Distinct Classes of Complex Structural Variation Uncovered across Thousands of Cancer Genome Graphs.

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.

Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.

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 cancer1-6. 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.

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.

Genetic clonal diversity predicts progression to esophageal adenocarcinoma.

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.

Somatic whole genome dynamics of precancer in Barrett's esophagus reveals features associated with disease progression.

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.

Within-patient phylogenetic reconstruction reveals early events in Barrett's Esophagus.

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.

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.

Cell proliferation, cell cycle abnormalities, and cancer outcome in patients with Barrett's esophagus: a long-term prospective study.

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.

Correction to: NSAID use and somatic exomic mutations in Barrett's esophagus.

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

Evolution of Barrett's esophagus through space and time at single-crypt and whole-biopsy levels.

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.

NSAID use and somatic exomic mutations in Barrett's esophagus.

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.

NSAIDs modulate CDKN2A, TP53, and DNA content risk for progression to esophageal adenocarcinoma.

BACKGROUND: Somatic genetic CDKN2A, TP53, and DNA content abnormalities are common in many human cancers and their precursors, including esophageal adenocarcinoma (EA) and Barrett's esophagus (BE), conditions for which aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as possible chemopreventive agents; however, little is known about the ability of a biomarker panel to predict progression to cancer nor how NSAID use may modulate progression. We aimed to evaluate somatic genetic abnormalities with NSAIDs as predictors of EA in a prospective cohort study of patients with BE. METHODS AND FINDINGS: Esophageal biopsies from 243 patients with BE were evaluated at baseline for TP53 and CDKN2A (p16) alterations, tetraploidy, and aneuploidy using sequencing; loss of heterozygosity (LOH); methylation-specific PCR; and flow cytometry. At 10 y, all abnormalities, except CDKN2A mutation and methylation, contributed to EA risk significantly by univariate analysis, ranging from 17p LOH (relative risk [RR] = 10.6; 95% confidence interval [CI] 5.2-21.3, p < 0.001) to 9p LOH (RR = 2.6; 95% CI 1.1-6.0, p = 0.03). A panel of abnormalities including 17p LOH, DNA content tetraploidy and aneuploidy, and 9p LOH was the best predictor of EA (RR = 38.7; 95% CI 10.8-138.5, p < 0.001). Patients with no baseline abnormality had a 12% 10-y cumulative EA incidence, whereas patients with 17p LOH, DNA content abnormalities, and 9p LOH had at least a 79.1% 10-y EA incidence. In patients with zero, one, two, or three baseline panel abnormalities, there was a significant trend toward EA risk reduction among NSAID users compared to nonusers (p = 0.01). The strongest protective effect was seen in participants with multiple genetic abnormalities, with NSAID nonusers having an observed 10-y EA risk of 79%, compared to 30% for NSAID users (p < 0.001). CONCLUSIONS: A combination of 17p LOH, 9p LOH, and DNA content abnormalities provided better EA risk prediction than any single TP53, CDKN2A, or DNA content lesion alone. NSAIDs are associated with reduced EA risk, especially in patients with multiple high-risk molecular abnormalities.

Chromosomal instability in Barrett's esophagus is related to telomere shortening.

Barrett's esophagus is a useful model for the study of carcinogenesis, as the metaplastic columnar epithelium that replaces squamous esophageal epithelium is at elevated risk for development of adenocarcinoma. We examined telomere length and chromosomal instability (CIN) in Barrett's esophagus biopsies using fluorescence in situ hybridization. To study CIN, we selected centromere and locus-specific arm probes to chromosomes 17/17p (p53), 11/11q (cyclin D1), and 9/9p (p16 INK4A), loci reported to be involved in early stages of Barrett's esophagus neoplasia. Telomere shortening was observed in Barrett's esophagus epithelium at all histologic grades, whereas CIN was highest in biopsies with dysplastic changes; there was, however, considerable heterogeneity between patients in each variable. Alterations on chromosome 17 were strongly correlated with telomere length (r = 0.55; P < 0.0001) and loss of the 17p arm signal was the most common event. CIN on chromosome 11 was also associated with telomere shortening (r =0.3; P = 0.05), although 11q arm gains were most common. On chromosome 9p, arm losses were the most common finding, but chromosome 9 CIN was not strongly correlated with telomere length. We conclude that CIN is related to telomere shortening in Barrett's esophagus but varies by chromosome. Whether instability is manifested as loss or gain seems to be influenced by the chromosomal loci involved. Because telomere shortening and CIN are early events in Barrett's esophagus neoplastic progression and are highly variable among patients, it will be important to determine whether they identify a subset of patients that is at risk for more rapid neoplastic evolution.

Mutagen sensitivity and neoplastic progression in patients with Barrett's esophagus: a prospective analysis.

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.

Genetic mechanisms of TP53 loss of heterozygosity in Barrett's esophagus: implications for biomarker validation.

BACKGROUND AND AIMS: 17p (TP53) loss of heterozygosity (LOH) has been reported to be predictive of progression from Barrett's esophagus to esophageal adenocarcinoma, but the mechanism by which TP53 LOH develops is unknown. It could be (a) DNA deletion, (b) LOH without copy number change, or (c) tetraploidy followed by genetic loss. If an alternative biomarker assay, such as fluorescence in situ hybridization (FISH), provided equivalent results, then translation to the clinic might be accelerated, because LOH genotyping is presently limited to research centers. METHODS: We evaluated mechanisms of TP53 LOH to determine if FISH and TP53 LOH provided equivalent results on the same flow-sorted samples (n = 43) representing established stages of clonal progression (diploid, diploid with TP53 LOH, aneuploid) in 19 esophagectomy specimens. RESULTS: LOH developed by all three mechanisms: 32% had DNA deletions, 32% had no copy number change, and 37% had FISH patterns consistent with a tetraploid intermediate followed by genetic loss. Thus, FISH and LOH are not equivalent (P < 0.000001). CONCLUSIONS: LOH develops by multiple chromosome mechanisms in Barrett's esophagus, all of which can be detected by genotyping. FISH cannot detect LOH without copy number change, and dual-probe FISH is required to detect the complex genetic changes associated with a tetraploid intermediate. Alternative biomarker assay development should be guided by appreciation and evaluation of the biological mechanisms generating the biomarker abnormality to detect potential sources of discordance. FISH will require validation in adequately powered longitudinal studies before implementation as a clinical diagnostic for esophageal adenocarcinoma risk prediction.

Crypt dysplasia with surface maturation: a clinical, pathologic, and molecular study of a Barrett's esophagus cohort.

Little is known regarding the significance of esophageal biopsies that show dysplasia-like atypia limited to the bases of the crypts, without involvement of the surface epithelium in Barrett's esophagus (BE). The aim of this study was to evaluate the clinical, pathologic, immunohistochemical, and molecular characteristics of basal crypt dysplasia-like atypia (BCDA) with surface maturation in surveillance endoscopic mucosal biopsies to gain insight into its biologic significance. The Seattle Barrett's Esophagus Project is a prospective cohort study in which patients and their biopsies have been evaluated prospectively for clinical, pathologic, and molecular markers. As part of continued surveillance of the cohort, 206 consecutive BE patients were evaluated prospectively for BCDA between July 1, 2001 and August 13, 2003; 15 patients had BCDA (prevalence rate = 7.3%). These 15 patients were evaluated for clinical, pathologic, and immunohistochemical (p53 and MIB-1) features during the study period (2001-2003) as well as associations with clinical, pathologic, and molecular markers [17p(TP53) loss of heterozygosity (LOH), 9p(p16) LOH, tetraploidy, and aneuploidy] that were detected previously in the same patients in the cohort study (1983-2001). All BE patients with BCDA (male-to female ratio, 12:3; mean age, 72 years; mean length of BE, 7.0 cm; mean duration of BE, 95.1 months), except 2 (87%), had dysplasia or adenocarcinoma detected in biopsies either prior to or concurrent to the one that contained BCDA. In contrast, only 112 of 191 (59%) controls had neoplasia during the same time period (59%, P = 0.05). The difference between BCDA and controls was particularly significant with regard to the association with high-grade dysplasia (P = 0.004). Compared with adjacent nonatypical and nondysplastic (metaplastic) BE, areas of BCDA showed a significantly elevated prevalence rate of p53 positivity (60% vs. 13%, P<0.02) and a significantly elevated total crypt and basal crypt MIB-1 proliferation rate (P<0.001). Indeed, the MIB-1 proliferation rate in the basal portion of the crypts in BCDA was similar to that detected in conventional low- or high-grade dysplasia. Patients with BCDA showed a significantly increased rate of 17p(TP53) LOH (P = 0.016), aneuploidy (P = 0.004), and a trend in increased 9p(p16) LOH (P = 0.08), compared with control patients without BCDA. The clinical, pathologic, immunohistochemical, and molecular abnormalities were similar in BCDA cases that were considered low-grade versus those considered high-grade by histologic evaluation, except that high-grade cases tended to be older (79 years vs. 68 years, P = 0.06). BCDA with surface maturation, in mucosal biopsies from patients with BE, is an uncommon but significant pathologic change that shows a variety of proliferative and molecular abnormalities and has a high association with conventional dysplasia and/or adenocarcinoma. Based on these findings, BCDA warrants further investigation as a possible subtype of true dysplasia despite the morphologic appearance of surface maturation.

Mucin core polypeptide expression in the progression of neoplasia in Barrett's esophagus.

We have previously demonstrated a specific pattern of mucin (MUC) core polypeptide expression in Barrett's esophagus (BE) characterized by MUC1 and MUC6 positivity in goblet cells in a proportion of cases. The aim of this study was to determine the pattern of MUC expression associated with the development and progression of dysplasia in BE. Endoscopic mucosal biopsies from 35 patients with BE (10 with no dysplasia, 6 with indefinite for dysplasia, 12 with low-grade dysplasia [LGD], and 7 biopsies with high-grade dysplasia [HGD]) were immunostained (ABC method) with antibodies against MUC1, MUC2, MUC3, MUC5AC, and MUC6. The extent and pattern of staining for each marker was evaluated in intestinalized Barrett's epithelium and in the various grades of dysplasia. For cases with dysplasia, staining was evaluated separately in nondysplastic epithelium adjacent to (<1 cm) and distant from (>2 cm) areas of dysplasia. In nondysplastic BE, MUC1, MUC2, MUC3, MUC5AC, and MUC6 stained 40%, 100%, 100%, 100%, and 90% of cases, respectively, in goblet or nongoblet columnar epithelium. With the progression of dysplasia (from metaplasia to indefinite, LGD and HGD), there was a significant decrease in expression of MUC1, MUC2, and MUC3, and alterations in the staining patterns of MUC5 and MUC6. In fact, MUC1 and MUC3 were entirely absent from all cases of HGD. Interestingly, BE-associated adenocarcinomas showed an MUC phenotype distinct from that of HGD, with expression of MUC1 and MUC3 in 47% and 67% of cases, and expression of MUC1 in a membranous pattern. There was no significant difference in MUC staining in nondysplastic BE between patients with and without dysplasia. Alterations in MUC expression occur in the progression of dysplasia in BE. However, none of these markers helps identify a subgroup of patients at increased risk for neoplasia.

Selectively advantageous mutations and hitchhikers in neoplasms: p16 lesions are selected in Barrett's esophagus.

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.

The combination of genetic instability and clonal expansion predicts progression to esophageal adenocarcinoma.

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.

Low-fat, high fruit and vegetable diets and weight loss do not affect biomarkers of cellular proliferation in Barrett esophagus.

Risk factors for esophageal adenocarcinoma include obesity, high fat intake, and low consumption of fruits and vegetables. This trial tested whether an intervention to reduce these risk factors in patients with Barrett esophagus, a preneoplastic condition for esophageal adenocarcinoma, could reduce biomarkers of cellular proliferation and, by inference, the risk of neoplastic progression. Eighty-seven men and women with Barrett esophagus were randomized to an intensive dietary intervention or control group. At baseline, 18 and 36 months after intervention, biopsies were obtained at 2-cm intervals throughout the length of the Barrett segment. Ki67/DNA content flow cytometry was used to assess (a) % Ki67-positive proliferating diploid G(1) cells, (b) % total Ki67-positive proliferating cells, (c) presence of aneuploidy, and (d) presence of >6% of cells in the 4N (G(2)/tetraploid) fraction of the cell cycle. We also assessed re-epithelialization and length of the Barrett segment, reflux symptoms, and medication use. The intervention effects for energy, fat, fruits and vegetables, and weight were, respectively, -314 kcal, -12.2% energy, 1.8 servings/d, and -4.0 kg at 18 months (all P < 0.005) and were smaller but remained significant at 36 months. There were no significant effects of the intervention on any biomarker of cellular proliferation. The intervention effects +/- SE for mean %G(1) Ki67+ cells were 0.98 +/- 1.58 at 18 months and 1.79 +/- 1.31 at 36 months; the relative risks (95% confidence interval) for developing >6% of cells in 4N were 0.5 (0.1-2.6) at 18 months and 0.75 (0.2-3.1) at 36 months. A single control participant developed aneuploidy. There were no significant effects on re-epithelialization, segment length, or reflux medication use. We conclude that substantial dietary change has no short-term effects on biomarkers of cellular proliferation in Barrett esophagus or on clinical observations of the Barrrett segment.