Diseases of the Esophagus (2009) 22, 588–595 DOI: 10.1111/j.1442-2050.2009.00953.x
Original article
P53 and Ki-67 overexpression in gastroesophageal reflux disease – Barrett’s esophagus and adenocarcinoma sequence des_953
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M. Binato,2,3,6 R. R. Gurski,1,3,5 R. B. Fagundes,2,4,5,6 L. Meurer,3,5 M. I. Edelweiss3,5 Federal University of Rio Grande do Sul, 2Federal University of Santa Maria, 3Postgraduate Course in Surgery – Famed-UFRGS, 4Postgraduate Course in Gastro – Famed-UFRGS, 5Hospital de Clinicas de Porto Alegre, 6Hospital Universitário de Santa Maria (HUSM), Cirurgia Geral e Serviço de Gastroenterologia, Rua Professor José Mariano da Rocha Filho, Bairro Camobi, Santa Maria, Rio Grande do Sul, Brazil 1
SUMMARY. Gastroesophageal reflux disease (GERD) is a major risk factor for the development of esophageal adenocarcinoma (ACE). Many molecular alterations occur in esophageal carcinogenesis, yet the exact mechanism of ACE development remains unknown. This study aims to determine p53 protein and Ki-67 expression in esophageal mucosa of patients with GERD and study the correlation between these markers and the progression from normal squamous epithelium to esophagitis, columnar epithelium with or without intestinal metaplasia and ACE. We analyzed p53 protein and Ki-67 expression in biopsies of 200 patients with GERD and 35 patients with ACE. Those biopsies were classified into five groups: (i) G1 normal squamous epithelium (58); (ii) G2 esophagitis (80); (iii) G3 columnar epitheliums without intestinal metaplasia (30); (iv) G4, columnar epitheliums with intestinal metaplasia (32); and (v) G5 ACEs (35). p53 protein overexpression was found in 7% (4) of G1, 37.5% (30) of G2, 30% (9) of G3, 62.5% (20) of G4, and 71.4% (25) of G5 (p < 0.001). Ki-67 index increased according to the severity of histopathological diagnoses. Ki67 index was 21.3 ⫾ 19.5% in G1, 38.8 ⫾ 24.9% in G2, 37.7 ⫾ 26.3% in G3, 52.8 ⫾ 24.6% in G4, and 57.1 ⫾ 25.1% in G5 (P < 0.001). Linear correlation between p53/Ki67 expression and the multistep progression from squamous epithelium to ACE was observed (P < 0.001 and P < 0.05). Our results indicate that overexpression of p53 and increased Ki-67 could be associated with the development and progression to ACE in patients with GERD. KEY WORDS: adenocarcinoma, Barrett’s esophagus, gastroesophageal reflux disease, Ki-67 (MIB-1), p53.
INTRODUCTION Gastroesophageal reflux disease (GERD) is one of the most frequent clinical conditions observed in medical practice, affecting more than 30% of the adult population.1,2 A population-based study estimated that approximately 12% of the Brazilian population has GERD.3 Esophageal mucosa in patients with GERD has a wide range of endoscopic findings varying from esophageal mucosa with no lesions (non-erosive reflux disease), erosive disease, Barrett’s esophagus, stenosis, to carcinoma.4,5 Experimental studies and clinical essays support the theory that the columnar mucosa in the distal esophagus is a consequence of the squamous epithelium exposure to gastroduodenal content.6,7 The Address correspondence to: Dr Marcelo Binato, MD, PhD, Rua Francisco Manoel N° 01/AP 202 CEP 97015-260, Santa Maria, Rio Grande do Sul, Brasil. Email:
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development of intestinal metaplasia characterizes Barrett’s epithelium, which is a risk factor for adenocarcinoma (ACE).3 It is estimated that 6 to 12% of patients with gastroesophageal reflux symptoms submitted to endoscopy have Barrett’s esophagus.8–10 Approximately 64 to 86% of esophageal ACEs arise from columnar epithelium with intestinal metaplasia, indicating that Barrett’s esophagus is by far the most important risk factor for esophageal ACE.11–13 Patients with Barrett’s esophagus are at a 0.5 to 2% increased annual risk of developing esophageal ACE, which is 30 to 125 times greater than the general population.14–17 Because of this risk, early identification of malignant changes is of utmost importance.18–25 Associated to the Tp53 gene malfunction, an increase in cell proliferation may precede dysplastic changes in Barrett’s esophagus.26–39 The interactions of p53 protein expression with the increase in Ki-67 index in esophageal carcinogenesis are still being studied.40
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We examined prospectively and consecutively patients aged 30 years or older who were referred to the Digestive Endoscopy Unit between January 2003 and December 2006 with symptoms of GERD (pyrosis and/or regurgitation) at least once a week for a minimum of 5 years, and patients with esophageal ACE. We excluded patients with cardiac diseases or coagulation disorders, use of Nonsteroidal antiinflamatory drugs (NSAIDs), gastroesophageal varices, hepatopathy, recent upper gastrointestinal hemorrhage, previous radiotherapy or chemotherapy treatment, hypersensitiveness to iodine, use of acid suppressor therapy over the last 60 days, and those who had prior esophageal or stomach surgery. The research was approved by Internal Board Review of, Hospital de Clínicas de Porto Alegre). Patients were required to sign an informed consent.
equate samples; normal squamous epithelium; chronic esophagitis (mild, moderate, or severe); columnar epithelium without intestinal metaplasia; columnar epithelium with intestinal metaplasia; or ACE. Samples were adequate when two well-oriented intact crypts with or without lamina propria were observed. Chronic esophagitis was diagnosed when one or more of the following criteria was observed: (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.42 The columnar epithelium in the distal esophagus was classified according to the absence or presence of caliciform cells after staining with Alcian blue, pH 2.5 in: (i) columnar epithelium without intestinal metaplasia; and (ii) columnar epithelium with intestinal metaplasia. ACE was defined by the presence of atypical glands with significant loss of the normal structure, nuclear atypia, loss of normal cell polarity, and abnormal tissue maturation invading or not the submucosal layer.43 Two independent pathologists with experience in gastrointestinal pathology examined the slides, and a diagnosis was reached when they agreed. In case of disagreement, the final result was obtained by a consensus as described by Weston et al.44 Their agreement rate was measured with the kappa test.
Upper gastrointestinal endoscopy
Immunohistochemical analysis
We used a video–endoscope (Fujinon 2200, Tokyo, Japan). After a complete examination of esophagus, stomach, and duodenum, we placed the tip of the endoscope at 30 cm from the front teeth and sprayed the esophageal mucosa with Lugol solution 3% (12 g I + 24 g KCl in 1000 mL/water), with the purpose of highlighting the Z line and esophageal mucosal lesions. After Lugol spraying, we performed the esophageal biopsies as follows. At least two samples were taken 2 cm above the Z line from normal stained mucosa, at the erosion site or unstained area, and at intervals of 1 to 2 cm in the four quadrants if the columnar mucosa was visible above the gastroesophageal junction according to Seattle protocol.41 The specimens were placed on filter paper, fixed in 10% formalin solution, and embedded in paraffin.
Biopsy specimens were evaluated by immunohistochemistry using monoclonal antibodies for p53 DO-7 (DAKO code M7001 mouse anti-p53) and Ki-67 nuclear antigen (DAKO clone MIB-1). The DO-7 antibody detects wild-type and mutant forms of p53 protein. Immunohistochemical analysis was performed with the avidin–biotin–peroxidase complex method. In brief, after de-waxing, endogenous peroxidase activity was inactivated and the specimen was blocked with normal serum. Then, the sections were incubated overnight at 4C with a 1 : 700 dilution of DO-7 antibody and a 1 : 400 dilution of the Ki-67 antibody. The streptavidine–biotin complex was used to stain for both antigens, whereas the diaminobenzidine–tetrahydrochloride was used as chromogen. As a negative control, a slide was similarly processed, except without the inclusion of the primary antibodies. Positive controls for p53 and Ki-67 consisted breast cancer and cecal appendix specimens, respectively. The parameter used to define p53 protein expression was the presence of staining in the nuclear
The identification of tumor markers in patients with GERD may allow defined subgroups of patients more prone to develop dysplasia and/or cancer. We studied the immunoexpression of the p53 protein and the Ki-67 (MIB-1) proliferation index in biopsies obtained from the esophageal mucosa of individuals with GERD who presented at endoscopy examination normal mucosa, mucosa with esophagitis, columnar mucosa with and without intestinal metaplasia, and esophageal ACE.
MATERIALS AND METHODS Study population
Histological diagnostic criteria For the histological examinations, all sections were stained with hematoxylin and eosin. The histological findings were classified as follows: adequate or inad-
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region, as shown by Wang et al.45 Positive cases were considered to be those with more than 10% stained cells in the microscopic field at high magnification. Ki-67 (MIB-1) was considered positive with the staining of the cells’ nuclei. Ki67 index was calculated for each specimen by counting the number of reactive cells per a minimum count of 500 cells, as previously described by Feith et al.46 Immunohistochemical slides were analyzed by two independent observers who were blinded to the histopathological diagnosis.
Statistical analysis Statistical analysis was conducted with the Statistical Package for Social Science (SSPS) version 12.0 (SPSS Inc., Chicago, IL USA). Statistical differences were analyzed using the c2 test for linear trends for the p53 results, and the analysis of variance and Tukey tests were used to analyze the Ki-67 results (mean and standard deviations). Linear correlations of both variables with their respective groups were analyzed by Kendall and Pearson correlation coefficients. Significant differences were considered when P < 0.05. Kappa test was used to control interobserver agreement.
RESULTS Histological analysis of specimens from the 235 patients showed that 58 (24.6%) had normal squamous epithelium, 80 (34%) had esophagitis, 30 (12.7%) had columnar epithelium without intestinal metaplasia, 32 (13.6%) had columnar epithelium with intestinal metaplasia, and 35 (14.8%) had ACE. Kappa test was 0.9 for the histological diagnosis. The mean patient age was 55 ⫾ 14.5 years. The mean age was higher in patients with ACE compared with the other groups (P < 0.05), except for patients with metaplastic columnar mucosa. Compared with the other groups, most patients with normal squamous epithelium were female (P < 0.05). p53 Immunoexpression was positive in 88/235 individuals (37.4%). p53 Protein expression was found in 4 (7%) patients with normal epithelium, in 30 (37.5%) with esophagitis, in 9 (30%) with columnar epithelium without intestinal metaplasia, in 20 (62.5%) with columnar epithelium with intestinal metaplasia, and in 25 (71.4%) patients with ACE (Fig. 1). The agreement rate between the two pathologists in analysis of immunohistochemical slides was 0.6 (kappa). p53-Positive immunoexpression showed a statistically significant difference between all groups in relation to the normal epithelium group (Fig. 2). The esophagitis group also differed from the ACE group. Differences were also found between the group with
columnar epithelium without intestinal metaplasia and the ACE group (P < 0.01). We observed a positive linear correlation between the p53 expression and the severity of multistep stage of histological diagnosis (normal epithelium, esophagitis, columnar without intestinal metaplasia, columnar with intestinal metaplasia, and ACE; P < 0.001) (Fig. 3). Ki-67 (MIB-1) index showed mean stained cells of 39 ⫾ 26.7%. The mean was 21.3 ⫾ 19.5% in patients with normal squamous epithelium, 38.8 ⫾ 24.9% in patients with esophagitis, 37.7 ⫾ 26.3% in patients with columnar epithelium without intestinal metaplasia, 52.8 ⫾ 24.6% in patients with columnar epithelium with intestinal metaplasia, and 57.1 ⫾ 25.1% in patients with ACE (Fig. 4). There was a significant difference in the Ki-67 index between the normal squamous epithelium group and the other groups (P < 0.001). Esophagitis group differed from all other groups except from the columnar epithelium without intestinal metaplasia group (P = 1.000). There was a significant difference in the Ki-67 index between patients with ACE and those with columnar epithelium without intestinal metaplasia (P < 0.05) (Fig. 5). A positive linear correlation was identified between the increase of Ki-67 staining and the progression from normal epithelium to esophagitis, columnar without intestinal metaplasia, columnar with intestinal metaplasia, and finally to ACE (P < 0.05) (Fig. 6).
DISCUSSION GERD constitutes one of the most important and specific risk factors for carcinogenesis in the esophagus, particularly esophageal ACE.47–49 The current strategy to anticipate progression to ACE of the esophagus is endoscopic surveillance of patients with Barrett’s esophagus to detect dysplastic alterations.8,20 The medical literature suggests dysplasia as the better marker for cancer risk, particularly that related to Barrett’s esophagus. For many reasons, such as intra- and interobserver divergence in the interpretation of dysplastic alterations and the low predictive value of dysplasia evolution (progression, persistency, or regression), it is an imperfect marker.26,50 Consequently, the search for new biological markers and a better understanding of the pathogenesis of GERD can help identify patients at increased risk of malignant transformation. The present study was directed toward analyzing p53 protein expression and the Ki-67 (MIB-1) proliferation index in the multistep sequence from squamous mucosa to esophagitis, columnar epithelium without intestinal metaplasia, columnar epithelium with intestinal metaplasia, and ACE.
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Fig. 1 Photomicrograph of p53 protein expression determined by immunohistochemistry: (A) esophagitis (27 ¥ 20 mm [300 ¥ 300 DPI]); (B) columnar epithelium without intestinal metaplasia (27 ¥ 20 mm [300 ¥ 300 DPI]); (C) arrows, columnar epithelium with intestinal metaplasia (27 ¥ 20 mm [300 ¥ 300 DPI]); and (D) adenocarcinoma (91 ¥ 70 mm [150 ¥ 150 DPI]).
Immunohistochemistry has been used in the investigation of various genes, including the study of the Tp53 tumor suppressor gene and the Ki-67 nuclear antigen in gastrointestinal carcinogenesis.21,51–53
Under normal conditions, the Tp53 gene generates a short-lived protein (wild p53) that cannot be detected by immunohistochemistry. In cases with mutations, the altered Tp53 gene produces a more stable protein
Fig. 2 Sequence of increased p53 activity in normal epithelium, esophagitis, columnar without and with intestinal metaplasia (IM), and adenocarcinoma (ACE): P < 0.001 for group 1 (G1) compared with G2, G3, G4, and G5; P < 0.05 for G2 compared with G5; P < 0.01 for G3 compared with G5. 366 ¥ 204 mm (96 ¥ 96 DPI). © 2009 Copyright the Authors Journal compilation © 2009, Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus
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Fig. 3 Correlation between p53 protein expression and the progression of normal epithelium to esophagitis, columnar without and with intestinal metaplasia (IM), and adenocarcinoma (ACE). Kendall coefficient = 0.4.
that extends the protein’s half life by 6 h, and the intra-nuclear accumulation makes it possible to detect p53 by immunohistochemistry.20,25,44,51 However, the use of immunohistochemistry should be carefully considered, as the absence of staining
Fig. 5 Ki-67 activity in the normal epithelium- esophagitis – columnar epithelium without and with IM – adenocarcinoma sequence: « P < 0.001 G1 between G2, G3, G4 and G5; ® P < 0.05 G2 between G4 and G5; ¥ P < 0.05 between G3 and G5.
does not necessarily exclude the existence of mutations.22,26 We identified p53 protein expression associated to the severity of the histological findings, that is, the immunoexpression of p53 occur in ascendant fashion
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Fig. 4 Photomicrograph of Ki-67 (MIB-1) expression determined by immunohistochemistry: (A) esophagitis (27 ¥ 20 mm [300 ¥ 300 DPI]); (B) columnar epithelium without intestinal metaplasia (27 ¥ 20 mm [300 ¥ 300 DPI]); (C) columnar epithelium with intestinal metaplasia; and (D) adenocarcinoma (169 ¥ 127 mm [96 ¥ 96 DPI]). © 2009 Copyright the Authors Journal compilation © 2009, Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus
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Fig. 6 Correlation between Ki-67 and evolution normal epithelium – esophagitis – columnar epithelium without and with IM – adenocarcinoma. Pearson’s coefficient = 0.4.
from normal squamous epithelium to ACE passing through esophagitis, columnar epithelium without intestinal metaplasia, and columnar epithelium with intestinal metaplasia. A linear correlation of increasing positive p53 protein expression is supported by large number of patient samples studied and the high index of statistical significance. The outstanding data that p53 protein expression significantly increased after the occurrence of intestinal metaplasia, could point out to the progression from metaplasia to ACE of the esophagus. Segal et al.54 found that p53 protein was expressed in Barrett’s epithelium in 60.9% of the patients and 39.1% in those with cardia intestinal metaplasia. Although the authors pointed to limitations in the immunohistochemical methods for p53 immunoreactivity, they concluded that the immunohistochemical expression correlated very well with Tp53 mutations detected by protein chain reaction. Our study found 7% (four) positive reactivity for p53 in histologically normal patients. The distribution of the immunoreactivity in this group was restricted to the basal layer of the squamous epithelium, and in all cases had a faded color when examined under an optical microscope. In a similar way, Krishnadath et al.19 studied the expression of the p53 protein (DO-7) by immunohistochemistry in normal mucosa and found a faded nuclear color in the basal layer in 24 samples of esophageal mucosa used as control group. Positive p53 staining in the nonesophagitis squamous group may occur because of rapidly dividing cells and inactivation of wild-type p53 through binding another protein, which prolongs its half-life55. Moreover, differences in the detection rate with various immunohistochemical antibodies and between immunohistochemical staining have been described21. This study also showed positive p53 protein reactivity that was similar between groups with esophagitis and columnar mucosa without intestinal metaplasia. Gastroesophageal reflux causes inflammation and may explain the positive reactivity
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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 p53.51,55 Casson et al.56 identified p53 overexpression in 50% of patients with intestinal metaplasia without dysplasia, and Krishnadath et al.19 found that 71.4% of ACE patients had modified p53 expression. Our results showing a 62.5% (25) positive reactivity of p53 in patients with columnar epithelium with intestinal metaplasia and 71.4% among patients with ACE corroborate these results. In addition data, we studied the other the proposed steps of esophageal carcinogenesis, esophagitis, and columnar epithelium without intestinal metaplasia. Clearly our study has the limitations of immunohistochemistry method. Our results could be underestimated if we did not identified p53 immunoreactivity because of variations in the immunohistochemical methods or the interpretation of the nuclear color intensity, as reported by Rice et al.57 On the other hand, we might overestimating our findings because most of the patients had GERD and therefore the esophageal mucosa was under the aggression of the gastroduodenal reflux and as different degrees of inflammatory response, but in this case the p53 expression was restricted to the basal layer. The proliferation index (Ki-67) was progressively higher with the severity of the histological alterations. The mean Ki-67 index in abnormal epithelium (inflammatory and malignant) was higher than that in normal epithelium (P < 0.001). The comparison of the nuclear expression of Ki-67 showed a significant difference (P < 0.01) between normal squamous mucosa, esophagitis, and columnar epithelium with intestinal metaplasia. Additionally, high cell proliferation indexes were observed, particularly when intestinal metaplasia was present, indicating that this proliferative activity was also related to the intestinalization of the columnar mucosa in the distal esophagus. In this study, we demonstrated that molecular alterations measured as increased p53 protein expression were progressively greater when histological alterations caused by GERD grew progressively worse. Likewise, proliferative alterations measured by the Ki-67 expression exhibited the same pattern. The progressive expression of both markers presented a positive correlation with the histological severity. It suggests that the mechanisms that increase proliferative activity in the esophageal mucosa produce the molecular alterations that lead to cancer. Prospective studies including longitudinal follow-ups are required to determine if both immunohistochemical markers can identify those patients who may be at a higher risk of developing ACE and may benefit from more strict endoscopic surveillance.
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