Barrett's esophagus is the condition in which columnar epithelium replaces the squamous epithelium that normally lines the distal esophagus[22]. The endoscopic appearance is characteristic, with a salmon-pink color and velvety texture. This upward extension may be circumferential, or limited to finger-like projections, or even islands of columnar epithelium that only involve part of the distal esophageal wall[23]. In healthy individuals, the squamo-columnar junction (SCJ) does not always correspond with the anatomic EGJ. The juxtaposition of pale squamous epithelium and reddish columnar epithelium forms a visible line called the Z line, or the SCJ. The EGJ is the most proximate part of gastric folds. The Z line and gastroesophageal junction often coincide. When the Z line is located above the EGJ, there is a columnarly lined segment of esophagus. Barrett's esophagus is diagnosed endoscopically if columnar mucosa extending greater than 3 cm above the EGJ, or by histologic finding of intestinal metaplasia (specialized mucosa) existing at any level of the tubular esophagus[22].

The pathogenesis of Barrett's esophagus is still unclear. Most Barrett's esophagus develops from approximately 10% of persons who have chronic gastroesophageal reflux[24]. In addition to gastroesophageal reflux diseases (GERD), Barrett's esophagus is associated with several other esophageal disorders, such as hiatal hernia[25], and occasionally familial groupings are reported[26-28].

The significance of Barrett's esophagus is its association with the development of esophageal malignancy[29,30]. Patients with Barrett's esophagus have (by conservative estimates) a 30- to 40-fold higher risk of developing esophageal adenocarcinoma than those without[23]. Since the diagnostic criteria for Barrett's esophagus now include the histological finding of intestinal metaplasia, a columnar-lined esophagus less than 3 cm in length is now recognized as short-segment Barrett's esophagus (SSBE), in contrast to the traditional long-segment Barrett's esophagus (LSBE)[31]. The risk of malignancy in SSBE still needs to be determined[32].

In China, Barrett's esophagus used to be regarded as a rare condition, and consequently has not been studied in detail. However, based on an endoscopic survey performed by our research group in the high-risk population in Linzhou, Barrett's esophagus was detected with a frequency of 0.7% (3/402); not as rare as it was considered previously[33]. It has been identified that there is an apparent change of Z line upgrowth among symptom free subjects in the high-incidence area for esophageal cancer in Henan Province. Z line upgrowth is related with epithelial cell hyperproliferation both in gastric cardia and lower segment of esophagus. These results suggest that Z line upgrowth may be one of the characteristic changes, or precancerous lesions related with EGJ carcinogenesis in the high-risk population for GCA in Henan[11]. Further characterizing Barrett's Esophagus, Z line upgrowth, and esophageal and gastric reflux esophagitis in this population may provide new insights into understanding of cardia carcinogenesis[34].

Since the diagnostic criteria for Barrett's esophagus based on endoscopy are arbitrary, recent definitions of Barrett's esophagus have included the histologic finding of intestinal metaplasia, regardless of the length of the columnar-lined esophagus[35]. Histologically, three types of columnar epithelium have been observed in Barrett's esophagus: gastric fundic type, junctional type and specialized intestinal metaplasia (IM). Based on the mucin content of the columnar and goblet cells, intestinal metaplasia is histopathologically divided into three types: the complete form (Type I), in which the columnar cells are absorptive and contain neutral mucins, and the two incomplete forms, in which the columnar cells are partly secretory and contain acidic sialomucins (Type II) or sulphomucins (Type III)[36,37]. Type I is the predominant form of intestinal metaplasia found in the stomach, including both benign and malignant conditions. Type III is the least common form in the stomach, but the one most strongly associated with gastric cancer[38]. The incomplete form of intestinal metaplasia also termed specialized columnar epithelium (SCE), is the histological mark of Barrett's esophagus[39].

IM is also associated with GERD, and the frequency of IM is increased with the extention of columnar lining in the esophagus[40]. However, gastritis rather than GERD seems to be a risk factor of cardiac intestinal metaplasia[41]. While complete intestinal metaplasia is a manifestation of multifocal atrophic gastritis, the incomplete form may result from carditis and GERD[42].

It has been hypothesized that Barrett's adenocarcinoma follows the metaplasia-dysplasia-adenocarcinoma sequence (MCS)[43]. Whether intestinal metaplasia is a premalignant lesion for adenocarcinoma of the gastric cardia is still not clear[44]. Several studies showed that dysplasia was infrequently observed in EGJ or gastric cardia with intestinal metaplasia[42,44,45]. These suggest that intestinal metaplasia at the EGJ or gastric cardia is not a high-risk precursor lesion for adenocarcinoma. However, progression from intestinal metaplasia at the EGJ or gastric cardia to dysplasia still requires further characterization[46].

Examination of the adjacent gastric cardia cancer tissue by our research group showed that IM was frequently observed in as high as 20% of the specimens[47]. In contrast, only 0.7% symptom-free subjects in Linzhou were identified with IM in gastric cardia biopsies[33]. Goblet cell was invariably identified on HE stained specimen. Half of the IM lesion was found to have dysplasia. These suggest that IM may be a precursor of gastric cardia adenocarcinoma in Henan, which is not consistent with that in Western countries. The reason is not clear. Standard histologic evaluation fails to reliably differentiate between IM in the esophagus and IM in native cardiac mucosa. Further characterization for subtypes of IM with biohistochemistry and immunohistochemistry will provide important evidence for elucidating the significance of IM in gastric cardia carcinogenesis.

IM in the distal stomach is usually a consequence of chronic inflammation, mainly caused by H. pylori infection[48], while the intestinal metaplasia of Barrett's esophagus has been demonstrated to be associated with GERD[49]. Furthermore, recent studies suggested that gastric infection with H. pylori might protect the esophagus from developing of reflux esophagitis and Barrett's esophagus[50-54]. It is reasonable to hypothesize that intestinal metaplasia at the gastric cardia may develop as a consequence of chronic inflammation due to either GERD or infection. Several recent studies have focused on the relative contribution of H. pylori infection and GERD to gastric carditis, however, results have been inconsistent, to date[55-58]. One possible reason may be related to the use of different histopathologic criteria for identification of the gastric cardia. A study of 33 consecutive autopsies in children showed that the length of cardiac epithelium ranged from 1 to 4 mm, with a mean extent of 1.8 mm[59]. Ormsby expanded the study to 223 adult autopsies, finding that the mean extent of cardiac epithelium in patients aged < 18, 19-50, and > 50 years was 1.7, 2.6, and 3.3 mm, respectively[60]. Such a narrow range makes accurate sampling extremely difficult. Therefore, biopsies from the gastric cardia may have two origins: esophagus or stomach.

LOH has been demonstrated to play an important role in tumorigenesis, and to be frequently associated with the loss of tumor suppressor gene function. LOH was initially observed at several tumor suppressor gene loci (17p, 13q, 5q, 18q and 9p for p53, Rb, APC, MSH2, DCC and p16, E-cadherin, 19p, respectively) in human esophageal and gastric cancer[61-67]. At present, LOH has been used to localize putative tumor suppressor genes. LOH has also been reported at 1p, 3p, 4q, 9q, 11p and 17q in esophageal adenocarcinomas[68,69]. Allelic loss of the tumor suppressor gene p73 on 1p is frequently observed in neuroblastoma[70,71], and hMLH-1, a mismatch repair gene associated with hereditary non-polyposis colorectal cancer, is located on 3q21[72]. The tylosis esophageal cancer (TOC) gene, initially reported in families with autosomal dominant tylosis families who developed esophageal cancer, has been located on chromosome 17q25. Allelic loss at this region has been implicated in both sporadic SCC and Barrett's adenocarcinoma[73]. BCRA1, associated with susceptibility to breast and ovarian cancer, is located on 17q[74]. Using comparative genomic hybridization (CGH), deletion of a specific region at 14q31-32.1 occurred significantly more frequently in Barrett's adenocarcinomas in the distal esophagus than in gastric cardia cancers, suggesting genetic divergence in this group of closely related cancers[75]. Allelotype analysis performed on 38 gastric cardia adenocarcinomas even differentiated this allelic imbalance between the intestinal-type and diffuse-type adenocarcinomas, and a higher frequency of allelic imbalance on chromosome 16q was detected in the diffuse-type adenocarcinomas[76].

Tumor suppressor gene p53 P53 is located on chromosome 17p13, encoding a phosphoprotein with a molecular weight of 53 kd. Abnormalities in p53 gene function cause uncontrolled cell cycles and abnormal cell proliferation. P53 mutations are the most frequent genetic alterations found in human cancers[77]. Over 90 percent of p53 mutations are observed in exons 5-8, which is the highly conserved region for DNA binding. Comparison of p53 mutations in premalignant (basal cell hypertrophy, BCH; dysplasia, DYS; carcinoma in situ, CIS) esophageal tissues matched with corresponding invasive SCC tumor tissues, showed similar p53 mutations in DYS, CIS and tumors[78]. Similar studies with Barrett's adenocarcinoma showed that p53 mutations in Barrett's epithelia (metaplasia, non-dysplastic) did not necessarily correspond to the matched adenocarcinoma[20]. The high coincident alterations for P53 in SCC and GCA from the same patient indicate the possibility of similar molecular mechanisms, which provides important molecular basis and etiological clue for similar geographic distribution and risk factors in SCC and GCA[79,80]. These results suggest that some p53 mutations may have a selective tumorigenic advantage during tumor progression. And some findings suggest p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells[81]. In the course of the formation of gastric carcinoma, proliferation of gastric mucosa can be greatly increased by H. pylori infection, which can strengthen the expression of mutated p53 gene[82]. Less aggressive mutations may increase the genetic instability of Barrett's mucosa, promoting abnormal cellular proliferation, but as they are incapable of transformation independently, additional molecular or epigenetic events are required for tumorigenesis.

About 90 percent of p53 mutations are located within the DNA binding domain (exons 5 to 8). They may occur either at the binding surface or at the hydrophobic core, disrupting the structure for DNA binding. P53 mutations do not distribute in a random manner through the whole encoding region. Several "hot spot" mutations have been located such as codon 175, 176, 245, 248, 249, 273 and 282 in all the human cancers[83], implying that there may be a general pathway for the pathogenesis of tumors with a high percentage of p53 mutations. However, mutation sites also appear to be organ specific and cell-dependent. In esophageal adenocarcinomas, p53 mutations occur most frequently at codon 175 (9%), 248 (16%) and 273 (16%), which are also hot spot mutations for all human cancers recorded, while mutations at codon 248 and 273 are much less frequently observed, even though the mutation at codon 175 accounts for 8% in SCC. Relatively high frequencies of p53 mutations at codons 193, 194, 195 and 270 are unique in comparison to other human cancers[77].

P53 mutations may result from endogenous processes or exogenous carcinogens[84], and the spectrum of mutations may be indicative of specific carcinogenic mechanisms. Mutation profiles of p53 also help to identify the DNA damage arising from environmental carcinogens. Approximately 31% of p53 mutations occur at the A:T base pairs in the SCC, which are usually caused by exogenous carcinogenic compounds. But in esophageal adenocarcinoma, p53 mutations have a very high frequency of transition at the CpG dinucleotides. This alteration is thought to result from spontaneous deamination of 5-methylcytosine, suggesting a defective DNA mismatch repair[77,85]. Differences in p53 mutation spectra between SCC and esophageal adenocarcinoma are consistent with the results of epidemiological studies, suggesting that SCC and esophageal adenocarcinoma have different etiology.

Adenocarcinomas of gastric cardia share similar epidemiological and histological features to esophageal adenocarcinomas in North America and some European countries. Gleeson et al[86] compared the p53 abnormalities in adenocarcinoma of the distal esophagus and gastric cardia, and reported that p53 mutations were detected in 70% and 63% of adenocarcinoma of esophagus and gastric cardia, respectively. 85% of the p53 mutations in esophageal adenocarcinoma occurred as G:C→A:T transitions, with 69% at the CpG dinucleotides. Similar p53 mutations were observed in adenocarcinoma of the gastric cardia, in which 82% were base transitions with 55% occurring at the CpG dinucleotides. One study from Linzhou based on the p53 gene mutation analysis identified 6 mutations among 14 adenocarcinomas of the gastric cardia, 3 of which were G to T transversions, a mutation that is rarely observed in Barrett's adenocarcinoma[87]. Further investigation and comparison of mutation analysis of p53 gene between SCC and adenocarcinoma of the gastric cardia in this population are needed so to provide more insights into the etiology of both SCC and adenocarcinomas of esophagus and the gastric cardia.

p53 gene family Recently, discovery of p53 homologues such as p73 and p63 suggests a more complex pathway. p73 and p63, similar to p53, may result in transactivation, DNA binding and oligomerization domains[88], capable of activating the transcription of p53-responsive genes and inducing apoptosis[89-91]. p73 is located on chromosome 1p36, a region that is frequently deleted in neuroblastoma[73,74], but unlike p53, p73 mutations have been detected infrequently in other types of tumor, including esophageal and gastric carcinoma[92-100]. Silencing of p73 gene due to hypermethylation at its promoter region was observed in a subset of lymphoblastic leukaemia and Burkett's lymphoma[101,102], but increased level of mRNA was more commonly observed in other tumors[95,103-105]. p73 is characterized by loss of imprinting (LOI), resulting in monoallelic expression in normal tissues. It has been observed that p73 is overexpressed in several tumors, which is thought to be due to the switching from monoallellic to biallelic expression[93,95]. Meanwhile, p73 isoforms, caused by alternative splicing, have been observed in rare types of brain tumors, suggesting tissue-specificity in the regulation of p73 gene transcription[106]. However, the precise function of p73 isoforms in tumor development is still unclear. Specific mutations of p73 or p63 causing amino acid substitutions are not identified. Neither p53, p73 nor p63 is related to prognosis. p73 and p63 have rarely been found to be mutated in gastric carcinomas, but both proteins are expressed in only a subset of tumors. The status of these p53 homologues is discordant among all patients with multiple simultaneous gastric carcinomas. The increased expression of p63 (TAp63 and black triangleNp63) in less well-differentiated gastric carcinomas may indicate that p63 can act to promote neoplastic growth in the gastric epithelium[107]. In 15 SCC samples from Henan, no p73 mutations were found in exons 4-7, but a high frequency of LOI and LOH was observed in these samples. The SCC samples with p53 defects were significantly correlated with those, which had elevated expression of p73. These results suggest that increased expression of p73, including that by LOI, could be a partial compensatory mechanism for defective p53[100].

The p63 gene also encodes multiple isoforms through alternative splicing with different abilities to transactivate p53-responsive genes. However, p63 is more closely related to p73 than to p53, and p63 mutations have been rarely observed in human tumors[108,109]. The predominant isoform of p63, lacking in an acidic N-terminus corresponding to the transactivation domain of p53, has been detected in many kinds of epithelial tissue. This truncated protein may act as a dominant-negative agents toward transactivation by p53[110]. Up to date, p63 has not been studied in esophageal adenocarcinoma or adenocarcinomas of the cardia.

Rb gene One study compared LOH with the expression of Rb gene in SCC, finding that 90% of the tumor samples containing LOH showed low or no Rb expression, while only 20% of those without LOH had altered expression of Rb, suggesting LOH is the principal molecular alteration of Rb in SCC[111]. Recent studies of SCC cell lines demonstrated that the hypophosphorylated Rb protein was also associated with G2 arrest[112]. Besides inhibition of the cell cycle progression, Rb protein appears to have other distinct mechanisms to suppress cellular proliferation[113]. In the gastric carcinogenesis, H pylori might cause severe imbalance of proliferation and apoptosis in the precancerous lesions (IMIII and DysIII) first, leading to p53-Rb tumor-suppressor system mutation and telomerase reactivation, and finally causing gastric cancer[114]. Wang et al[115] suggested the alteration of Rb protein might play a role in the early stages of gastric cardia carcinogenesis.

p16INK4a, p15INK4b and p14ARF LOH of p16INK4a has been detected with high frequencies in both SCC (65%) and esophageal adenocarcinoma (69%)[116]. Several studies have demonstrated that hypermethylation of the promoter region of p16INK4a is the main mechanism causing gene silencing rather than point mutation, which rarely occurrs in esophageal carcinomas[116-119]. Distinct from p16INK4a, p15INK4b, which inhibits the cell growth in response to extracellular stimuli such as TGF-β[120], is more frequently deleted, at a frequency of 40%[118]. Though p16INK4a regulates cell cycle through inhibiting the phosphorylation of Rb protein, p14ARF is closely associated with p53 by attenuating mdm2-mediated degradation of p53[121,122]. Similar to p15INK4b, p14ARF is also deleted frequently in SCC, losing its function to protect p53 from ubiqitin-dependent degradation[123].

Abnormalities of oncogenes Point mutation, amplification, rearrangement and overexpression are the most frequent mechanisms for oncogene activation. Amplification of cyclinD1, HER-2/neu(c-erbB2), c-myc, c-ras, Int-2/hst-1 and c-erbB (EGFR) has been observed in gastric and esophageal carcinomas[124-130]. The amplification of c-erbB2 was followed by overexpression in the same gastric adenocarcinoma tissue[125,129]. No amplification of c-erbB2 was detected in SCC. Amplification of oncogenes that encode growth factor receptors is more commonly found in adenocarcinomas. Int-2 and hst-1, encoding FGF-3 and FGR-4 respectively, are located at chromosome 11q13 with the oncogene cyclinD1. Coamplification of int-2 and hst-1 was observed at a frequency of 28%-47% in primary esophageal carcinomas and much higher in metastatic tumors, suggesting an association with tumor progression and distal metastasis[131,132]. CyclinD1, a cell cycle protein, facilitates cell cycle progression from G1 to S phase through combining with CDK4. Both the amplification and overexpression of cyclinD1 were detected in esophageal carcinomas[133,134]. Abnormal expression of cyclin E and p27 may be one of the important molecular changes in the early stage of esophageal carcinogenesis, and the high-expression of cyclin E and low-expression of p27 may be one of the mechanisms driving the mild lesion towards carcinogenesis[135]. In contrast to other gastrointestinal tumors, the ras oncogene is rarely mutated in human esophageal adenocarcinoma[127]. However, overexpression of ras and ras-regulated genes (osteopontin, cathepsin L) has been reported in 58% of primary esophageal adenocarcinomas[136].

Because of the highly concurrent incidence of gastric cardia adenocarcinoma and esophageal squamous cell carcinoma in Linzhou[140], it is highly desirable to characterize the molecular differences between these tumors of different histological types to explore the possible clues related with etiological risk factors. However, useful information concerning this topic is very limited. In Linzhou, it is not uncommon clinically to identify patients with both gastric cardia adenocarcinoma and esophageal squamous cell carcinoma[141], which is the most common pattern of multiple primary malignant neoplasm (MPN) with an incidence of 0.4%-2.5%[142]. Recent studies from Wang's laboratory showed that there was a highly consistent positive immunostaining rate for p53 in SCC and GCA from the same patient (60%, 12/25 vs 40%, 10/25)[79] similar results were observed for PCNA. p53 mutation analysis in patients with either SCC or GCA from Linzhou indicated that G:C to A:T transition was the most frequent mutation pattern both in SCC and GCA. The consistency of P53 gene mutation was as high as 64%[143]. G to A mutation pattern may be resulted from DNA methylation induced by nitrosamine[141]. These results are of important value in explaining the similar geographic distribution of SCC and GCA in this area. Protein file analysis also showed similar expression pattern in SCC and GCA, such as PTEN[144], c-erbB-2 and c-myc[145], MUC1[146], CYP1A1 and 2E1[147], MUC3[148], mEH[149], GSTM1, GSTT1 and GSTP[150], EGFR[151], etc from the samples in Linzhou.