Multistage carcinogenesis in Barrett's esophagus

The multistage carcinogenesis of esophageal adenocarcinoma is a process of clonal evolution within Barrett's esophagus neoplasms. The initiating event for Barrett's esophagus is unknown, but is associated with chronic gastric reflux which probably also promotes progression. Inactivation of both alleles of CDKN2A appear to be early events causing clonal expansion. Clones with TP53 inactivated expand if they have already inactivated CDKN2A. After TP53 has been inactivated, tetraploid and aneuploid clones tend to develop. The final events that lead to invasion and metastasis are unknown. Evolutionary biology provides important tools to understand clonal evolution in progression and cancer prevention.     

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.     

Dedifferentiation precedes invasion in the progression from Barrett's metaplasia to esophageal adenocarcinoma.

PURPOSE: Adenocarcinoma arises in Barrett's esophagus by progression from metaplasia to cancer through grades of dysplasia. Our aim in this exploratory study was to characterize the broad changes in gene expression that underlie this histologic progression to cancer and assess the potential for using these gene expression changes as a marker predictive of malignant progression in Barrett's epithelium. EXPERIMENTAL DESIGN: Microarray analysis was used to obtain individual gene expression profiles from endoscopic biopsies of nine esophageal adenocarcinomas and the Barrett's epithelia from which three of the cancers had arisen. Pooled samples from the Barrett's epithelia of six patients without cancer or dysplasia served as a reference. RESULTS: Barrett's epithelia from which cancer had arisen differed from the reference Barrett's epithelia primarily by underexpression of genes, many of which function in governing cell differentiation. These changes in gene expression were found even in those specimens of Barrett's epithelia from which cancer had arisen that lacked dysplasia. Each cancer differed from the Barrett's epithelium from which it had arisen primarily by an overexpression of genes, many of which were associated with tissue remodeling and invasiveness. Cancers without identifiable Barrett's epithelium differed from cancers that had arisen from a Barrett's epithelium by having an even greater number of these overexpressed genes. CONCLUSIONS: Histologic progression from Barrett's epithelium to cancer is associated with a gradient of increasing changes in gene expression characterized by an early loss of gene function governing differentiation that begins before histologic change; gain in function of genes related to remodeling and invasiveness follows later. This correlation of histologic progression with increasing changes in gene expression suggests that gene expression changes in biopsies taken from Barrett's epithelium potentially could serve as a marker for neoplastic progression that could be used to predict risk for developing cancer.     

Hypermethylation of tachykinin-1 is a potential biomarker in human esophageal cancer.

PURPOSE: Our aim was to investigate whether and at what stage hypermethylation of the tachykinin-1 (TAC1) gene is associated with human esophageal neoplastic transformation. EXPERIMENTAL DESIGN: TAC1 promoter hypermethylation was examined by real-time methylation-specific PCR in 258 human esophageal specimens and 126 plasma samples from patients or tissues at various stages of neoplastic evolution. RESULTS: TAC1 hypermethylation in tissue samples showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) from normal esophagus (P < 0.0001). Both frequencies and normalized methylation values of TAC1 tissue methylation were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's esophagus, EAC, and ESCC than in normal esophagus (P < 0.01). The frequency of TAC1 hypermethylation increased dramatically and early during neoplastic progression, from 7.5% in normal esophagus to 55.6% in BE from patients with Barrett's metaplasia alone, 57.5% in dysplastic Barrett's esophagus, and 61.2% in EAC. There was a significant relationship between TAC1 hypermethylation and BE segment length, a known clinical risk factor for neoplastic progression. Twelve (50%) of 24 ESCC exhibited TAC1 hypermethylation. Overall patient survival correlated significantly with TAC1 methylation status in ESCC patients (mean survival, 22 versus 110 months; P = 0.0102, log-rank test), but not in EAC patients. Both mean normalized methylation values and frequency of TAC1 hypermethylation in plasma samples were significantly higher in EAC patients than in control subjects. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2'-deoxycytidine reduced TAC1 methylation and increased TAC1 mRNA expression. CONCLUSIONS: TAC1 promoter hypermethylation is a common event in both major histologic types of human esophageal carcinoma, occurs early, correlates with other progression risk factors in esophageal adenocarcinogenesis, and is a tissue biomarker of a poor prognosis in ESCC. Circulating methylated TAC1 promoter DNA also offers potential as a biomarker for the diagnosis of EAC.     

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.     

Clinical use of p53 in Barrett's esophagus.

Barrett's esophagus is an established precursor to esophageal adenocarcinoma. Whereas most patients with Barrett's esophagus do not progress to adenocarcinoma, patients with progression have a poor prognosis. Current management strategies use frequent endoscopic surveillance and multiple nontargeted biopsies. This approach, however, may miss dysplastic areas. Furthermore, given the relatively high prevalence of Barrett's esophagus but low incidence of progression, this invasive and expensive approach has not been shown to be cost-effective. Thus, there is intense interest in using biomarkers to identify patients at increased risk of progressing to adenocarcinoma. This has included examination of mutations in the tumor suppressor gene, p53. In this report, we discuss the biology of p53 and the incidence of p53 mutations in Barrett's esophagus and review relevant studies regarding the ability of p53 to predict neoplastic progression. Additionally, we report our results of the expression of p53 by immunohistochemistry in a group of 18 patients that have undergone endoscopic esophageal mucosal resection for dysplasia. Although the presence of a p53 mutation increases the risk of neoplastic progression, the absence of this mutation does not abrogate the risk. Continuing efforts, therefore, are needed to define and prospectively validate a panel of biomarkers to risk-stratify patients with Barrett's esophagus. Determination of p53 mutational status may ultimately be a component of such a panel.     

Hypermethylation of the AKAP12 promoter is a biomarker of Barrett's-associated esophageal neoplastic progression

The A-kinase anchoring protein 12 (AKAP12) is a kinase scaffold protein with known tumor suppressor activity. Recently, AKAP12 promoter hypermethylation was reported in gastric and colorectal cancers. We examined AKAP12 promoter hypermethylation using real-time methylation-specific PCR in 259 human esophageal tissues. AKAP12 hypermethylation showed highly discriminative receiver-operator characteristic (ROC) curve profiles, clearly distinguishing esophageal adenocarcinoma (EAC) from esophageal squamous cell carcinoma and normal esophagus (P < 0.0001). AKAP12-normalized methylation values were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's, and EAC than in normal esophagus (P < 0.0000001). AKAP12 hypermethylation frequency was zero in normal esophagus but increased early during neoplastic progression, to 38.9% in BE from patients with Barrett's alone, 52.5% in dysplastic Barrett's metaplasia, and 52.2% in EAC. AKAP12 hypermethylation levels were significantly higher in normal esophageal epithelia from patients with EAC (mean = 0.00082) than in normal esophagi from patients without Barrett's or esophageal cancer (mean = 0.00007; P = 0.006). There was a significant correlation between AKAP12 hypermethylation and BE segment length, a known clinical neoplastic progression risk factor. In contrast, only 2 (7.7%) of 26 esophageal squamous cell carcinomas exhibited AKAP12 hypermethylation. Treatment of BIC and OE33 EAC cells with 5-aza-2'-deoxycytidine reduced AKAP12 methylation and increased AKAP12 mRNA expression. AKAP12 mRNA levels in EACs with unmethylated AKAP12 (mean = 0.1663) were higher than in EACs with methylated AKAP12 (mean = 0.0668). We conclude that promoter hypermethylation of AKAP12 is a common, tissue-specific event in human EAC, occurs early during Barrett's-associated esophageal neoplastic progression, and is a potential biomarker for the early detection of EAC.     

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.     

Barrett's esophageal cancer

Barrett's esophageal cancer is defined as carcinoma developing in Barrett's esophagus. The esophagogastric junction is located at the distal end of a network of fine longitudinal vessels, and the columnar epithelium existing above it is Barrett's mucosa. Barrett's mucosa, especially specialized columnar epithelium is considered as precancerous lesion, and malignant potential is examined in various ways. For the surveillance of malignant lesions from Barrett's esophagus, periodic endoscopic examination is necessary with chromoendoscopy or magnifying endoscopy. Treatment strategies are EMR and other endoscopic treatment for mucosal cancer, and surgical treatment for submucosal and advanced cancer. Several surgical modalities are employed depending on the stage of cancerous progression, the location of the cancer in Barrett's esophagus, and the length of Barrett's esophagus. There remain many unexplained problems in Barrett's esophagus and Barrett's cancer.     

The molecular biology of esophageal adenocarcinoma

BACKGROUND: Barrett's esophagus is an acquired metaplastic change that occurs in the distal esophagus secondary to chronic gastroesophageal reflux. This premalignant condition forms the most important risk factor for developing esophageal adenocarcinoma, which is an extremely aggressive tumor with a 5-year survival rate of less than 25%. Carcinomas that arise in the setting of Barrett's esophagus are thought to develop as part of the metaplasia-dysplasia-carcinoma sequence. OBJECTIVE: To review the current knowledge on the genomic alterations involved in the development of Barrett's esophagus and its progression to dysplasia and/or cancer. RESULTS: Several changes in gene structure, gene expression, and protein structure are associated with the progression of Barrett's esophagus to adenocarcinoma. Accumulation of these changes seems to be essential, rather than the exact sequence of these changes. Multiple molecular pathways are involved and interact with each other. Alterations in tumor suppressor genes, amongst which p53 and p16, are early events in the metaplasia-dysplasia-adenocarcinoma sequence, followed by loss of cell cycle checkpoints. Ongoing genomic instability leads to cumulative genetic errors and thereby the generation of multiple clones of transformed cells. CONCLUSIONS: Within the multistep process of esophageal adenocarcinogenesis, to date no single molecular marker came forward able to predict who will and who will not develop cancer in the setting of Barrett's esophagus. Instead, panels of markers need to be developed in the future allowing to indicate disease progression. Identification of crucial molecular pathways involved in esophageal adenocarcinogenesis would ultimately improve therapy and facilitate development of new treatment strategies.     

Progression of Barrett's metaplasia to adenocarcinoma is associated with the suppression of the transcriptional programs of epidermal differentiation

We did expressional profiling on 24 paired samples of normal esophageal epithelium, Barrett's metaplasia, and esophageal adenocarcinomas. Matching tissue samples representing the three different histologic types were obtained from each patient undergoing esophagectomy for adenocarcinoma. Our analysis compared the molecular changes accompanying the transformation of normal squamous epithelium with Barrett's esophagus and adenocarcinoma in individual patients rather than in a random cohort. We tested the hypothesis that expressional profiling may reveal gene sets that can be used as molecular markers of progression from normal esophageal epithelium to Barrett's esophagus and adenocarcinoma. Expressional profiling was done using U133A GeneChip (Affymetrix), which represent approximately two thirds of the human genome. The final selection of 214 genes permitted the discrimination of differential gene expression of normal esophageal squamous epithelium, Barrett's esophagus, and adenocarcinoma using two-dimensional hierarchical clustering of selected genes. These data indicate that transformation of Barrett's esophagus to adenocarcinoma is associated with suppression of the genes involved in epidermal differentiation, including genes in 1q21 loci and corresponding to the epidermal differentiation complex. Correlation analysis of genes concordantly expressed in Barrett's esophagus and adenocarcinoma revealed 21 genes that represent potential genetic markers of disease progression and pharmacologic targets for treatment intervention. PCR analysis of genes selected based on DNA array experiments revealed that estimation of the ratios of GATA6 to SPRR3 allows discrimination among normal esophageal epithelium, Barrett's dysplasia, and adenocarcinoma.     

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.     

p16(INK4a) lesions are common, early abnormalities that undergo clonal expansion in Barrett's metaplastic epithelium.

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.     

Proton pump inhibitors reduce cell cycle abnormalities in Barrett's esophagus.

Neoplastic progression in Barrett's esophagus is a multi-step process in which the metaplastic columnar epithelium sequentially evolves through a metaplasia-dysplasia-carcinoma sequence. The expression and DNA copy number of key cell cycle regulatory genes in paired normal and Barrett's esophagus samples was evaluated. Protein levels were evaluated in 60 formalin-fixed, paraffin-embedded human tissues by immunohistochemistry. DNA copy number from 20 fresh tissue pairs was analysed by Southern blot analysis. All normal mucosal samples expressed the p27(kip1) protein, but did not display appreciable nuclear staining for p16(kip4), p21(cip1) or cyclins D1 and E. Barrett's metaplastic specimens displayed increased expression levels of p16(kip4) (74%), p21(cip1) (89%) and cyclins D1 (43%) and E (37%). p27 protein was absent in three cases. There was a significant correlation between the expression of p16(kip4) and cyclin E, and p21(cip1) and p27(kip4) with cyclin D1. DNA analysis did not reveal any amplification or deletion of these genes. Acid suppression, however, was associated with significantly lower expression levels of key cell cycle proteins. Increased expression of key cell cycle regulatory genes appears to occur early in the neoplastic progression associated with Barrett's esophagus. Treatment with proton pump inhibitors appears to alter this increased expression.     

Endoscopic therapy of esophageal premalignancy and early malignancy

Esophageal adenocarcinoma (EAC) is an often deadly cancer with a rising incidence in Western countries. Chronic gastroesophageal reflux disease is associated with the metaplastic transformation of normal squamous epithelium to premalignant specialized intestinal metaplasia within the esophagus (Barrett's esophagus). Barrett's esophagus may progress to low-grade dysplasia (LGD), high-grade dysplasia (HGD), or even EAC. Although nondysplastic Barrett's esophagus progresses to EAC at a rate of 0.5% per year, rates of progression for true LGD and HGD are significantly higher. Treatment is mandatory for HGD and may be appropriate in select patients with nondysplastic Barrett's esophagus and many with LGD. Thus, accurate pathologic assessment is necessary before considering endoscopic therapy. Previously, only esophagectomy was offered to patients with HGD or EAC. However, esophagectomy has significant morbidity and mortality, and therefore endoscopic therapies have been advocated for early Barrett's neoplasia. These methods include endoscopic mucosal resection (EMR) and ablative techniques. Ablation techniques include argon plasma coagulation, multipolar electrocoagulation, laser therapy, photodynamic therapy, radiofrequency ablation, and cryotherapy. Of these, radiofrequency ablation has experienced the greatest adoption for the treatment of dysplastic Barrett's esophagus because of excellent published outcomes. The use of EMR to resect suspicious areas or raised lesions is mandatory to provide histology. In contrast, ablation techniques such as radiofrequency ablation have been shown to effectively eradicate large areas of dysplastic tissue with relative ease but do not allow for histologic assessment of the treated area. Combination EMR with radiofrequency ablation is thus advocated to resect visible lesions via EMR (providing histology) and ablate the remainder of the Barrett's esophagus. As always, the appropriate treatment is best determined after careful discussion with patients in a multidisciplinary environment. However, endoscopic therapy offers an attractive alternative to esophagectomy for early Barrett's neoplasia.     

p53-mutant clones and field effects in Barrett's esophagus.

Previous studies have demonstrated multifocal neoplasia in Barrett's esophagus. We evaluated 213 mapped, flow-purified, endoscopic biopsies to determine the distribution of p53-mutant clones in the Barrett's segments of 58 patients who had high-grade dysplasia without cancer. Twenty-nine patients (50%) had p53 mutations in their Barrett's segments, including 3 patients with multiple distinct p53 mutations. p53-mutant clones, including diploid cell populations, underwent expansion from 1 to 9 cm in the Barrett's segment. In 12 of 29 patients (41%) with a p53 mutation, the same mutation was found at every evaluated level of the metaplastic epithelium. This extensive p53-mutant clonal expansion suggests a somatic genetic basis for previous observations of field effects in Barrett's esophagus.