Is there p53 over-expression in Barrett's patients with or without dysplasia?

G. Tougas, C. Dienenfeld (Hamilton)

Barrett's esophagus, a consequence of chronic gastroesophageal acid reflux is characterized by the presence of intestinal metaplasia (IM) replacing the normal squamous epithelium of the esophagus. Its primary clinical interest resides in the fact that it is associated with a substantially higher risk of esophageal adenocarcinoma. This concern is not trivial, as the incidence of adenocarcinoma of the esophagus has grown almost exponentially in the last two decades. While present in only 16% of patients with esophageal cancer in the 70s, Barrett's esophagus was found to be present in nearly 34% in the mid 1980s [1] and as much as 50% of all cases of esophageal cancer in 1990 [2].

Given that reflux is common (between 6-10% of the adult population in most surveys) and that Barrett's esophagus is present in as many as 10% of all chronic gastroesophageal reflux disease (GERD) patients, the markedly increased odds of adenocarcinoma certainly justifies the high degree of interest in this entity. Still, even if the risk of developing an esophageal neoplasm has been estimated in some analyses to increase about 30- to 125-fold in patients with Barrett's esophagus [3, 4], it nevertheless remains relatively uncommon, with an incidence about 10-15X > than that of colon cancer.

The sequence of events between IM in the distal esophagus and the subsequent development of neoplasia involve a transformation of the IM into dysplasia. Difficult when applied to all patients with GERD, endoscopic surveillance with histologic sampling remains an important consideration, aiming to identify the patients with Barrett's esophagus in whom there is progression from metaplasia to either dysplasia or neoplasia. The hope is clearly to identify these changes early enough that resection is possible.

A number of biological markers have been proposed as useful markers in Barrett's patients. These include c-erbB-2 oncoprotein over-expression, DNA content, and p53 overexpression. The latter is particularly reviewed in the present manuscript.

The role of p53 in oncogenesis

The p53 tumor-suppressor gene (16 to 20 kb of DNA on the short arm of chromosome 17) encodes a 393-amino acid protein important in the regulation of the cell cycle, where it plays an important role in the G1 phase [5, 6]. Under normal circumstances, the wild-type p53 protein suppresses cell transformation whereas in certain situations including gastrointestinal malignancies, it can mutate into a protein capable of enhancing tumor progression. The mutation functionally converts the p53 tumor suppressor gene into a dominant oncogene. Such p53 gene mutation occurs in several human solid tumors including several gastrointestinal tumors [7]. Several different genetic alterations have been described, including missence base substitution, frameshift and chain-terminating (nonsense) mutation [8, 9]. The exact clinical relevance of these changes is to date not fully elucidated but there is increasing evidence that p53 expression or over-expression in the context of neoplastic growth may have important prognostic implications.

Under circumstances leading to DNA damage such as occurring with chronic mucosal inflammation associated with GERD, the p53 suppressor gene is activated and in turn activates a number of other genes whose product all have biological activity and contribute to the cellular response to DNA damage. One major p53 suppressor gene-mediated response is to arrest or delay the G1 phase of the cell cycle. This allows time for the cell to repair the DNA damage that has occurred prior to the start of the S phase of the cell cycle.

Cells lacking normal p53 suppressor gene activity, such as most neoplastic cells, suffer from genomic instability resulting from loss of checkpoint activity during the G1 phase, eventually culminating in gene amplification, aneuploidy and other chromosomal aberrations. These abnormalities then contribute to the clonal evolution of cancer cells and tumor progression [8, 9].

In chronic inflammation such as GERD, there is an intracellular overload of oxygen radicals. This leads to a number of changes and mutations of several cancer related genes including the p53 tumor suppressor gene [10].

This is in part related to nitric oxide (NO) metabolism. Under normal circumstances, Ca2+ dependent isoforms (NOS1 and NOS3) of the nitric oxide-synthase are expressed constitutively, producing NO in pico- to nanomolar concentrations. On the other hand, there is another isoform of the nitric oxide synthase that is Ca2+ independent, the NOS2 isoform, which is inducible and capable of producing NO concentrations in the micromolar range. The p53 suppressor gene normally is an important repressor of the expression of this inducible NOS2 isoform. In vivo, this repression will attenuate excessive NO production, through a negative feedback loop. Chronic inflammatory mucosal damage activates the inducible NOS3 isoform. This in turn leads to excessive NO production or release that probably contributes to neoplastic transformation by selecting against wild-type p53 suppressor gene and instead favors the emergence of the mutant p53 containing cells. This transformation then leads to the loss of the normally occurring negative feedback loop.

The predominant mutation in the p53 gene appears to be transition G:C>A:T. Such G:C>A:T transitions have been associated with increased activity of the NOS2 isoform of the NO synthase. Furthermore NO may also lead to tumor progression through its actions as an endothelial growth factor mediating increased tumor vascularization and blood flow.

NOS2 expression can be induced by factors such as hypoxia and by cytokines [10, 11]. Changes in the cellular micro-environment of premalignant and malignant tumor cells is felt to lead to sustained increases in NO production in a variety of tumor cells, thereby supporting clonal selection and tumor growth [10, 12, 13]. In this scenario however, tumor progression and mutation of the p53 tumor suppressor gene occurs after the initiating events and are not the initiator of carcinogenesis.

As tumors increase in size, hypoxia occurs. It has been found to select for mutant forms of the p53 suppressor gene in cells that are resistant to hypoxia-induced apoptosis [10, 14]. If hypoxia and/or cytokines induce NOS2 expression, only cell clones with nonfunctional p53 would tolerate the stress by sustained NO production and escape growth arrest and apoptosis while tumor growth is supported [10, 15].

p53 and esophageal cancer

Several studies have examined the role of the p53 suppressor gene in Barrett's esophagus and its implication in the metaplasia-carcinoma sequence (MCS). An older study initially reported that while p53 over-expression could be demonstrated in only 5% of patients IM, it was found in 15% of Barrett's patients with low-grade dysplasia (LGD), in 45% of patients with high-grade dysplasia (HGD) and in 55% of cases with adenocarcinoma [8, 16, 17]. This had led to the suggestion that p53 mutation is a late-phase phenomenon in tumor development and thereby constitutes a negative prognostic factor.

These observations were confirmed by other investigators, further supporting the concept that p53 over-expression occurs later in tumor development and progression [18, 19].

With the advent of improved methods, accurate and specific immuno-staining for p53 is now possible. This has confirmed that p53 mutations, such as seen in Barrett's patients with adenocarcinoma are highly correlated with positive immuno-staining [20, 21].

In a provocative study using immuno-staining, Younes et al. furthermore showed overexpression of p53 protein in esophageal mucosal samples from Barrett’s esophagus that occurred before the development of HGD or carcinoma in two out of three patients [20, 22]. This implies that this approach and p53 over-expression may be used to predict the development of neoplasia in Barrett’s patients. Subsequent studies using a specific antip53 monoclonal antibody (BP-53-12, Bio Genex) have even found p53 over-expression in nondysplastic cells adjacent to neoplastic or dysplastic cells. Importantly, p53 over-expression could be detected as early as 48 months prior to the development of HGD or adenocarcinoma. When 61 patients with Barrett’s esophagus were prospectively followed, p53 over-expression in those patients with LGD was associated with a high risk of subsequently developing HGD or neoplasia, whereas absence of p53 over-expression was associated with a favorable prognosis. When compared with the current gold standard, which is histology, p53 over-expression appears to be a sensitive marker of the malignant potential in Barrett’s.

As patients with LGD and p53-positive biopsies are more likely to develop either HGD and/or adenocarcinoma, they should be followed up more closely than those with p53 negative biopsies [20].

Gimenez et al. subsequently obtained similar results [23], and a significant correlation between the percentage of samples with p53 expression and the severity of dysplasia. They also observed that in a subset of patients with mild dysplasia there was a statistically significant difference in the percentage of p53-positive samples between the group that progresses to more severe dysplasia and the group that did not progress. They suggested that the technique, which is simple, economical, and rapid, could play a role in the evaluation and follow-up of patients with Barrett's esophagus.

Using an animal model, Fein et al. [24] induced esophagitis in mice. Both knockout mice with a mutant p53 gene, and p53 wild type mice were studied. The p53 knockout mice all eventually developed severe dysplasia or adenocarcinoma, whereas the p53 wild type did not. Other control mice, whether Apc mutant mice or non-inflamed knockout developed dysplasia.

There is good evidence that p53 over-expression is occurring in patients with Barrett's metaplasia at risk of progressing to either HGD and/or carcinoma. Its detection is technically easy and economically affordable. It may turn out be an attractive diagnostic option and improve the current approach. As it has been estimated that the cost of annual endoscopic surveillance in patient with intestinal metaplasia will lead to cost as high as $ 62,000.00 US for each carcinoma detected, the technique also offers the possibility of important cost savings [20, 25].

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