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Background & Aims: The aim of this study was to characterize baseline morphologic features of crypts in nondysplastic Barrett's esophagus and correlate them with DNA content abnormalities and risk of progression to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC)., Methods: The morphologic features of nondysplastic crypts in baseline biopsy specimens from 212 BE patients (2956 biopsy specimens) were graded histologically using a 4-point scale (crypt atypia levels, 0-3). DNA content abnormalities were detected using flow cytometry., Results: In patients who had dysplasia in their baseline biopsy specimens, dysplasia was associated significantly with increasing grades of crypt atypia in the background nondysplastic Barrett's esophagus (P < .001). In a subset of patients without dysplasia at baseline (N = 149), a higher grade of crypt atypia was associated with longer Barrett's esophagus segment length (5.5 vs 3.3 cm; P = .0095), and a higher percentage of cells with 4N DNA content (3.67 ± 1.27 vs 2.93 ± 1.22; P = .018). Crypt atypia was associated with the development of any neoplasia (low-grade dysplasia and HGD/EAC). Although no significant association was noted between the grade of crypt atypia and increased 4N, aneuploidy, or progression to HGD/EAC, only patients with grade 2 or 3 crypt atypia showed increased 4N, aneuploidy, or progression to HGD/EAC., Conclusions: Patients with Barrett's esophagus likely develop dysplasia via a progressive increase in the level of crypt atypia before the onset of dysplasia, and these changes may reflect some alteration of DNA content., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer 1-6 . At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett's oesophagus. These data included 206 biopsies in Barrett's oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case-control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett's-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection., (© 2023. The Author(s).)

While the genomes of normal tissues undergo dynamic changes over time, little is understood about the temporal-spatial dynamics of genomes in premalignant tissues that progress to cancer compared to those that remain cancer-free. Here we use whole genome sequencing to contrast genomic alterations in 427 longitudinal samples from 40 patients with stable Barrett's esophagus compared to 40 Barrett's patients who progressed to esophageal adenocarcinoma (ESAD). We show the same somatic mutational processes are active in Barrett's tissue regardless of outcome, with high levels of mutation, ESAD gene and focal chromosomal alterations, and similar mutational signatures. The critical distinction between stable Barrett's versus those who progress to cancer is acquisition and expansion of TP53-/- cell populations having complex structural variants and high-level amplifications, which are detectable up to six years prior to a cancer diagnosis. These findings reveal the timing of common somatic genome dynamics in stable Barrett's esophagus and define key genomic features specific to progression to esophageal adenocarcinoma, both of which are critical for cancer prevention and early detection strategies., (© 2022. The Author(s).)

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis., Competing Interests: Declaration of Interests J.S.R.-F. reports receiving personal/consultancy fees from VolitionRx, Paige.AI, Goldman Sachs, REPARE Therapeutics, GRAIL, Ventana Medical Systems, Roche, Genentech, and InviCRO outside of the scope of the submitted work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Barrett's Esophagus is a neoplastic condition which progresses to esophageal adenocarcinoma in 5% of cases. Key events affecting the outcome likely occur before diagnosis of Barrett's and cannot be directly observed; we use phylogenetic analysis to infer such past events. We performed whole-genome sequencing on 4-6 samples from 40 cancer outcome and 40 noncancer outcome patients with Barrett's Esophagus, and inferred within-patient phylogenies of deconvoluted clonal lineages. Spatially proximate lineages clustered in the phylogenies, but temporally proximate ones did not. Lineages with inferred loss-of-function mutations in both copies of TP53 and CDKN2A showed enhanced spatial spread, whereas lineages with loss-of-function mutations in other frequently mutated loci did not. We propose a two-phase model with expansions of TP53 and CKDN2A mutant lineages during initial growth of the segment, followed by relative stasis. Subsequent to initial expansion, mutations in these loci as well as ARID1A and SMARCA4 may show a local selective advantage but do not expand far: The spatial structure of the Barrett's segment remains stable during surveillance even in patients who go on to cancer. We conclude that the cancer/noncancer outcome is strongly affected by early steps in formation of the Barrett's segment., Competing Interests: None declared., (© 2020 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

It was highlighted that in the original article [1] the Availability of data and materials section was incorrect.

Background: Use of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) has been shown to protect against tetraploidy, aneuploidy, and chromosomal alterations in the metaplastic condition Barrett's esophagus (BE) and to lower the incidence and mortality of esophageal adenocarcinoma (EA). The esophagus is exposed to both intrinsic and extrinsic mutagens resulting from gastric reflux, chronic inflammation, and exposure to environmental carcinogens such as those found in cigarettes. Here we test the hypothesis that NSAID use inhibits accumulation of point mutations/indels during somatic genomic evolution in BE., Methods: Whole exome sequences were generated from 82 purified epithelial biopsies and paired blood samples from a cross-sectional study of 41 NSAID users and 41 non-users matched by sex, age, smoking, and continuous time using or not using NSAIDs., Results: NSAID use reduced overall frequency of point mutations across the spectrum of mutation types, lowered the frequency of mutations even when adjusted for both TP53 mutation and smoking status, and decreased the prevalence of clones with high variant allele frequency. Never smokers who consistently used NSAIDs had fewer point mutations in signature 17, which is commonly found in EA. NSAID users had, on average, a 50% reduction in functional gene mutations in nine cancer-associated pathways and also had less diversity in pathway mutational burden compared to non-users., Conclusions: These results indicate NSAID use functions to limit overall mutations on which selection can act and supports a model in which specific mutant cell populations survive or expand better in the absence of NSAIDs.

The low risk of progression of Barrett's esophagus (BE) to esophageal adenocarcinoma can lead to over-diagnosis and over-treatment of BE patients. This may be addressed through a better understanding of the dynamics surrounding BE malignant progression. Although genetic diversity has been characterized as a marker of malignant development, it is still unclear how BE arises and develops. Here we uncover the evolutionary dynamics of BE at crypt and biopsy levels in eight individuals, including four patients that experienced malignant progression. We assay eight individual crypts and the remaining epithelium by SNP array for each of 6-11 biopsies over 2 time points per patient (358 samples in total). Our results indicate that most Barrett's segments are clonal, with similar number and inferred rates of alterations observed for crypts and biopsies. Divergence correlates with geographical location, being higher near the gastro-esophageal junction. Relaxed clock analyses show that genomic instability precedes and is enhanced by genome doubling. These results shed light on the clinically relevant evolutionary dynamics of BE.

Cancer development is driven by genomic alterations, including copy number aberrations. The detection of copy number aberrations in tumor cells is often complicated by possible contamination of normal stromal cells in tumor samples and intratumor heterogeneity, namely the presence of multiple clones of tumor cells. In order to correctly quantify copy number aberrations, it is critical to successfully de-convolute the complex structure of the genetic information from tumor samples. In this article, we propose a general Bayesian method for estimating copy number aberrations when there are normal cells and potentially more than one tumor clones. Our method provides posterior probabilities for the proportions of tumor clones and normal cells. We incorporate prior information on the distribution of the copy numbers to prioritize biologically more plausible solutions and alleviate possible identifiability issues that have been observed by many researchers. Our model is flexible and can work for both SNP array and next-generation sequencing data. We compare our method to existing ones and illustrate the advantage of our approach in multiple datasets.

Technological advances in genome sequencing and copy number analysis have allowed researchers to catalog the wide variety of genomic alterations that occur across diverse cancer types. For most cancer types, the lack of high-frequency alterations and the heterogeneity observed both within and between tumors suggest neoplastic progression proceeds through a branched evolutionary pathway as proposed by Nowell in 1976, as opposed to the linear pathway that has dominated medical science for the last century. To understand how cancer evolves over time and space in the body, new study designs are needed that can distinguish between alterations that develop in patients who progress to cancer from to those who don't. Here we present approaches developed in the study of Barrett's esophagus, a premalignant precursor of esophageal adenocarcinoma, and discuss strategies for applying the results from these analyses to address the critical clinical problems of overdiagnosis of benign disease, early detection of life-threatening cancer, and effective risk stratification.

Beginning in the 1980s, an alarming rise in the incidence of esophageal adenocarcinoma (EA) led to screening of patients with reflux to detect Barrett's esophagus (BE) and surveillance of BE to detect early EA. This strategy, based on linear progression disease models, resulted in selective detection of BE that does not progress to EA over a lifetime (overdiagnosis) and missed BE that rapidly progresses to EA (underdiagnosis). Here we review the historical thought processes that resulted in this undesired outcome and the transformation in our understanding of genetic and evolutionary principles governing neoplastic progression that has come from application of modern genomic technologies to cancers and their precursors. This new synthesis provides improved strategies for prevention and early detection of EA by addressing the environmental and mutational processes that can determine "windows of opportunity" in time to detect rapidly progressing BE and distinguish it from slowly or nonprogressing BE., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

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., (©2015 American Association for Cancer Research.)

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., (©2013 AACR.)

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., Competing Interests: The authors have declared that no competing interests exist.

Identifying molecular markers of endometrial hyperplasia (neoplasia) progression is critical to cancer prevention. To assess RNA and DNA quantity and quality from routinely collected endometrial samples and evaluate the performance of RNA- and DNA-based arrays across endometrial tissue types, we collected fresh frozen (FF) Pipelle, FF curettage, and formalin-fixed paraffin-embedded (FFPE) hysterectomy specimens (benign indications) from eight women. Additionally, neoplastic and uninvolved tissues from 24 FFPE archival hysterectomy specimens with endometrial hyperplasias and carcinomas were assessed. RNA was extracted from 15 of 16 FF and 51 of 51 FFPE samples, with yields >1.2  μ g for 13/15 (87%) FF and 50/51 (98%) FFPE samples. Extracted RNA was of high quality; all samples performed successfully on the Illumina whole-genome cDNA-mediated annealing, selection, extension, and ligation (WG-DASL) array and performance did not vary by tissue type. While DNA quantity from FFPE samples was excellent, quality was not sufficient for successful performance on the Affymetrix SNP Array 6.0. In conclusion, FF Pipelle samples, which are minimally invasive, yielded excellent quantity and quality of RNA for gene expression arrays (similar to FF curettage) and should be considered for use in genomic studies. FFPE-derived DNA should be evaluated on new rapidly evolving sequencing platforms.

Background: Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett's esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis., Methodology/principal Findings: We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D) in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption., Conclusions/significance: Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells.

Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates., (© 2012 Society of Photo-Optical Instrumentation Engineers (SPIE).)

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.

Purpose: Elevated cellular proliferation and cell cycle abnormalities, which have been associated with premalignant lesions, may be caused by inactivation of tumor suppressor genes. We measured proliferative and cell cycle fractions of biopsies from a cohort of patients with Barrett's esophagus to better understand the role of proliferation in early neoplastic progression and the association between cell cycle dysregulation and tumor suppressor gene inactivation., Experimental Design: Cell proliferative fractions (determined by Ki67/DNA multiparameter flow cytometry) and cell cycle fractions (DNA content flow cytometry) were measured in 853 diploid biopsies from 362 patients with Barrett's esophagus. The inactivation status of CDKN2A and TP53 was assessed in a subset of these biopsies in a cross-sectional study. A prospective study followed 276 of the patients without detectable aneuploidy for an average of 6.3 years with esophageal adenocarcinoma as an end point., Results: Diploid S and 4N (G(2)/tetraploid) fractions were significantly higher in biopsies with TP53 mutation and loss of heterozygosity. CDKN2A inactivation was not associated with higher Ki67-positive, diploid S, G(1), or 4N fractions. High Ki67-positive and G(1)-phase fractions were not associated with the future development of esophageal adenocarcinoma (P=0.13 and P=0.15, respectively), whereas high diploid S-phase and 4N fractions were (P=0.03 and P<0.0001, respectively)., Conclusions: High Ki67-positive proliferative fractions were not associated with inactivation of CDKN2A and TP53 or future development of cancer in our cohort of patients with Barrett's esophagus. Biallelic inactivation of TP53 was associated with elevated 4N fractions, which have been associated with the future development of esophageal adenocarcinoma.

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.

Single nucleotide polymorphisms (SNPs) have been increasingly utilized to investigate somatic genetic abnormalities in premalignancy and cancer. LOH is a common alteration observed during cancer development, and SNP assays have been used to identify LOH at specific chromosomal regions. The design of such studies requires consideration of the resolution for detecting LOH throughout the genome and identification of the number and location of SNPs required to detect genetic alterations in specific genomic regions. Our study evaluated SNP distribution patterns and used probability models, Monte Carlo simulation, and real human subject genotype data to investigate the relationships between the number of SNPs, SNP HET rates, and the sensitivity (resolution) for detecting LOH. We report that variances of SNP heterozygosity rate in dbSNP are high for a large proportion of SNPs. Two statistical methods proposed for directly inferring SNP heterozygosity rates require much smaller sample sizes (intermediate sizes) and are feasible for practical use in SNP selection or verification. Using HapMap data, we showed that a region of LOH greater than 200 kb can be reliably detected, with losses smaller than 50 kb having a substantially lower detection probability when using all SNPs currently in the HapMap database. Higher densities of SNPs may exist in certain local chromosomal regions that provide some opportunities for reliably detecting LOH of segment sizes smaller than 50 kb. These results suggest that the interpretation of the results from genome-wide scans for LOH using commercial arrays need to consider the relationships among inter-SNP distance, detection probability, and sample size for a specific study. New experimental designs for LOH studies would also benefit from considering the power of detection and sample sizes required to accomplish the proposed aims.

Chromosomal instability is regarded as an underlying mechanism of neoplastic progression, integral to the clonal selection and evolution that leads to cancer. We evaluated chromosomal instability in premalignant Barrett's esophagus tissue using high resolution Affymetrix mapping 100K SNP arrays as patients progressed through three molecular stages of disease-CDKN2A(LOH) only, CDKN2A(LOH)/TP53(LOH), and CDKN2A(LOH)/TP53(LOH) with aneuploidy. Within individuals over time, we observed increases in both numbers and sizes of regions of LOH or copy number change. In the earliest CDKN2A(LOH) only samples, we detected few regions with both copy change and LOH, whereas copy loss and LOH were highly correlated in more advanced samples. These data indicate that genomic instability increases in severity and changes character during neoplastic progression. In addition, distinct patterns of clonal evolution could be discerned within a segment of Barrett's esophagus. Overall, this study illustrates that pre-malignant disease can be associated with extensive instability and clonal dynamics that evolve from an initial stage characterized by small recombination-based alterations to one with larger copy change events likely associated with mitotic instability., ((c) 2007 Wiley-Liss, Inc.)

Background: Defects in DNA damage recognition and repair have been associated with a wide variety of cancers. We conducted a prospective study to determine whether mutagen sensitivity, as determined by an in vitro assay, was associated with the future development of cancer in patients with Barrett's esophagus, which is associated with increased risk of progression to esophageal adenocarcinoma., Methods: We measured sensitivity to bleomycin in peripheral blood lymphocytes in a cohort of 220 patients with Barrett's esophagus. We followed these patients for 1,230 person-years (range, 3 months to 10.1 years; median, 6.4 years), using development of cancer and aneuploidy as end points. A subset of these patients was evaluated for inactivation of tumor-suppressor genes CDKN2A/p16 and TP53 [by mutation and loss of heterozygosity (LOH)] in their Barrett's segments at the time of, or before, the bleomycin test, and the patients were stratified by CDKN2A/p16 and TP53 status in an analysis of mutagen sensitivity and progression., Results: Bleomycin-sensitive patients were found to be at significantly greater risk of developing aneuploidy (adjusted hazard ratio, 3.71; 95% confidence interval, 1.44-9.53) and nonsignificantly greater risk of cancer (adjusted hazard ratio, 1.63; 95% confidence interval, 0.71-3.75). Among patients with detectable LOH at the TP53 locus (on chromosome 17p), increasing bleomycin sensitivity was associated with increased risk of developing cancer (P(trend) < 0.001) and aneuploidy (P(trend) = 0.005)., Conclusions: This study supports the hypothesis that sensitivity to mutagens increases the risk of neoplastic progression in persons with Barrett's esophagus, particularly those with 17p LOH including TP53.

We have constructed a high-speed, two-dimensional capillary electrophoresis system with a compact and high-sensitivity fluorescence detector. This instrument is used for the rapid and reproducible separations of Barrett's esophagus tissue homogenates. Proteins and biogenic amines are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled biomolecules are separated sequentially in two capillaries. The first capillary employs capillary sieving electrophoresis using a replaceable sieving matrix. Fractions are successively transferred to a second capillary where they undergo additional separation by micellar electrokinetic capillary chromatography. The comprehensive two-dimensional separation requires 60 min. Within-day migration time reproducibility is better than 1% in both dimensions for the 50 most intense features. Between-day migration time precision is 1.3% for CSE and better than 0.6% for MECC. Biopsies were obtained from the squamous epithelium in the proximal tubular esophagus, Barrett's epithelium from the distal esophagus, and fundus region of the stomach from each of three Barrett's esophagus patients with informed consent. We identified 18 features from the homogenate profiles as biogenic amines and amino acids. For each of the patients, Barrett's biopsies had more than 5 times the levels of phenylalanine and alanine as compared to squamous tissues. The patient with high-grade dysplasia shows the highest concentrations for 13 of the amino acids across all tissue types. Concentrations of glycine are 40 times higher in squamous biopsies compared to Barrett's and fundal biopsies from the patient with high-grade dysplasia. These results suggest that two-dimensional capillary electrophoresis may be of value for the rapid characterization of endoscopic and surgical biopsies.

Neoplasms are thought to progress to cancer through genetic instability generating cellular diversity and clonal expansions driven by selection for mutations in cancer genes. Despite advances in the study of molecular biology of cancer genes, relatively little is known about evolutionary mechanisms that drive neoplastic progression. It is unknown, for example, which may be more predictive of future progression of a neoplasm: genetic homogenization of the neoplasm, possibly caused by a clonal expansion, or the accumulation of clonal diversity. Here, in a prospective study, we show that clonal diversity measures adapted from ecology and evolution can predict progression to adenocarcinoma in the premalignant condition known as Barrett's esophagus, even when controlling for established genetic risk factors, including lesions in TP53 (p53; ref. 6) and ploidy abnormalities. Progression to cancer through accumulation of clonal diversity, on which natural selection acts, may be a fundamental principle of neoplasia with important clinical implications.

Purpose: Neosquamous epithelium (NSE) can arise within Barrett's esophagus as a consequence of medical or surgical acid reduction therapy, as well as after endoscopic ablation. Morphologic studies have suggested that NSE can develop from adjacent squamous epithelium, submucosal gland ducts, or multipotent progenitor cell(s) that can give rise to either squamous or Barrett's epithelium, depending on the luminal environment. The cells responsible for Barrett's epithelium self-renewal are frequently mutated during neoplastic progression. If NSE arises from the same cells that self-renew the Barrett's epithelium, the two tissues should be clonally related and share genetic alterations; if NSE does not originate in the self-renewing Barrett's, NSE and Barrett's esophagus should be genetically independent., Experimental Design: We isolated islands of NSE and the surrounding Barrett's epithelium from 20 patients by microdissection and evaluated each tissue for genetic alterations in exon 2 of CDKN2A or exons 5 to 9 of the TP53 gene. Nine patients had p16 mutations and 11 had TP53 mutations within the Barrett's epithelium., Results: In 1 of 20 patients, a focus of NSE had a 146 bp deletion in p16 identical to that found in surrounding Barrett's epithelium. The NSE in the remaining 19 patients was wild-type for p16 or TP53., Conclusion: Our mutational data support the hypothesis that, in most circumstances, NSE originates in cells different from those responsible for self-renewal of Barrett's epithelium. However, in one case, NSE and Barrett's epithelium seem to have arisen from a progenitor cell that was capable of differentiating into either intestinal metaplasia or NSE.

There is debate in the literature over the relative importance of genetic instability and clonal expansion during progression to cancer. Barrett's esophagus is a uniquely suited model to investigate neoplastic progression prospectively because periodic endoscopic biopsy surveillance is recommended for early detection of esophageal adenocarcinoma. We hypothesized that expansion of clones with genetic instability would predict progression to esophageal adenocarcinoma. We measured p16 (CDKN2A/INK4A) lesions (loss of heterozygosity, mutations, and CpG island methylation), p53 (TP53) lesions (loss of heterozygosity, mutation) and ploidy abnormalities (aneuploidy, tetraploidy) within each Barrett's esophagus segment of a cohort of 267 research participants, who were followed prospectively with cancer as an outcome. We defined the size of a lesion as the fraction of cells with the lesion multiplied by the length of the Barrett's esophagus segment. A Cox proportional hazards regression indicates that the sizes of clones with p53 loss of heterozygosity (relative risk = 1.27(x) for an x cm clone; 95% confidence interval, 1.07-1.50) or ploidy abnormalities (relative risk = 1.31(x) for an x cm clone; 95% confidence interval, 1.07-1.60) predict progression to esophageal adenocarcinoma better than the mere presence of such clones (likelihood ratio test, P < 0.01). Controlling for length of the Barrett's esophagus segment had little effect. The size of a clone with a p16 lesion is not a significant predictor of esophageal adenocarcinoma when we controlled for p53 loss of heterozygosity status. The combination of clonal expansion and genetic instability is a better predictor of cancer outcome than either alone. This implies that interventions that limit expansion of genetically unstable clones may reduce risk of progression to cancer.

Neoplastic progression is an evolutionary process characterized by genomic instability and waves of clonal expansions carrying genetic and epigenetic lesions to fixation (100% of the cell population). However, an evolutionarily neutral lesion may also reach fixation if it spreads as a hitchhiker on a selective sweep. We sought to distinguish advantageous lesions from hitchhikers in the premalignant condition Barrett's esophagus. Patients (211) had biopsies taken at 2-cm intervals in their Barrett's segments. Purified epithelial cells were assayed for loss of heterozygosity and microsatellite shifts on chromosomes 9 and 17, sequence mutations in CDKN2A/MTS1/INK4a (p16) and TP53 (p53), and methylation of the p16 promoter. We measured the expanse of a lesion in a Barrett's segment as the proportion of proliferating cells that carried a lesion in that locus. We then selected the lesion having expanses >90% in the greatest number of patients as our first putative advantageous lesion. We filtered out hitchhikers by removing all expanses of other lesions that did not occur independent of the advantageous lesion. The entire process was repeated on the remaining expanses to identify additional advantageous lesions. p16 loss of heterozygosity, promoter methylation, and sequence mutations have strong, independent, advantageous effects on Barrett's cells early in progression. Second lesions in p16 and p53 are associated with later selective sweeps. Virtually all of the other lesion expansions, including microsatellite shifts, could be explained as hitchhikers on p16 lesion clonal expansions. These techniques can be applied to any neoplasm.

As there has been no previous information on the consequences of telomerase expression in genetically altered, mortal cells derived from pre-malignant tissue, we sought to determine the effect of hTERT (human catalytic subunit of telomerase reverse transcriptase) transduction of pre-malignant cell strains from Barrett's esophagus that do not contain telomerase activity and possess a finite lifespan. Primary cultures of Barrett's esophageal epithelium transduced with a retrovirus containing hTERT were characterized by growth factor requirements, cytogenetics and flow cytometry. Expression of telomerase lengthened telomeres and greatly extended the lifespan of hTERT transduced (hTERT+) Barrett's esophagus cells. Growth factor dependency of the hTERT+ cultures remained largely similar to the parental cultures, although there was a modest increase in the ability to grow in agar. Chromosomal instability, measured by both karyotypic and FISH (fluorescence in situ hybridization) analyses, was reduced but not abrogated by hTERT transduction, suggesting that telomerase expression can enhance genomic stability. However, the persistence of residual instability gave rise to new clonal and non-clonal genetic variants, and in one hTERT+ culture a new DNA aneuploid population was observed, the only time such a ploidy shift has been seen in Barrett's cell strains in vitro. These in vitro observations are analogous to the clinical progression to aneuploidy that often precedes cancer in Barrett's esophagus, and suggest that reactivation of telomerase may be permissive for continued genetic evolution to cancer. Long-lived Barrett's esophagus epithelial cultures should provide a useful in vitro model for studies of neoplastic evolution and chemopreventive therapies.

Barrett's esophagus (BE) is the only known precursor to esophageal adenocarcinoma, a cancer of which the incidence has been increasing at an alarming rate in Western countries. p16(INK4a) lesions occur frequently in esophageal adenocarcinomas but their role in neoplastic progression is not well understood. We detected 9p21 loss of heterozygosity, p16 CpG island methylation, and p16 mutations in biopsies from 57%, 61%, and 15%, respectively, of 107 patients with BE. In contrast, no mutations were found in p14(ARF) or p15, and methylation was found in only 4% and 13%, respectively. >85% of Barrett's segments had clones with one (p16+/-) or two (p16-/-) p16 lesions. Both p16+/- and p16-/- clones underwent extensive expansion involving up to 17 cm of esophageal mucosa. The prevalence of established biomarkers in BE, such as 17p (p53) loss of heterozygosity, aneuploidy, and/or increased 4N (tetraploid) populations, increased from 0% to 20% to 44% in patients whose biopsies were p16+/+, p16+/-, and p16-/-, respectively (P < 0.001). Barrett's segment lengths also increased with change in p16 status with a median of 1.5, 6.0, and 8.0 cm for patients with p16+/+, p16+/-, and p16-/- biopsies, respectively (P < 0.001). We conclude that most Barrett's metaplasia contains genetic and/or epigenetic p16 lesions and has the ability to undergo clonal expansion, creating a field in which other abnormalities can arise that can lead to esophageal adenocarcinoma.

It has been hypothesized that neoplastic progression develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal populations with accumulated genetic errors. Accordingly, human cancers and some premalignant lesions contain multiple genetic abnormalities not present in the normal tissues from which the neoplasms arose. Barrett oesophagus (BE) is a premalignant condition which predisposes to oesophageal adenocarcinoma (EA) that can be biopsied prospectively over time because endoscopic surveillance is recommended for early detection of cancer. In addition, oesophagectomy specimens frequently contain the premalignant epithelium from which the cancer arose. Neoplastic progression in BE is associated with alterations in TP53 (also known as p53) and CDKN2A (also known as p16) and non-random losses of heterozygosity (LOH). Aneuploid or increased 4N populations occur in more than 90-95% of EAs, arise in premalignant epithelium and predict progression. We have previously shown in small numbers of patients that disruption of TP53 and CDKN2A typically occurs before aneuploidy and cancer. Here, we determine the evolutionary relationships of non-random LOH, TP53 and CDKN2A mutations, CDKN2A CpG-island methylation and ploidy during neoplastic progression. Diploid cell progenitors with somatic genetic or epigenetic abnormalities in TP53 and CDKN2A were capable of clonal expansion, spreading to large regions of oesophageal mucosa. The subsequent evolution of neoplastic progeny frequently involved bifurcations and LOH at 5q, 13q and 18q that occurred in no obligate order relative to each other, DNA-content aneuploidy or cancer. Our results indicate that clonal evolution is more complex than predicted by linear models.

Loss of heterozygosity (LOH) is a common genetic lesion found in many human neoplasms. Extending investigation of LOH to large-scale clinical and public health science studies has proven difficult because of the small size and cellular and genetic heterogeneity of human neoplasms, in addition to the challenges associated with increasing throughput. Our approach to LOH analysis was developed using clinical biopsy samples from patients with Barrett's esophagus (BE) and uses flow cytometric cell sorting to increase sample purity, whole genome amplification to increase sample amount, and automated fluorescent genotyping to increase sample throughput. This approach allows LOH assessment at 20 loci in DNA extracted from 1000 flow-purified cells while maintaining accurate and reproducible allele ratios compared with the standard method of using genomic DNA. This method of analysis should allow accurate, reproducible determination of allele ratios in a variety of human tumors and premalignant conditions.

Amplification of genes involved in signal transduction and cell cycle control occurs in a significant fraction of human cancers. Loss of p53 function has been proposed to enable cells with gene amplification to arise spontaneously during growth in vitro. However, this conclusion derives from studies employing the UMP synthesis inhibitor N-phosphonacetyl-L-aspartate (PALA), which, in addition to selecting for cells containing extra copies of the CAD locus, enables p53-deficient cells to enter S phase and acquire the DNA breaks that initiate the amplification process. Thus, it has not been possible to determine if gene amplification occurs spontaneously or results from the inductive effects of the selective agent. The studies reported here assess whether p53 deficiency leads to spontaneous genetic instability by comparing cell cycle responses and amplification frequencies of the human fibrosarcoma cell line HT1080 when treated with PALA or with methotrexate, an antifolate that, under the conditions used, should not generate DNA breaks. p53-deficient HT1080 cells generated PALA-resistant variants containing amplified CAD genes at a frequency of >10(-5). By contrast, methotrexate selection did not result in resistant cells at a detectable frequency (<10(-9)). However, growth of HT1080 cells under conditions that induced DNA breakage prior to selection generated methotrexate-resistant clones containing amplified dihydrofolate reductase sequences at a high frequency. These data demonstrate that, under standard growth conditions, p53 loss is not sufficient to enable cells to produce the DNA breaks that initiate amplification. We propose that p53-deficient cells must proceed through S phase under conditions that induce DNA breakage for genetic instability to occur.

TP53 serves as a key relay for signals elicited by cellular stresses arising from diverse environmental or therapeutic insults. This relay then activates a cell cycle arrest or cell death program, depending on the stimulus and cell type. The absence of TP53 function disables the cell death or arrest programmes, thereby allowing the emergence of variants with various types of genomic alterations. The data discussed focus on two different types of signals that trigger the TP53 relay system. Firstly, TP53 arrests cell cycle progression in response to the types of DNA damage most commonly detected in cells undergoing tumour progression. Secondly, TP53 is activated by specific depletion of ribonucleotide pools, which prevent cells from entering S phase under conditions that could lead to chromosome breakage. The contribution of both responses limits the emergence of genetic variants. The DNA damage induced arrest appears to be triggered by as few as one double strand break in normal human fibroblasts. Analysis of the arrest kinetics after ionizing radiation shows that TP53 activates a prolonged arrest response in cells with irreparable DNA damage and that high efficiency cell elimination is achieved by a process that can be activated over multiple cell cycles. These data indicate that the primary function of the TP53 arrest/apoptosis pathway in response to double strand break is to eliminate damaged cells from the proliferating population, not to allow additional time for lesion repair. However, it remains possible that repair of other types of damage may benefit from TP53 mediated arrest. Analyses in model genetic systems indicate that the absence of TP53 function allows, but does not ensure, a high intrinsic rate of genetic variation and that instability is increased substantially when cells proceed through S phase under inappropriate growth conditions. This implies that inactivation of TP53 function in combination with other genetic alterations, such as oncogene activation, could accelerate genomic instability and tumour progression.

Size changes in microsatellite sequences have been detected in many types of cancer, but the influence of this form of genetic instability on disease progression remains unclear. We determined the incidence of microsatellite instability in breast cancer by comparing PCR-amplified sequences from paraffin-embedded samples of normal and tumor tissue from affected individuals. This analysis showed that at least 30% of breast cancers exhibit microsatellite instability (MI). Of importance, MI correlated with indicators commonly associated with poor disease prognosis, including lymph node status, tumor size, and advanced tumor stage. Individuals with MI+ tumors also showed significantly reduced disease-free and overall survival. These data contrast with studies showing that MI correlates with improved prognosis in colon and gastric cancers. We propose that defects resulting in MI promote disease progression and result in a poor prognosis in breast cancer.

The stability of the mammalian genome depends on the proper function of G1 and G2 cell cycle control mechanisms. Two tumor suppressors, p53 and retinoblastoma (Rb), play key roles in progression from G1 into S-phase. We address the mechanisms by which these proteins mediate a G1 arrest in response to DNA damage and limiting metabolic conditions. Gamma-irradiation induced a prolonged, p53-dependent G1 arrest associated with a long-term increase in the levels of the cdk-inhibitor p21WAFl/Cipl (p21). Microinjection of linear plasmid DNA also caused a G1 arrest. The p53-dependent arrest induced by inhibitors of UMP biosynthesis was reversible and occurred in the absence of detectable DNA damage. Both arrest mechanisms contribute to limiting the formation and propagation of damaged genomes. Cells containing mutant p53 but wild-type Rb do not generate methotrexate (Mtx) resistant variants. However, pre-treatment with DNA damaging agents prior to drug selection resulted in resistant clones containing amplified dihydrofolate reductase (DHFR) genes, suggesting that DNA breakage is a rate limiting step for gene amplification. The Mtx-induced arrest did not occur in cells with non-functional Rb. Rb acts as a negative regulator of the E2F transcription factors, and Rb-deficient primary mouse embryo fibroblasts (MEFs) produced elevated levels of mRNA and protein for key E2F target genes. Failure to prevent entry into S-phase in Rb-/- MEFs exposed to DNA-damaging or nutrient limiting conditions caused apoptosis and correlated with p53 induction. Taken together, these findings indicate a link between p53 and Rb function and suggest that their coordination insures correct entry into S-phase, minimizing the emergence of genetic variants.