Section 3 -

Molecular Basis of Barrett's Esophagus Associated Dysplasia and Adenocarcinoma Robert D. Odze, M.D. Jonathan Glickman, M.D., Ph.D. Mark Redston, M.D.

The molecular characteristics of neoplastic progression in Barrett's esophagus have been extensively studied (reviewed in 1, 2, 3, 4, 5, 6, 7). A summary of the findings is presented in Table 1. Genetic Alterations in Non-dysplastic Barrett's Esophagus It has become clear in recent years that non-dysplastic columnar-lined esophageal mucosa may harbor genetic alterations. Acquired genetic alterations lead to clonal expansion, and these abnormal cells may occupy large regions of esophageal mucosa. In addition to abnormal DNA content [8] and p53 mutations [9], a number of chromosomal deletions (loss of heterozygosity) have also been found [10]. Several studies have mapped out the distribution of clonal populations, and their expansion and progression to dysplasia and carcinoma [11]. While it seems intuitive that genetic aberrations could be useful as biomarkers of risk of progression, careful prospective studies are required to validate the clinical utility of these alterations [12, 13, 14]. Factors that May Predict the Risk of Neoplastic Progression in Barrett's Esophagus 1. Aneuploidy Aneuploidy is a characteristic finding in adenocarcinoma. It is present not only in cancer, but may also be detected in dysplasia, and in non-dysplastic Barrett's mucosa. Aneuploidy is a risk factor for progression of low grade dyslpasia to cancer. Several studies have also found that aneuploidy in non-dysplastic Barrett's mucosa increases the risk of neoplastic progression. In one study, 6/13 patients with aneuploid non-dysplastic Barrett's mucosa progressed to high grade dysplasia or cancer during a 13 year follow-up [15]. In contrast, only 1/21 diploid patients progressed, and this patient developed a non-dysplastic aneuploid population prior to the identification of adenocarcinoma. The predictive role of aneuploidy has been most extensively studied in the Seattle Barrett's Esophagus Project, where aneuploidy and increased 4N fractions were both associated with a high risk of progression to cancer in a 5 year follow-up period, even in patients without high grade dysplasia [16]. In these studies, dysplasia, aneuploidy, or increased 4N populations were present at baseline in 38/42 patients who progressed to cancer, and in 34/34 who progressed to cancer within 5 years [17]. Recent refinements have found that a 4N fraction cut point of 6% and aneuploid DNA content >2.7N were optimal for discriminating cancer risk (relative risk = 11.7, 95% CI = 6.2-22, and relative risk 9.5, 95% CI = 4.9-18, respectively) [17]. The 4N fraction cut point of 6% is also associated with the presence of histologic dysplasia [18], p53 LOH [19], and the subsequent development of aneuploidy [19]. The presence of a 4N fraction of 6% and an aneuploid DNA population with content >2.7N in a single individual was highly predictive of cancer (relative risk = 23, 95% CI = 10-50) [17]. In contrast, there was no difference in the risk of progression for negative, indefinite, and low grade dysplastic biopsies in the absence of these cytometric features [16, 17]. Other recent investigations have also found aneuploidy as determined by image analysis to be predictive of progression of dysplasia grade [20]. Although the predictive role of flow cytometric analyses is well supported in the literature, widespread application remains somewhat limited due to methodologic requirements. Similarly, care must be taken when performing these analyses. Artifactual 4N elevations may be obtained from aggregation of cells or nuclei [8, 16, 18]. 2. 17p Deletion/p53 Mutation and Inactivation The p53 gene prevents cells with DNA breaks from entering DNA synthesis, where the breaks could lead to chromosomal damage and genetic instability ( reviewed in [21] ). In human neoplasia, p53 behaves as a tumor suppressor gene, and this normal cellular function is lost by gene inactivation. In esophageal adenocarcinoma, as in many other human cancers, p53 is inactivated by a two-hit mechanism; one copy is inactivated by mutation, and the other copy is deleted (usually referred to as loss of heterozygosity). 17p LOH is the most frequent genetic alteration in esophageal adenocarcinoma, present in 85-100% of tumors [22, 23, 24, 25, 26, 27]. As further support that p53 is the major target of 17p LOH, in some studies p53 mutations are found in about 90% of esophageal adenocarcinomas [25, 28]. In addition to adenocarcinoma, 17p LOH has been found in non-dysplastic Barrett's mucosa surrounding cancers [25, 29] and in Barrett's without cancer [30, 31]. p53 inactivation occurs early in neoplastic progression, being present in normal diploid cells [10, 11, 29, 32]. In one of the largest studies to date, 17p LOH at baseline was found in 6% of negative biopsies, 6% of indefinite biopsies, 20% of those with low grade dysplasia, and 57% with high grade dysplasia [33]. In a 5 year follow-up period, 20/54 patients with 17p LOH progressed to adenocarcinoma, compared to only 6/202 without LOH (relative risk 16, 95% CI = 6.2-39). Of the 6 that progressed to cancer without 17p LOH at baseline, 3 had 17p LOH detected after baseline but prior to carcinoma. Even amongst patients with histologic high grade dysplasia, 17p LOH was associated with an increased risk of progression to carcinoma. In patients whose initial biopsies were negative, indefinite, or showed only low grade dysplasia, 5/19 with 17p LOH progressed to high grade dysplasia or cancer compared to only 16/178 without LOH (relative risk = 3.6, 95% CI = 1.3-10) [33]. Finally, in patients without abnormalities in DNA content at baseline, 17p LOH was associated with progression to increased 4N content and aneuploidy (relative risk = 6.1, 95% CI = 3.0-12, and relative risk = 7.5, 95% CI = 3.5-16, respectively) [33]. In other studies, p53 abnormalities detected by p53 immunohistochemistry were associated with an increased risk of progression of dysplasia grade [20, 34, 35]. These findings suggest that p53 may have an important role in generating genomic instability. p53 immunohistochemistry is only about 70% sensitive for p53 mutations in esophageal adenocarcinoma. False positives may also occur [36, 37, 38]. Methods have also been developed to identify rare p53 mutations in mixed cell samples of esophageal biopsies [39]. Unfortunately, analysis of p53 remains challenging in small biopsy specimens. Although methods have been developed to successfully analyze flow sorted cell populations [10, 40], these have not been implemented by many laboratories, even in a research setting. 3. p16 Loss Allelic loss of 9p21, the site of the p16 gene, is found in up to 75% of non-dysplastic Barrett's mucosa [41, 42]. Loss precedes identification of aneuploidy, suggesting that it may provide a novel biomarker for progression [42]. Loss of p16 is also more prevalent than p53 loss. It is present throughout metaplastic mucosa [10, 30, 41], suggesting that it is one of the earliest molecular changes. Hypermethylation is a common mechanism of p16 silencing, and is found in nearly 50% of Barrett's cases [41, 43, 44, 45]. Furthermore, hypermethylation is associated with loss of p16 immunoreactivity [41]. Additional studies are required to test the utility of p16 as a marker for progression. 4. Cyclin D1 Nuclear accumulation of cyclin D1 is found in many non-dysplastic Barrett's cases [46]. In one study, patients with cyclin D1 overexpression were 6-7 times more likely to progress to adenocarcinoma compared to those without overexpression [36]. 5. Methylation of APC and Other Loci Aberrant CpG island methylation is a common occurrence in esophageal neoplasms. In addition, methylation of APC and other loci are found in large fields of non-dysplastic Barrett mucosa [47]. Additional studies are required to test the utility of these other methylation events as markers for progression. 6. Cell Proliferation Proliferative rate measured by S phase analysis has only modest power for predicting progression to cancer [17, 48]. A high proliferation rate is common to many non-dysplastic Barrett's cases. Therefore it does not strongly indicate which cases are likely to progress to cancer. 7. Other Several studies have examined the use of immunohistochemical markers to detect Barrett's metaplasia, including CK7, CK20, MUC2, other mucins, hepatocyte antigen (Hep), Fas, and CDX2 [49, 50, 51, 52, 53, 54], particularly in the setting of absent goblet cells, though these have not been well charaterized as predictive markers. Flow sorting has identified the presence of specific subsets of abnormal 4N p53 mutant populations that have characteristic gene expression profiles [55]. Expression of secreted protein acidic and rich in cysteine (SPARC) mRNA has been reported at higher levels in Barrett's associated with adenocarcinoma compared to those without dysplasia or adenocarcinoma [56], while expression of glutathione S-transferase-pi (GSTPI) mRNA, and other glutathiones, is reported to be down-regulated as an early event in high risk non-dysplastic mucosa [57, 58]. Differences in mucin subtype staining and minichromosome maintenance proteins are observed, but additional studies are required to relate this to risks of neoplastic progression [59, 60]. Expression of the leucine rich fibroblast growth factor receptor-1 (CFR-1/PAM-1) is also abnormally upregulated in non-dysplastic and dysplastic Barrett's esophagus, however this has not yet been assessed as a predictive marker [61]. Comparative genomic hybridization approaches have idetnified amplification of chromosomes 4 and 8q as possible markers of progression to high grade dysplasia and adenocarcinoma [62], further supported by the presence of these alterations at high levels in non-dysplastic Barrett's mucosa [63]. Genomic deletions of 3p21 and 5q21 have also been identified in non-dysplastic Barrett's mucosa adjacent to cancer [64]. Mitochondrial DNA mutations are present in 40% of cancers and 10% of Barrett's mucosa, providing another potential screening target, as well as further supporting the possible role of oxidative damage in etiology [65]. Raman spectra and tissue fluorescence have been found to predict histologic diagnoses, raising the possibility of in vivo approaches [66, 67, 68]. High serum selenium levels have been shown to be associated with lower grade histologic lesions and lower frequencies of many genetic abnormalities in Barrett's patients [69]. Abnormal distribution of body fat (male-pattern obesity) has also been associated with a higher prevalence of genetic alterations in Barrett's patients [70]. 8. Biomarkers and Therapeutic Intervention There has been significant interest in the role of oxidative damage in generating DNA damage. For instance, acidic pH has been associated with TOP-2 dependent DNA damage [71]. In one study, acid suppression therapy had no effect on decreasing DNA damage in patients whose Barrett's tissues had immunohistochemically stabilized p53 or aneuploidy as determined by image analysis [20]. In another study, possible adverse effects on DNA damage were detected in association with endoscopy [72]. The role of COX-2 inhibition is also of intense interest. In one study, the COX-2 inhibitor Rofecoxib was associated with decreased COX-2, PGE2, and PCNA expression in Barrett's biopsies, suggesting the possibility of a clinically effective approach to chemoprevention [73]. Summary Investigators in the Seattle Barrett's Esophagus Project have concluded that flow cytometry is a useful adjunctive technique to histology in evaluating patients with Barrett's esophagus [17]. In particular, their findings suggest that in the absence of high grade dysplasia, flow cytometry can stratify patients into a high and low risk groups, depending on the presence or absence of 4N fractions and aneuploid content >2.7N. Using these criteria, high risk groups have increased surveillance and follow-up, whereas low risk groups may have surveillance deferred for 5 years [17]. Finally, many other genetic alterations, such as 17p LOH and p53 mutation, occur independently, therefore it is likely that a panel of biomarkers may have the greatest clinical utility [33]. Further studies will be required to better define such a panel, and to evaluate the clinical effectiveness of patient stratification. Table 1. Molecular alterations in Barrett's esophagus Molecular Alteration Biologic Role Non-dysplastic Barrett's SRC overexpression tyrosine kinase in EGF signalling pathway 11q13 amplification/Cyclin D1 overexpression stimulates cell cycle progression Bcl2 overexpression inhibits apoptosis 13q14 deletion/Rb inactivation de-regulation of cell cycle progression and apoptosis 9p21 deletion/p16 inactivation CDK inhibitor; de-regulation of cell cycle progression 5q21 deletion/APC inactivation WNT signalling; cell adhesion 18q deletion/DCC (and other) gene loss some genes involved in TGFB signalling 17p13 deletion/p53 inactivation DNA damage response; de-regulation of cell cycle arrest and apoptosis Aneuploidy end result of chromosomal instability Dysplastia KRAS activating mutations signal transduction; growth stimulation p21 overexpression CDK inhibitor; may reflect underlying p53 abnormalities telomerase overexpression maintains chromosomes; important for cell immortality p27 loss or cytoplasmic localization CDK inhibitor; de-regulation of cell cycle Adenocarcinoma 17q21 amplification/HER2 overexpression transmembrane receptor; external growth signals 7p12-13 amplification/EGFR overexpression transmembrane receptor; external growth signals 2p13 amplification/TGF-α overexpression ligand for EGFR; stimulates cell division MDM2 overexpression negative regulation of p53 19q12 amplification/Cyclin E overexpression stimulates cell cycle progression E and P cadherin loss increased cell migration; metastasis Other chromosomal deletions (including 3p, 4p, 4q, 7q, 12q, 17q, 22q) and amplifications (including 2p, 8q, 20q) unknown tumor suppressor genes and oncogenes Table 2. Molecular alterations in squamous neoplasia of the esophagus Molecular Alteration Biologic Role Non-dysplastic squamous epithelium p53 immunoreactivity DNA damage response; de-regulation of cell cycle arrest and apoptosis Squamous Dysplasia 17p13 LOH/p53 mutation DNA damage response; de-regulation of cell cycle arrest and apoptosis Squamous Cell Carcinoma 11q13 amplification/Cyclin D1 overexpression stimulates cell cycle progression 7p12-13 amplification/EGFR overexpression transmembrane receptor; external growth signals 8q24.1 amplification/c-myc overexpression transcriptional activation 9p21 deletion/p16 inactivation CDK inhibitor; de-regulation of cell cycle progression 13q14 deletion/Rb inactivation de-regulation of cell cycle progression and apoptosis Other chromosomal deletions (including 1p, 3p, 5q, 11q, and 18q) unknown tumor suppressor genes and oncogenes References Beilstein, M. and D. 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