|Year : 2014 | Volume
| Issue : 4 | Page : 691-698
Immunohitochemical study of p53 protein expression in gastroesophageal reflux disease
Atef Abu Al-SoudAly1, GamalSaad El-Deeb1, Sawsan El-sayedAnees1, Ayman Mohammed El-Lehleh1, Mohammed Ebrahim Shaaban2, Mervat Ragab Abd El-Rahman Nassar MSc 1
1 Tropical Medicine Department, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||09-Sep-2013|
|Date of Acceptance||10-Dec-2013|
|Date of Web Publication||22-Jan-2015|
Mervat Ragab Abd El-Rahman Nassar
Bemam, Tala, Menoufia
Source of Support: None, Conflict of Interest: None
This study aimed to determine p53 protein expression by immunohistochemistry in the esophageal mucosa of patients with gastroesophageal reflux disease (GERD).
GERD is a common clinical disorder that can significantly impact the patient's quality of life and pose a significant burden on healthcare systems worldwide. The incidence of esophageal adenocarcinoma has been increasing rapidly over the past few decades. The major risk factors predisposing to the development of adenocarcinoma are long-standing GERD and Barrett's esophagus. P53 protein expression in esophageal carcinogenesis is still being studied. The identification of tumor markers in patients with GERD may enable recognition of subgroups of patients who are more at risk of developing dysplasia and/or cancer.
Patients and methods
This study was carried out on 200 patients with symptoms of GERD. They were recruited from the endoscopy unit of Menoufia University Hospital between August 2010 and March 2013, and were included in group 1. According to the results of upper endoscopy and histopathology, they were subdivided as follows: group 1a included patients with GERD with erosive esophagitis ( n = 180), group 1b included patients with GERD and Barrett's esophagus ( n = 20), and group 2 included 20 patients with other GIT symptoms with no reflux and normal esophageal squamous epithelium; this was the control group. Written valid consent was obtained from every patient for examination and upper endoscopy; the clinical sheet for every case was completed, including assessment of history, examination, and the investigations required.
We found negative reactivity for p53 in 20 histologically normal patients (0%), and positive p53 protein expression in 76 patients (40%) in the esophagitis group and in 14 patients (70%) with Barrett's metaplasia ( n = 20). According to the Los Angeles classification grade, p53 was overexpressed in 33.3% ( n = 24) of esophagitis grade A (mild esophagitis) patients, 46.3% ( n = 32) of esophagitis grade B (moderate esophagitis) patients, 56.4% ( n = 22) of esophagitis grade C patients, and 60% ( n = 12) of esophagitis grade D (severe esophagitis) patients.
GERD grades C and D were more prevalent in Barrett's esophagus. P53 expression was higher in Barrett's esophagus than in erosive esophagitis compared with the normal group. P53 protein expression increases gradually with the severity of esophagitis caused by GERD.
Keywords: Barrett′s esophagus, erosive esophagitis, gastroesophageal reflux disease, p53
|How to cite this article:|
Al-SoudAly AA, El-Deeb G, El-sayedAnees S, El-Lehleh AM, Shaaban ME, Abd El-Rahman Nassar MR. Immunohitochemical study of p53 protein expression in gastroesophageal reflux disease. Menoufia Med J 2014;27:691-8
|How to cite this URL:|
Al-SoudAly AA, El-Deeb G, El-sayedAnees S, El-Lehleh AM, Shaaban ME, Abd El-Rahman Nassar MR. Immunohitochemical study of p53 protein expression in gastroesophageal reflux disease. Menoufia Med J [serial online] 2014 [cited 2020 Feb 17];27:691-8. Available from: http://www.mmj.eg.net/text.asp?2014/27/4/691/149686
| Introduction|| |
Gastroesophageal reflux disease (GERD) is a common medical problem with a broad spectrum of symptoms and varying degrees of severity . The first consensus definition of GERD is known as the Montreal definition, which defines the condition as 'a condition which develops when the reflux of stomach contents causes troublesome symptoms and/or complications' . It usually manifests as heartburn, regurgitation, or dysphagia and predisposes to the development of esophagitis, stricture, Barrett's metaplasia, and esophageal adenocarcinoma, in addition to a marked decrease in the quality of life .
Barrett's esophagus (BE) is a change in the distal esophageal epithelium of any length that can be recognized as columnar-type mucosa at endoscopy and intestinal metaplasia can be confirmed by a biopsy of the tubular esophagus . BE is a recognized precursor of esophageal adenocarcinoma, which arises ultimately after progression through a sequence of increasing degrees of dysplasia . Patients with BE are at a 0.5-2% increased risk of developing esophageal adenocarcinoma, which is 30-125 times greater than that in the general population .
Because of this risk, early identification of malignant changes is very important; the p53 gene malfunction may precede dysplastic changes in BE . p53 protein expression in esophageal carcinogenesis is still being studied. The identification of tumor markers in patients with GERD may enable recognition of subgroups of patients who may be more at risk of developing dysplasia and/or cancer . Studies have suggested that certain molecular biomarkers including p53 mutations may aid in this risk stratification. Furthermore, an understanding of intermediate molecular biomarkers may aid in the development and testing of pharmaceutical agents in cancer chemoprevention trials .
The normal p53 gene acts as a tumor suppressor and its wild type inhibits proliferation and transformation. Cells that contain p53 genes of the wild type can delay cell cycle to allow the repair of damaged DNA or divert the cell into apoptosis. When the protein is mutated or absent, the cells replicate the damaged DNA, which will result in more mutations and chromosome rearrangement. These mutations may result in the formation of defective, highly stabilized protein with an increased half-life in tissues compared with the 20 min for the wild-type protein. This is the basis for the use of immunohistochemistry, which can detect mutant p53 products that are indicative of mutant protein .
Although BE and esophageal adenocarcinoma have been studied extensively using immunohistochemical methods, there is a lack of immunohistochemical studies of benign disorders such as erosive esophagitis or non erosive reflux disease ,. However, the association between GERD and carcinogenesis has been constantly suggested, and in another study, Shaheen et al.  reported that the risk of esophageal adenocarcinoma is increased five-fold in patients with previously diagnosed esophagitis. However, most of the adenocarcinomas occurred among patients with BE. Numerous studies and animal models have shown the carcinogenetic effect and mutagenic potential of GERD in the esophageal mucosa. The mechanism is probably indirect, involving the induction of oxidative stress and the production of reactive oxygen species that damage DNA. Repeated DNA damage likely increases the mutation rate, including the mutation rate of tumor-suppressor genes and oncogenes .
| Patients and methods|| |
The present study was carried out on 200 patients with symptoms of GERD; the patients were asked questions on the basis of a standardized questionnaire by an independent individual. Patients with one or more reflux symptoms (heartburn, acid regurgitation, water brash, and eructation) and more than three reflux episodes per week were defined as having GERD. They were recruited from the endoscopy unit of Menoufiya university hospital between August 2010 and March 2013. There were 104 men (52%) and 96 women (48%), ranging in age from 18 to 60 years, mean age 42.84 ± 11.53 years; these patients were included in group 1. According to the results of upper endoscopy and histopathology, they were subdivided as follows: group 1a: patients with GERD with erosive esophagitis (n0 = 180). Group 1b: patients with GERD and BE (n = 20). Group 2: this group included 20 patients  males (55%) and nine females (45%) ranging in age from 15 to 53 years, mean age 38.50 ± 8.04 years] with GIT symptoms other than GERD (dyspepsia, upper GIT bleeding, persistent vomiting, and epigastric pain) and had normal esophageal squamous epithelium. This group was included in the study as a control group.
We excluded patients with cardiac diseases or coagulation disorders, those taking nonsteroidal anti-inflammatory drugs, those with gastroesophageal varices and liver diseases, those who had received previous radiotherapy or chemotherapy treatment, those on acid suppressor therapy over the last 60 days, and those who had undergone previous esophageal or stomach surgery.
Patients and control groups were subjected to the following: full and detailed assessment of history, complete clinical examination, laboratory investigations, and abdominal ultrasonography.
Upper gastrointestinal endoscopy
Biopsies were taken from the distal esophageal mucosa (we used Olympus Evis CV 100 Videoscope; Olympus, Japan). Multiple esophageal biopsies were taken 2 cm above the Z line at intervals of 1-2 cm from the four quadrants of the esophagus. The specimens were placed on filter paper, fixed in a 10% formalin solution, and embedded in paraffin. BE was diagnosed on the basis of the presence of columnar-lined esophagus at endoscopy. We recorded the presence of esophagitis or any esophageal lesions, gastritis, duodenitis, gastric or duodenal ulcer, and hiatus hernia. Esophagitis is best defined by the Los Angeles classification system and identifies the degree to which mucosal breaks (erosions or ulcerations) occur, which are graded in severity from A to D, with D being the most severe .
Los Angeles classification of esophagitis 
Grade A: more than one mucosal break of less than 5 mm long confined to the mucosal folds.
Grade B: more than one mucosal break of more than 5 mm long confined to the mucosal folds, but not continuous between the tops of two-folds.
Grade C: mucosal breaks continuous between the tops of two or more folds involving less than 75% of the esophageal circumference.
Grade D: mucosal breaks involving more than 75% of the esophageal circumference.
Histopathological examination of all biopsies
The histological findings were classified into reflux esophagitis, BE, and normal squamous epithelium. BE was defined by the presence of intestinal metaplasia with goblet cells; the histopathologic diagnosis of esophagitis was defined by the presence of one or more of the following criteria: (i) the presence of basal layer cells that reached more than 15% of the total epithelial layer or elongation of papillae up to 2/3 of the total epithelial layer; (ii) epithelial infiltration by polymorphonuclear leukocytes (neutrophils and/or eosinophils); and (iii) mononuclear infiltration into the cells of the lamina propria .
P53 protein expression was determined in biopsies by immunohistochemical studies
All sections were stained with hematoxylin and eosin stain. Immunostaining for p53 was performed on formalin-fixed, paraffin-embedded tissues using the avidin-biotin-peroxidase complex technique. Sectioned tissue (3 μm thick) was deparaffinized with xylene and rehydrated with descending strengths of alcohol. Endogenous peroxidase activity was blocked by incubating specimens in 2% hydrogen peroxide in methanol for 5 min. An antigen retrieval step using a heat-induced epitope retrieval procedure was performed by boiling the sections for 15 min in 10 mmol/l citrate buffer at pH 6.0.
The slides were then incubated overnight at 4°C in a humidified chamber with a primary antibody. The primary antibody used for the detection of p53 protein was monoclonal p53.
After rinsing with a PBS solution for 15 min, the biotinylated secondary antibody was applied for 30 min at room temperature. After an incubation step with an avidin-biotin-horseradish peroxidase conjugate for 30 min, sections were stained with the chromogen. The chromogen used was diaminobenzidinetetrahydrochloride until a brown color was obtained. Counterstaining was performed using Mayer hematoxylin.
P53 immunostaining interpretation
p53 expression was evaluated as follows: P53 expression was classified semiquantitatively according to the following criteria: 0 if less than 1% of neoplastic cells discretely expressed P53 in their nucleus; 1+ if less than 10% of morphologically unequivocal neoplastic cells discretely expressed P53 in their cytoplasm; and 2+ if 10% or more of morphologically unequivocal neoplastic cells discretely expressed P53 in their nucleus. Samples scored as 1+ or 2+ were considered positive .
- Descriptive statistics: percentage, mean (x), and SD.
- Analytic statistics: As c2 -test, student (t) test, Z-tests with the following levels of significance: P value less than 0.05 is significant, P value less than 0.001 is highly significant, and P value more than 0.05 is nonsignificant.
| Results|| |
There was no significant difference between the groups studied in age, although patients with BE were older as shown in [Table 1].
|Table 1: Comparison between the groups studied in age, sex, and upper endoscopic findings|
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There was a nonsignificant difference between the groups studied in terms of sex. Men and women were equally distributed in the reflux esophagitis group (G1a), there were more males (70%) than females (30%) in Barrett's group (G1b), and in the control group (G2), 55% were males and 45% were females as shown in [Table 1].
GERD grade A and grade B were more prevalent in patients with GERD without BE, but GERD grades D and C were more prevalent in patients with GERD and BE. There was a significant difference between the groups studied in terms of the presence of hiatus hernia, which was more prevalent in patients with GERD and BE. There was a significant difference between the groups studied in gastritis, which was more prevalent in the normal squamous epithelium group as shown in [Table 1].
There was a significant difference between the groups studied in p53 expression by immunohistochemistry; p53 expression was significantly higher in group 1b (BE 70%) than in group 1a (erosive esophagitis 42.2%) compared with the normal controls (0%) as shown in [Table 2].
There was a significant difference in p53 expression in relation to the severity of esophagitis from the normal controls; p53 was overexpressed in 33% of esophagitis grade A (mild esophagitis) patients, 43.2% of esophagitis grade B (moderate esophagitis) patients, 53% of esophagitis grade C (severe esophagitis) patients, 65% of esophagitis grade D (more severe esophagitis) patients, and 0% in the control group as shown in [Table 3].
|Table 3: Correlation between p53 expression and degree of severity of esophagitis according to the Los Angeles grade classification|
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| Discussion|| |
The current study showed that there was no significant difference between the groups studied in the mean age, although patients with BE were older; this was in agreement with Lagergren et al. , who reported that older age is a risk factor for developing BE as well esophageal adenocarcinoma. This study showed that there was no significant difference between groups the studied in terms of sex. Barrett's group included more males than females. This finding was in agreement with Koek et al. , who found that BE is associated with male sex and exposure to both acid and duodenogastroesophageal reflux, but this was not in agreement with Banki et al. , who found that there was the prevalence of BE in men and women diagnosed with severe reflux by 24-h pH monitoring was equal. Patients with severe erosive esophagitis were equal in male and female. This was in agreement with Lin et al. , who reported an equal prevalence of erosive esophagitis in males and females. The prevalence of BE was 10% in patients with GERD and reflux esophagitis. This finding was in agreement with Hak et al. , who found a prevalence rate of 9.9% of BE in patients with GERD.
The current study showed that among the patients with erosive esophagitis, grade A erosions were established in 70 patients (38.8%), grade B erosions were established in 65 patients (36.1%), grade C erosions were established in 33 patients (18.3%), and grade D erosions were established in 12 patients (6.7%). All the patients with BE also had erosive esophagitis: grade A in two patients (10%), grade B in four patients (20%), grade C in six patients (30%), and grade D in eight patients (40%). Hiatus hernia was found in 17 patients with BE (85%), in 70 patients with reflux esophagitis (38.8%), and in three patients with normal squamous epithelium (15%). The prevalence of hiatus hernia in the patients with BE was significantly higher than that in the patients with erosive esophagitis (P < 0.001) and patients with normal squamous epithelium; this was in agreement with Avidan et al. , who reported that BE was more common in older patients, in males, and also in those with HH. There was a significant difference between the groups studied in terms of gastritis, which was more prevalent in the control group.
The current study found that p53-positive immune expression showed a statistically significant difference between the normal epithelium group and the other groups (P < 0.001). The esophagitis group also differed from the BE group. We observed a positive linear correlation between the p53 expression and the severity of the multistep stage of histological diagnosis (normal epithelium, esophagitis, and columnar epithelium with intestinal metaplasia). This was in agreement with Binato et al. , who reported that positive rates of p53 protein expression in esophageal mucosa increased gradually from normal to reflux esophagitis and further to Barrett's metaplasia and dysplasia. Our study found negative reactivity for p53 in histologically normal patients. This was in agreement with Gao et al. , who observed that p53 protein expression was seldom detected by immunohistochemistry in normal esophageal mucosa. This is also in agreement with Krishnadath et al. , who studied the expression of the p53 protein by immunohistochemistry in normal mucosa and found a faded nuclear color in the basal layer in 24 samples of esophageal mucosa used as a control group. This was not in agreement with Fagundes et al. , who reported the accumulation of p53 protein in the normal mucosa and in the basal layer hyperplasia of the esophageal epithelium in patients at a higher risk of developing esophageal adenocarcinom. Wang et al.  had already found p53 in esophageal basal cell hyperplasia in surgical specimens resected for squamous cell carcinoma and in biopsy samples taken from asymptomatic individuals living in high-incidence areas of esophageal cancer in China; they reported that even in the absence of mucosal lesions, chronic reflux may produce proliferative and molecular alterations. Positive p53 staining in the non esophagitis squamous group may occur because of rapidly dividing cells and inactivation of wild-type p53 through binding of another protein, which prolongs its half-life . This study also found positive p53 protein expression in 40% of the patients in the esophagitis group. This was in agreement with Mandard et al. , who reported p53 protein expression in mucosa with esophagitis at a distance from the esophageal tumor in surgical specimens. GERD causes inflammation and may explain the positive reactivity of p53 in these patients. However, inflammatory alterations are closely related to the process of esophageal carcinogenesis and are capable of modifying the function of many proteins that regulate the cell cycle such as p53 . In our study, p53 protein expression was found in 70% of patients with Barrett's metaplasia; this was not in agreement with Casson et al. , who identified p53 overexpression in 50% of patients with intestinal metaplasia without dysplasia. Segal et al.  found that p53 protein was expressed in 60.9% of the patients with Barrett's epithelium.
The present study showed that p53 was overexpressed in 33% of esophagitis grade A (mild esophagitis) patients, 43.2% of esophagitis grade B (moderate esophagitis) patients, 53% of esophagitis grade C patients, and 65% of esophagitis grade D (severe esophagitis) patients; p53 was not expressed in normal squamous epithelium. p53 overexpression was significantly higher in grade A to D patients than in normal individuals, which suggests a correlation between p53 protein overexpression and the histologic progression of chronic esophagitis. There was a clear correlation between the degree of esophagitis severity and initial alterations in the p53 marker even before the occurrence of premalignant morphologic alterations of the esophageal mucosa. This was in agreement with Fagundes et al. , who detected a progressive increase in p53 expression from normal esophageal mucosa through moderate esophagitis, severe esophagitis, metaplasia to carcinoma. Thus, they suggested that patients with chronic esophagitis and p53 protein overexpression have an increased risk of developing esophageal cancer.
Our results could have been underestimated if we had not identified p53 immunoreactivity because of variations in the immunohistochemical methods or the interpretation of the nuclear color intensity . However, we might have overestimated our findings because most of the patients had GERD and therefore the esophageal mucosa was affected by gastroduodenal reflux and by different degrees of inflammatory response, but in this case, the p53 expression was restricted to the basal layer. The P53 mutation cannot be directly detected by immunohistochemistry and false-negative or false-positive cases may occur. Indeed, false-negative results occur in about 10% of all p53 mutations because of point mutations in most cases; false positivity may develop because of different factors such as rapidly dividing cells (e.g. activated lymphocytes express p53) and inactivation of wild-type p53 through binding to another protein, which prolongs its half-life .
Molecular analysis by PCR may be a more sensitive test as it shows p53 mutations, whereas immunohistochemistry detects only the altered protein accumulation . However, the PCR technique is expensive and still too complex for routine screening; in contrast, the immunohistochemistry technique used to detect p53 protein accumulation is easier, less expensive, and can be carried out in most pathology laboratories .
The impact of pathophysiologic factors on molecular alterations of the esophageal mucosa affected by esophagitis is not completely clear. Few studies have addressed squamous mucosal abnormalities in patients with nonerosive reflux disease or erosive disease . Most studies have been carried out on BE and its progression to adenocarcinoma of the esophagus. A better understanding of molecular biologic events during the initial stages of GERD may lead to improved therapeutics for the control of inflammatory mediators in the esophageal mucosa. Such therapeutics, if administered before the occurrence of molecular alterations, may help to prevent the progression of GERD to metaplasia and then to esophageal adenocarcinoma .
GERD grades C and D were more prevalent in BE. P53 expression was higher in BE than in erosive esophagitis compared with the normal group. P53 protein expression increased gradually with the severity of esophagitis caused by GERD.
Prospective studies including longitudinal follow-ups are required to determine whether p53 can identify those patients who may be at a higher risk of developing esophageal adenocarcinoma and may benefit from a more strict endoscopic surveillance [Figure 1],[Figure 2] and [Figure 3].
|Figure 1: Normal esophagus with negative p53 expression (immunoperoxidase stain, ×100).|
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|Figure 2: A case of reflux esophagitis with mild to moderate positivity of p35 (immunostaining, ×200).|
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|Figure 3: A case of Barrett's esophagus with high positivity of p53 (immunostaining, �400).|
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| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]