CRP), interleukin-6 (IL-6), and soluble tumor necrosis factor receptors I and II (sTNFRI and II) in a subsample of the Seattle Barrett's esophagus study cohort. Two fasting blood samples were collected between 1995 and 2009 from 360 participants on average 1.8 years apart. CRP, IL-6, and sTNF receptor levels were measured by immunonephelometry, ELISA, and multiplex assays, respectively. Intra- and inter-batch coefficients of variation (CV) were estimated using blinded pooled samples within each batch. Intraclass correlations (ICCs) were computed using random effects ANOVA. RESULTS: Intra- and inter-batch CVs for the pooled plasma aliquots were low (2.4-8.9 %), suggesting little laboratory variability. Reliability over time was excellent for sTNF receptors (ICCsTNF-RI = 0.89, ICCsTNF-RII = 0.85) and fair-to-good for CRP and IL-6 (ICCCRP = 0.55, ICCIL-6 = 0.57). For samples stored for over 13 years, the ICCs for CRP and IL-6 were decreased but those for sTNF receptors were unaffected. CONCLUSION: sTNF receptor levels are more stable within person over time than CRP or IL-6. Long-term storage of samples appears to increase the variability of CRP and IL-6 measures, while the reliability of soluble TNF receptor measures was not affected by storage time.

Quantification of population benefit in evaluation of biomarkers: practical implications for disease detection and prevention.

BACKGROUND: With the rapid development of "-omic" technologies, an increasing number of purported biomarkers have been identified for cancer and other diseases. The process of identifying those that are most promising and validating them for use at the population level for prevention and early detection is a critical next step in achieving significant health benefits. METHODS: In this paper, we propose that in order to effectively translate biomarkers for practical clinical use, it is important to distinguish and quantify the differences between the use of biomarkers and other risk factors to identify preventive interventions versus their use in disease risk prediction and early detection. We developed mathematical models for quantitatively evaluating risk and benefit in use of biomarkers for disease prevention or early detection. Simple numerical examples were used to demonstrate the potential applications of the models for various types of data. RESULTS: We propose an index which takes into account potential adverse consequences of biomarker-driven interventions - the 'naïve' ratio of population benefit (RPB) - to facilitate evaluating the potential impact of biomarkers on cancer prevention and personalized medicine. The index RPB is developed for both binary and continuous biomarkers/risk factors. Examples with computational analyses are presented in the paper to contrast the differences in using biomarkers/risk factors for prevention and early detection. CONCLUSIONS: Integrating epidemiologic knowledge into clinical decision making is a key step to translate new biomarkers/risk factors into practical use to achieve health benefits. The RPB proposed in this paper considers the absolute risk of a disease in intervention, and takes into account the risk-benefit effects simultaneously for a marker/exposure at the population level. The RPB illustrates a unique approach to quantitatively assess the risk and potential benefits of using a biomarker/risk factor for intervention in both early detection and prevention.

Temporal and spatial evolution of somatic chromosomal alterations: a case-cohort study of Barrett's esophagus.

All cancers are believed to arise by dynamic, stochastic somatic genomic evolution with genome instability, generation of diversity, and selection of genomic alterations that underlie multistage progression to cancer. Advanced esophageal adenocarcinomas have high levels of somatic copy number alterations. Barrett's esophagus is a risk factor for developing esophageal adenocarcinoma, and somatic chromosomal alterations (SCA) are known to occur in Barrett's esophagus. The vast majority (∼95%) of individuals with Barrett's esophagus do not progress to esophageal adenocarcinoma during their lifetimes, but a small subset develop esophageal adenocarcinoma, many of which arise rapidly even in carefully monitored patients without visible endoscopic abnormalities at the index endoscopy. Using a well-designed, longitudinal case-cohort study, we characterized SCA as assessed by single-nucleotide polymorphism arrays over space and time in 79 "progressors" with Barrett's esophagus as they approach the diagnosis of cancer and 169 "nonprogressors" with Barrett's esophagus who did not progress to esophageal adenocarcinoma over more than 20,425 person-months of follow-up. The genomes of nonprogressors typically had small localized deletions involving fragile sites and 9p loss/copy neutral LOH that generate little genetic diversity and remained relatively stable over prolonged follow-up. As progressors approach the diagnosis of cancer, their genomes developed chromosome instability with initial gains and losses, genomic diversity, and selection of SCAs followed by catastrophic genome doublings. Our results support a model of differential disease dynamics in which nonprogressor genomes largely remain stable over prolonged periods, whereas progressor genomes evolve significantly increased SCA and diversity within four years of esophageal adenocarcinoma diagnosis, suggesting a window of opportunity for early detection.

NSAIDs modulate clonal evolution in Barrett's esophagus.

Cancer is considered an outcome of decades-long clonal evolution fueled by acquisition of somatic genomic abnormalities (SGAs). Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce cancer risk, including risk of progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). However, the cancer chemopreventive mechanisms of NSAIDs are not fully understood. We hypothesized that NSAIDs modulate clonal evolution by reducing SGA acquisition rate. We evaluated thirteen individuals with BE. Eleven had not used NSAIDs for 6.2±3.5 (mean±standard deviation) years and then began using NSAIDs for 5.6±2.7 years, whereas two had used NSAIDs for 3.3±1.4 years and then discontinued use for 7.9±0.7 years. 161 BE biopsies, collected at 5-8 time points over 6.4-19 years, were analyzed using 1Million-SNP arrays to detect SGAs. Even in the earliest biopsies there were many SGAs (284±246 in 10/13 and 1442±560 in 3/13 individuals) and in most individuals the number of SGAs changed little over time, with both increases and decreases in SGAs detected. The estimated SGA rate was 7.8 per genome per year (95% support interval [SI], 7.1-8.6) off-NSAIDs and 0.6 (95% SI 0.3-1.5) on-NSAIDs. Twelve individuals did not progress to EA. In ten we detected 279±86 SGAs affecting 53±30 Mb of the genome per biopsy per time point and in two we detected 1,463±375 SGAs affecting 180±100 Mb. In one individual who progressed to EA we detected a clone having 2,291±78 SGAs affecting 588±18 Mb of the genome at three time points in the last three of 11.4 years of follow-up. NSAIDs were associated with reduced rate of acquisition of SGAs in eleven of thirteen individuals. Barrett's cells maintained relative equilibrium level of SGAs over time with occasional punctuations by expansion of clones having massive amount of SGAs.

Association between markers of obesity and progression from Barrett's esophagus to esophageal adenocarcinoma.

BACKGROUND & AIMS: Individuals with Barrett's esophagus (BE) have an increased risk of developing esophageal adenocarcinoma (EA). Obesity contributes to the development of BE and its progression to cancer. We investigated the roles of obesity-induced hyperinsulinemia and dysregulation of adipokines in these processes. METHODS: We measured fasting levels of glucose, insulin, leptin, and adiponectin in 392 patients enrolled in the Seattle Barrett's Esophagus Study. We calculated homeostatic model assessment scores (a measure of insulin sensitivity) and identified subjects with metabolic syndrome. We evaluated the association between these measures and the risk of EA using Cox regression models adjusted for known risk factors. RESULTS: Increasing homeostatic model assessment scores were associated with an increasing risk for EA; the strongest association was observed within the first 3 years after participants entered the study (hazard ratio [HR], 2.45; 95% confidence interval [CI], 1.43-4.1; P trend = .001). Leptin level also was associated significantly with an increased risk of EA within 3 years (HR, 2.51; 95% CI, 1.09-5.81; P trend = .03) and 6 years (HR, 2.07; 95% CI, 1.01-4.26; P trend = .048) of baseline. The level of high-molecular-weight adiponectin had a nonlinear inverse association with risk of EA; the strongest associations were observed in the second tertile (HR, 0.34; 95% CI, 0.14-0.82). Metabolic syndrome was not associated with risk of EA. CONCLUSIONS: Among patients with BE, increased levels of leptin and insulin resistance are associated with increased risk for EA, whereas increased levels of high-molecular-weight adiponectin is associated inversely with EA. These biomarkers might be used to determine cancer risk among patients with BE.

The role of tobacco, alcohol, and obesity in neoplastic progression to esophageal adenocarcinoma: a prospective study of Barrett's esophagus.

BACKGROUND: Esophageal adenocarcinoma (EA) incidence in many developed countries has increased dramatically over four decades, while survival remains poor. Persons with Barrett's esophagus (BE), who experience substantially elevated EA risk, are typically followed in surveillance involving periodic endoscopy with biopsies, although few progress to EA. No medical, surgical or lifestyle interventions have been proven to safely lower EA risk. DESIGN: We investigated whether smoking, obesity or alcohol could predict progression to EA in a prospective cohort of 411 BE patients. Data were collected during personal interview. Adjusted hazard ratios (HR) were estimated using Cox regression. RESULTS: 39% had body mass index (BMI) over 30 and 64% had smoked cigarettes. Main analyses focused on those with at least 5 months of follow-up (33,635 person-months), in whom 45 developed EA. Risk increased by 3% per year of age (trend p-value 0.02), with approximate doubling of risk among males. EA risk increased with smoking pack-years (trend p-value 0.04) and duration (p-value 0.05). Compared to never-smokers, the HR for those in the highest pack-year tertile was 2.29 (95%CI 1.04-5.07). No association was found with alcohol or BMI, whereas a suggestion of increased risk was observed in those with higher waist-hip ratio, especially among males. CONCLUSION: EA risk significantly increased with increasing age and cigarette exposure. Abdominal obesity, but not BMI, was associated with a modest increased risk. Continued follow-up of this and other cohorts is needed to precisely define these relationships so as to inform risk stratification and preventive interventions.

Selenium, selenoenzymes, oxidative stress and risk of neoplastic progression from Barrett's esophagus: results from biomarkers and genetic variants.

Clinical trials have suggested a protective effect of selenium supplementation on the risk of esophageal cancer, which may be mediated through the antioxidant activity of selenoenzymes. We investigated whether serum selenium concentrations, selenoenzyme activity, oxidative stress and genetic variation in selenoenzymes were associated with the risk of neoplastic progression to esophageal adenocarcinoma (EA) and two intermediate endpoints, aneuploidy and tetraploidy. In this prospective cohort study, during an average follow-up of 7.3 years, 47 EA cases, 41 aneuploidy cases and 51 tetraploidy cases accrued among 361 participants from the Seattle Barrett's Esophagus Research Study who were free of EA at the time of blood draw and had at least one follow-up visit. Development to EA was assessed histologically and aneuploidy and tetraploidy by DNA content flow cytometry. Serum selenium concentrations were measured using atomic absorption spectrometry, activity of glutathione peroxidase (GPX) 1 and GPX3 by substrate-specific coupled test procedures, selenoprotein P (SEPP1) concentrations and protein carbonyl content by ELISA method and malondialdehyde concentrations by HPLC. Genetic variants in GPX1-4 and SEPP1 were genotyped. Serum selenium was not associated with the risk of neoplastic progression to EA, aneuploidy or tetraploidy (P for trend = 0.25 to 0.85). SEPP1 concentrations were positively associated with the risk of EA [hazard ratio (HR) = 3.95, 95% confidence intervals (CI) = 1.42-10.97 comparing the third tertile with the first] and with aneuploidy (HR = 6.53, 95% CI = 1.31-32.58), but not selenoenzyme activity or oxidative stress markers. No genetic variants, overall, were associated with the risk of neoplastic progression to EA (global p = 0.12-0.69). Our results do not support a protective effect of selenium on risk of neoplastic progression to EA. Our study is the first to report positive associations of plasma SEPP1 concentrations with the risk of EA and aneuploidy, which warrants further investigation.

Use of statin medications and risk of esophageal adenocarcinoma in persons with Barrett's esophagus.

BACKGROUND: Persons with Barrett's esophagus experience increased incidence of esophageal adenocarcinoma and may benefit from use of preventives. Studies suggest that statin medications may have chemopreventive properties; we therefore assessed the association between statin use and progression to esophageal adenocarcinoma. METHODS: In a prospective cohort of 411 persons with Barrett's, Cox regression was used to calculate HRs for nonsteroidal anti-inflammatory drug (NSAID) and statin use accounting for variation in use during follow-up and adjusting for age, sex, and smoking. RESULTS: The HRs for statin use among all participants were 0.59 [95% confidence interval (CI), 0.26-1.33] and 0.68 (95% CI, 0.30-1.54) before and after further adjustment for NSAID use, respectively. Among persons with high-grade dysplasia, the HRs for statin use were 0.31 (95% CI, 0.11-0.86) and 0.41 (95% CI, 0.13-1.26) before and after adding NSAIDs to the model, respectively. CONCLUSIONS: While the reduced risk of esophageal adenocarcinoma observed among statin users may be explained by chance, the point estimates are similar in magnitude to those previously reported for NSAID use in this cohort and are unlikely to be confounded by known risk factors. IMPACT: Further study in larger cohorts and meta-analyses of the potential for statins to reduce risk of esophageal adenocarcinoma is warranted.

Application of biomarkers in cancer risk management: evaluation from stochastic clonal evolutionary and dynamic system optimization points of view.

Aside from primary prevention, early detection remains the most effective way to decrease mortality associated with the majority of solid cancers. Previous cancer screening models are largely based on classification of at-risk populations into three conceptually defined groups (normal, cancer without symptoms, and cancer with symptoms). Unfortunately, this approach has achieved limited successes in reducing cancer mortality. With advances in molecular biology and genomic technologies, many candidate somatic genetic and epigenetic "biomarkers" have been identified as potential predictors of cancer risk. However, none have yet been validated as robust predictors of progression to cancer or shown to reduce cancer mortality. In this Perspective, we first define the necessary and sufficient conditions for precise prediction of future cancer development and early cancer detection within a simple physical model framework. We then evaluate cancer risk prediction and early detection from a dynamic clonal evolution point of view, examining the implications of dynamic clonal evolution of biomarkers and the application of clonal evolution for cancer risk management in clinical practice. Finally, we propose a framework to guide future collaborative research between mathematical modelers and biomarker researchers to design studies to investigate and model dynamic clonal evolution. This approach will allow optimization of available resources for cancer control and intervention timing based on molecular biomarkers in predicting cancer among various risk subsets that dynamically evolve over time.

A comprehensive survey of clonal diversity measures in Barrett's esophagus as biomarkers of progression to esophageal adenocarcinoma.

Neoplastic progression is an evolutionary process driven by the generation of clonal diversity and natural selection on that diversity within a neoplasm. We hypothesized that clonal diversity is associated with risk of progression to cancer. We obtained molecular data from a cohort of 239 participants with Barrett's esophagus, including microsatellite shifts and loss of heterozygosity, DNA content tetraploidy and aneuploidy, methylation, and sequence mutations. Using these data, we tested all major diversity measurement methods, including genetic divergence and entropy-based measures, to determine which measures are correlated with risk of progression to esophageal adenocarcinoma. We also tested whether the use of different sets of loci and alterations to define clones (e.g., selectively advantageous versus evolutionarily neutral) improved the predictive value of the diversity indices. All diversity measures were strong and highly significant predictors of progression (Cox proportional hazards model, P < 0.001). The type of alterations evaluated had little effect on the predictive value of most of the diversity measures. In summary, diversity measures are robust predictors of progression to cancer in this cohort.

Chromosomal instability and copy number alterations in Barrett's esophagus and esophageal adenocarcinoma.

PURPOSE: Chromosomal instability, as assessed by many techniques, including DNA content aneuploidy, loss of heterozygosity, and comparative genomic hybridization, has consistently been reported to be common in cancer and rare in normal tissues. Recently, a panel of chromosome instability biomarkers, including loss of heterozygosity and DNA content, has been reported to identify patients at high and low risk of progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA), but required multiple platforms for implementation. Although chromosomal instability involving amplifications and deletions of chromosome regions have been observed in nearly all cancers, copy number alterations (CNA) in premalignant tissues have not been well characterized or evaluated in cohort studies as biomarkers of cancer risk. EXPERIMENTAL DESIGN: We examined CNAs in 98 patients having either BE or EA using Bacterial Artificial Chromosome (BAC) array comparative genomic hybridization to characterize CNAs at different stages of progression ranging from early BE to advanced EA. RESULTS: CNAs were rare in early stages (less than high-grade dysplasia) but were progressively more frequent and larger in later stages (high-grade dysplasia and EA), including high-level amplifications. The number of CNAs correlated highly with DNA content aneuploidy. Patients whose biopsies contained CNAs involving >70 Mbp were at increased risk of progression to DNA content abnormalities or EA (hazards ratio, 4.9; 95% confidence interval, 1.6-14.8; P = 0.0047), and the risk increased as more of the genome was affected. CONCLUSIONS: Genome-wide analysis of CNAs provides a common platform for the evaluation of chromosome instability for cancer risk assessment as well as for the identification of common regions of alteration that can be further studied for biomarker discovery.

Intestinal differentiation in metaplastic, nongoblet columnar epithelium in the esophagus.

Barrett esophagus (BE) is defined by the presence of metaplastic esophageal columnar epithelium with goblet cells within endoscopically recognizable areas of the esophagus. However, some carcinomas in BE, or from the gastroesophageal junction region, develop within mucosa devoid of goblet cells. However, the biologic properties, pathogenesis, and risk of malignancy of metaplastic, esophageal nongoblet columnar epithelium, is, essentially, unknown. In this study, 89 patients with metaplastic esophageal columnar epithelium were evaluated immunohistochemically for markers of intestinal differentiation, such as MUC2, DAS-1, Villin, and CDX2, a marker of gastric differentiation (MUC5AC), and Ki67, a marker of cell proliferation. Of the 89 patients, 59 had columnar metaplasia with goblet cells (BE), which were further separated into low-density goblet cell and high-density goblet cell groups based on the percentage of crypts with goblet cells, and 30 patients had columnar metaplasia of the esophagus without goblet cells. As controls, gastric biopsies from 19 age and sex matched patients without esophageal or gastric pathology were used. The rate of positivity of the markers and the location of Ki67 staining was evaluated only in non-goblet columnar epithelium from all patient groups. Patients with metaplastic esophageal columnar epithelium without goblet cells showed positivity for MUC5AC, MUC2, DAS-1, Villin, and CDX2 in 100%, 0%, 30%, 17%, and 43% of cases, respectively. 17% of cases showed aberrant surface Ki67 positivity. These values were significantly higher than gastric controls, which showed absence of staining for all markers except MUC5AC (100%). In patients with metaplastic esophageal columnar epithelium with goblet cells (BE) a significant increased rate of staining was observed for all markers, except MUC5AC. In addition, both MUC2 and surface Ki67 staining were significantly increased in BE patients with high-density goblet cells versus those with low-density goblet cells. In a separate analysis in which metaplastic esophageal nongoblet epithelium was evaluated in areas of mucosa devoid of goblet cells compared with areas of mucosa with goblet cells, from patients who had goblet cells elsewhere in the mucosa (N=59), no significant differences were observed with regard to the percentage of cases that stained with any of the markers in the nongoblet epithelium in areas devoid of goblet cells, similar to the patient group with metaplastic esophageal epithelium without goblet cells (N=30). Similar to above, in all cases, expression of intestinal markers increased in areas of mucosa adjacent to goblet cells. This study provides evidence that metaplastic esophageal columnar epithelium without goblet cells shows phenotypic evidence of intestinal differentiation and supports the theory that squamous epithelium converts initially to nongoblet columnar epithelium before goblet cell metaplasia. Further prospective studies are needed to evaluate the pathogenetic sequence, natural history, and risk of malignancy of metaplastic esophageal nongoblet epithelium.

Nonadenomatous dysplasia in barrett esophagus: a clinical, pathologic, and DNA content flow cytometric study.

Rarely, dysplasia in Barrett's esophagus (BE) is composed of crypts lined by cuboidal-shaped cells that contain a centrally located nucleus, markedly increased nuclear/cytoplasmic ratio, but without nuclear stratification characteristic of conventional "adenomatous" dysplasia. The aim of this study was to evaluate the clinical and pathologic features, natural history, and DNA content flow cytometric abnormalities of BE patients with non-adenomatous dysplasia (NAD) in a cohort of BE patients enrolled in a prospective surveillance program. Eighteen patients with NAD identified over a 6 year period, in a cohort of 270 consecutive patients with BE and without esophageal adenocarcinoma (EA) at baseline, were evaluated for clinical and pathologic features, including association with conventional adenomatous dysplasia and EA, DNA content flow cytometric abnormalities (tetraploidy and aneuploidy) and outcome, over a mean follow-up period of 4.1 years. The findings in the 18 study patients were compared to those in the 252 remaining (control) patients without NAD. Control patients included 228 with metaplasia/indefinite for dysplasia, and 24 with conventional adenomatous dysplasia (13 low-grade, 11 high-grade). The prevalence rate of NAD in our BE cohort was 6.7% Of the 18 study patients, there were 17 were males and 1 female of mean age 66.7 years. The mean length of BE was 3.9 cm NAD foci were associated with goblet or non-goblet epithelium in 62% and 38% of cases, respectively. Ninety-four percent of patients with NAD (17/18) also had conventional adenomatous dysplasia (four with low-grade, 13 with high-grade) elsewhere in the esophagus at the same endoscopic procedure as the one that detected NAD. Patients with NAD had a significantly shorter length of BE compared to control patients with conventional adenomatous dysplasia (N=24) (p=0.03). Patients with NAD also showed a significantly higher rate of DNA content flow cytometric abnormalities compared to the entire cohort of control patients (38% vs. 11%, p=0.05). However, no significant differences regarding either flow cytometric abnormalities or progression to EA were found when the NAD patients were compared only to the 24 controls with conventional adenomatous dysplasia. NAD is a high grade histologic variant of intraepithelial neoplasia that is episodic in nature, and shows a high association with conventional adenomatous high-grade dysplasia.

p16 mutation spectrum in the premalignant condition Barrett's esophagus.

BACKGROUND: Mutation, promoter hypermethylation and loss of heterozygosity involving the tumor suppressor gene p16 (CDKN2a/INK4a) have been detected in a wide variety of human cancers, but much less is known concerning the frequency and spectrum of p16 mutations in premalignant conditions. METHODS AND FINDINGS: We have determined the p16 mutation spectrum for a cohort of 304 patients with Barrett's esophagus, a premalignant condition that predisposes to the development of esophageal adenocarcinoma. Forty seven mutations were detected by sequencing of p16 exon 2 in 44 BE patients (14.5%) with a mutation spectrum consistent with that caused by oxidative damage and chronic inflammation. The percentage of patients with p16 mutations increased with increasing histologic grade. In addition, samples from 3 out of 19 patients (15.8%) who underwent esophagectomy were found to have mutations. CONCLUSIONS: The results of this study suggest the environment of the esophagus in BE patients can both generate and select for clones with p16 mutations.