Expression of p 53 protein in Barrett ’ s adenocarcinoma and adenocarcinoma of the gastric cardia and antrum

Clinical Center of Serbia, Institute of Digestive Diseases, *Clinic of Gastroenterology and Hepatology, Department of Histopathology, Department of Esophagogastric Surgery, Belgrade; Institute of Medical Research, Department of Experimental Pathology and Cytology, Laboratory for Immunohistochemistry and Electron Microscopy; Belgrade, Serbia and Montenegro; University of Crete, Medical School, Heraclion, Crete, Greece


Introduction
Gastric adenocarcinomas comprise a heterogenous group of tumours.They can occur in the antrum, in the gastric body, and at the esophagogastric junction (EGJ).Adenocarcinomas of the gastroesophageal junction have many features in common with adenocarcinomas of the esophagus.They show a rapidly increasing incidence in most Western countries, a time trend different from those in the distal stomach (1,2).Both are more common in white males.The ratio of males to females appears to be a little higher for esophageal than for cardia adenocarcinoma, but the difference is of no statistical significance.The age of diagnosis is similar (60-65 years), with a tendency for adenocarcinoma of the EGJ to be diagnosed at a later age.Some authors have reported differences in risk factors, between patients with adenocarcinoma of the EGJ and esophageal adenocarcinoma.There may be a stronger history of smoking and alcohol consumption in patients with adenocarcinoma of the cardia (3).Most of these tumours are diagnosed in advanced stages, and the prognosis is worse than that for carcinomas located in the middle or distal third of the stomach (4,5).
Esophageal adenocarcionama develops most often on Barrett's metaplastic mucosa, a condition that arises in about 10% of patients with chronic gastro-esophageal reflux disease.This cancer develops through a histological and biological sequence from metaplasia to dysplasia and eventually through carcinoma (6).While the relationship of the specialized intestinal metaplasia and esophageal adenocarcinoma is well recognized (7,8), the pathogenesis of specialized intestinal metaplasia in the cardia and its importance in cancerogenesis is still not well defined.
Among the registrated genetic abnormalities in oesophageal and gastric adenocarcinomas, allelic losses on chromosome 17p and mutation of the p53 gene have been documented to be frequent events (9)(10)(11).p53 is a the tumour-suppressor gene that encodes p53 protein, which allowes necessary DNA repairs by slowing down the cell cycle at the G1-S transition, and suppresses tumour growth by inducing apoptosis (12).Among the oncogenes and tumour suppressor genes, p53 is an important one since the mutations of this gene are the most frequent genetic alterations so far identified in human tumours (12,13).p53 protein expression, either secondary to mutation or adhesion to other cellular or viral proteins, can be detected by immunohistochemistry, and therefore, is an indirect way of analyzing the functional status of p53 gene.As such, the accumulation of p53 protein may be considered an indicator of p53 mutation (14).The nuclear accumulation of p53 protein has been detected in a variety of malignant tumours including Barrett's adenocarcinoma and gastric cancer and the relationship between p53 overexpression and poor prognosis has been reported (9)(10)(11)14).
Most studies of esophageal and gastric adenocarcinomas have shown a very high rate of p53 gene mutation and/or protein overexpression, but the influence of the tumour site upon the frequency of p53 protein expression has not been evaluated in most of the published studies.The aim of our study was to the analyze the correlation between the selected clinico-pathological parameters and p53 protein overexpression in regards to particular tumour location.

Patients and tissues
The material comprised 66 sugical specimens which were retrieved from the files of the Department of Histopathology of the First Surgical Clinic of Clinical Center of Serbia, Belgrade, during a 4-year period (1997)(1998)(1999)(2000).Ten surgical specimens were collected from the patients with Barrett's carcinoma, 25 from the patients operated for adenocarcinoma of the gastric cardia (type II adenocarcinoma of the EGJ), and 31 surgical specimen from the patients operated for adenocarcinoma of the antrum.Adenocarcinoma of the cardia was defined as a tumour with a center located within 2 cm of the esophagogastric junction, with no surrounding metaplastic mucosa detected preoperatively or on gross examination of the surgical specimen (15).The Lauren's histological classification of gastric carcinoma (16), as well as the evaluation of tumour differentiation were performed.
For negative controls the primary antibody was omitted and for positive controls a Barrett's adenocarcinoma with positive immunostaining as well as gastric cardia and antrum adenocarcinoma were included with each batch.
The cases were considered positive for p53 if at least 5% of the tumour cells expressed this protein by immunostaining.Nuclear expression was considered positive for p53 and protein immunoexpression was counted in 20 randomly selected fields at ×40 magnification.

Sttistical analysis
Statistical analysis was performed with the SPSS program for Windows (6.0, 1993), using the χ 2 test.The Fisher's test was used when there were fewer than five cases in one group included in the table for statistical evaluation.The results were considered statistically significant when p < 0.05.

Results
Table 1 shows the major pathological features of resected tumours.There was no significant difference observed between studied groups in regards to age, sex, Lauren's classification and tumour differentiation.There was a significant difference observed in the depth of tumour invasion between Barrrett's adenocarcinoma and adenocarcinoma of the cardia compared to the adenocarcinoma of the antrum.Namely, at the time of surgery, both Barrett's adenocarcinomas and adenocarcinomas of the cardia, were significantly more advanced comparing to adenocarcinomas of the antrum.
Table 2 presents the correlation between p53 immunopositivity and the different clinico-pathological characteristics in regards to tumour location.p53 overexpression (nu-clear staining of 5% or more of tumour cells) was found in 40% (4/10) of Barrett's adenocarcinomas (Figure 1), 72% (18/25) of cases of adenocarcinoma of the cardia, and 65% (20/31) of adenocarcinoma of the antrum.No significant differences in p53 expression in relation to sex, type (Lauren) of tumour, the depth of invasion, the lymph node involvement, or the tumour differentiation were observed in any of the analyzed groups of tumours.Patients with the more advanced Barrett's adenocarcinoma and in the cases of lymph node invasion revealed the tendency for greater p53 positivity compared with the early forms and lymph node-negative cases; however, this difference was not significant according to the statistical analysis.With regard to adenocarcinoma of the cardia, the higher rates of p53 positivity were recorded in the poorly differentiated, more advanced cases with lymph node invasion.Neverthe- less, none of these differences was statistically significant.On the contrary, in the patients suffering from adenocarcinoma of the antrum, greater p53 positivity was revealed in early forms without lymph node involvement, but the observed difference was not statistically significant.

Discussion
Over the recent decades, there has been an alarming rise in the incidence and prevalence of adenocarcinoma of the esophagogastric junction in the Western world, in contrast with the decreasing prevalence of gastric cancer.It is the consequence, primarily of an increase in rate of adenocarcinoma of the distal esophagus.Nevertheless, the incidence of gastric tumours is shifting from the distal portion of the stomach to the proximal stomach and the gastric cardia.Adenocarcinomas of the esophagus and gastric cardia share similar epidemiological and pathological features (1,17).
Some investigators believe that carditis and subsequently intestinal metaplasia of the cardiac mucosa are the consequences of repeated injury of the cardiac epithelium by gastric and duodenal content, with duodenal components as the crucial agent (18,19).The reason why the process is limited to the cardia in some patients while, in others, it involves the esophagus, is due to the competence of the lower esophageal sphincter.On the other hand, histologically and functionally, the antrum and the cardia share similar epithelium (20) with mucus secreting cells and similar local pH that determines the growth behavior of Helicobacter pylori (21).So, it is not unreasonable to believe that the inflammatory changes on those sites can have similar consequences regarding the Helicobacter pylori infection.
Intestinal metaplasia was found in several studies in the peritumorous mucosa of the gastric cardia tumors, although, it was not always clear whether it has its origin in esophagus or the proximal stomach (8,22).Thus, it is believed that intestinal metaplasia may have a premalignant potential (23).The pathogenesis of both esophageal and gastric cardia adenocarcinomas is likely to be associated with the specialized intestinal metaplasia.While the relationship of the specialized intestinal metaplasia and esophageal adenocarcinoma is well recognized (7,8), the pathogenesis of specialized intestinal metaplasia in the cardia and its importance in the cancerogenesis is still not well defined.
Stein et al. have defined adenocarcinoma of the esophagogastric junction as tumours which have their center within 2 cm proximal or distal to the anatomical cardia and have differentiated three distinct tumour entities within this area.Barrett's esophagus with the subsequently developed dysplastic changes is the main precursor lesion for adenocarcinoma in the distal esophagus.Type II and III tumours may also rise from the so-called short segments of intestinal metaplasia at or below the cardia, but it happens in less than 40% of patients with carcinoma of the cardia and in less than 10% of those with subcardial carcinoma.These tumours maybe associated with Helicobacter pylori and intestinal metaplasia at or below the gastric cardia contrary to the specialized intestinal metaplasia in the distal esophagus that is reflux related (15).
Overexpression of p53 protein is frequently observed in the dysplastic areas and cancer in Barrett's esophagus.It Број 12

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Страна 883 has been suggested that this is an early event in the dysplasia-carcinoma sequence (6).A number of descriptive molecular studies in esophageal adenocarcinoma has reported on frequency of the mutation of the p53 tumour suppressor gene, and p53 protein overexpression as the most commonly described abnormality.The incidence of p53 mutation tended to increase with the progression along the metaplasia-dysplasia-carcinoma sequence, with the mutation being rare in nondysplastic tissue, but present in up to 90% of esophageal adenocarcinomas (20,(24)(25)(26).
On the other hand, stadies of p53 alterations in gastric cancer have reported a slight lower frequency of either allelic loss on chromosome 17p, p53 gene mutation, or p53 protein overexpression.p53 overexpression has been detected in 26-65% of gastric adenocarcinoma (27,28).In most studies, the frequency of p53 protein overexpression was not compared to the location of the tumours.
We investigated the frequency of p53 protein expression in a series of Barrett adenocarcinoma, adenocarcinoma arising in the cardia (type II adenocarcinoma of the esophagogastric junction) and the gastric antrum.We found no association between p53 protein overexpression and neither tumour stage or histological type nor tumour location.Similar results have been reported in most series of adenocarcinomas of the esophagus and stomach (14,24,25).Some studies have shown a higher frequency of p53 alterations in the gastric cancers of the intestinal type (Lauren's classification) when compared with the diffuse type (29), a feature that we were unable to confirm, which might be because of the relatively small number of cases.Furthermore, in most studies the frequency of p53 alterations was not compared with the tumour location.We revealed the tendency for greater p53 positivity in adenocarcinoma of the cardia and adenocarcinoma of the antrum, than in Barrett's adenocarcinoma; the result that is quite opposite to the previous reports in which greater p53 positivity can be attributes to a more proximal gastric tumours as well as in Barrett's adenocarcinoma (6,11,27,(30)(31)(32)(33). In the comparative study of p53 alterations in the consecutive series of oesophageal and cardia adenocarcinomas, a high frequency of gene mutation (70% and 63%, respectively), and p53 protein production (70% and 59%, respectively) was observed in both groups of tumours.In respect to the anatomical site of tumours, higher frequency of DNA aneuploidy (34), and lower frequency of microsatellite instability (32,35), were observed in adenocarcinomas of the cardia and the oesophagus when compared with adenocarcinomas of the antrum.In our study we found no other site specific differances.
The observed clinicopathological parameters were not associated with p53 protein overexpression except at the time of surgery, Barrett's adenocarcinomas and adenocarcinomas of the cardia, both were more significantly advanced comparing to adenocarcinomas of the antrum.