aper
ABSTRACT
Background/Aims: The present study was aimed to investigate the correlation between selected clinicopathological characteristics and p53 protein overexpression, in Indian gastric cancer patients.
Methodology: Here, we studied specimens from 103 patients with gastric carcinomas and cases were categorized by AJCC-TNM classification. The p53 expression was analyzed in all the specimens by immunohistochemistry (IHC) and was correlated with the clinicopathological characteristics of the patients as well as etiological factors for gastric cancer. The cases were considered p53 overexpressed if more than 25% of the tumor cells were found
to be expressing this protein. Results: We found significant association of p53 overexpression with the gender (p = 0.004) and histology grades (p
= 0.001). However, p53 overexpression was not found to be significantly associated with other clinicopathological characteristics like age, cell differentiation, stage and location and various etiological factors. Conclusions: Our observation showed that IHC-based detection of altered expression of p53 protein in gastric carcinomas may play an important role for diagnosing the progression of gastric carcinoma, even in the early stages.
Key Words:
Gastric cancer;
p53; Immunohis- tochemistry.
Hepato-Gastroenterology 2013; 60:2113-2118 doi 10.5754/hge 12158
© H.G.E. Update Medical Publishing S.A., Athens
Clinicopathological Characteristics and Chronology of p53 Expression in the
Development of Gastric Cancer
Sajjad Karim1,3, Zeenat Mirza2, Muhammad Imran Naseer1, Mohammed Hussain Al-Qahtani1 and Arif Ali3
1
Center of Excellence in Genomic Medicine Research (CEGMR) King Abdulaziz University, Jeddah, Saudi Arabia
2
King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
3
Department of Biosciences, Jamia Millia Islamia, New Delhi, India
Corresponding author: Sajjad Karim, PhD, Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Post Box No: 80216, Jeddah -21589, Saudi Arabia;
Tel.: +966-6401000 ext 25487, Fax: +966-6952521; E-mail: [email protected]
INTRODUCTION
In recent decades, gastric cancer incidence has declined markedly in most of the Western world, but still remains the second most common cause of cancer related deaths worldwide (1). Its pattern and incidence vary widely between different parts of world. Costa Rica and Japan have the first and second highest rate in the world with a death rate of 77.7 and 50.5 per 100,000, respectively (1). However, there are large geographic variations in incidence, which may be related to environmental and genetic factors. Countries from South East Asia, including India reported lower rates of gastric cancer. With the establishment of the National Cancer Registry Programme in India, gastric cancer incidence is reported to be 5th in men and 6th in women (2).
Tumor development, growth and progression are characterized by uncontrolled cell proliferation.
Experimental data supports the hypothesis that tumors may originate from the accumulation of genetic defects in oncogenes and tumor suppressor genes (3). The wild type acts as tumor suppressor gene while the mutated form acts as a dominant oncogene. Wild type p53 have a role in preventing replication of damaged DNA (4). P53 is a protein involved in growth regulation.
In a model of multi-step carcinogenesis, mutations of the p53 tumor suppressor gene with functional abnormalities of the proteins they encode may be involved in the development of a variety of tumors (5). In human gastric cancer, however, various genetic changes accumulate leading to morphological changes (6). The abnormalities in the p53 tumor suppressor gene have been found in 5% to 70% of cases (7-8).Mutations occur most often in four of the five phylogenetically conserved
regions of the p53 gene that results into altered p53 product (7). Although wild-type p53, because of its short half-life, is normally present at very low level in the cell, changes in the conformation brought about by mutation apparently stabilize the mutant protein (9). Furthermore, the mutant and stable p53 protein accumulated in the nucleus is easily detected by immunohistochemistry (10). Experimental data suggest a chronology for the loss of p53 function and similarities with the carcinogenetic model of colorectal cancer (11). Therefore, in this study we investigated the p53 abnormalities and the role of p53 gene in the development of gastric cancer.
METHODOLOGY Patients
One hundred and three consecutive patient tissues with gastric cancer were provided by general surgeons from three distinct geographical regions of India during April 2002 to Feb 2007. Specimens were grossly examined and portion of the tumor and adjacent normal mucosa were excised, and fixed in neutral buffered formalin and embedded in paraffin for histopathology and immunohistochemistry. Demographic and pathological data like age, sex, tumor site and stage were obtained by consulting the original pathological reports. By re-examining the histological samples, gastric cancer were classified as being of the intestinal or diffused type according to Lauren system (12).
Immunohistochemical staining
Serial 4mm thick formalin-fixed paraffin-embedded tissue sections were used for p53 imuunostaining as described previously (10,13). These were deparaffinized
Age (Years) n (%)
Mean ± SD 55.68
<40 13 (12.62)
≥40 90 (87.37)
Gender
Male 78 (75.72)
Female 25 (24.48)
Cell differentiation
Well differentiated 11 (10.67) Moderately differentiated 39 (37.86) Poor differentiated 53 (51.45) Histology
Intestinal 45 (43.68)
Diffused 49 (47.57)
Unclassified/Mixed 9 (8.73) Tumor site
Cardiac 21 (20.38)
Fundus 16 (15.53)
Body 23 (22.33)
Antrum 43 (41.74)
Stage
I 32 (31.06)
II 38 (36.89)
III 27 (26.21)
IV 6 (5.82)
p53 IHC
Negative 66 (64.07)
Overexpression 37 (35.92)
TABLE 1. Clinicopathological characteristics of the 103 gastric cancer patients included in the study
(2 changes of xylene for 5 min and 1 change of acetone for 1 min each), followed by rehydration in decreasing concentration of alcohol (95% ethanol for 3 min, 70%
ethanol for 3 min, and distilled water for 1 min). For the immunostaining, antigen retrieval was done by heating the section immersed in citrate buffer (10 mmol/L)
inside a 600-W microwave oven in full power for 35 min. The sections were incubated with a 1:100 dilution of biotinylated goat anti-mouse immunoglobulin G (DAKO- LSAB kit, Peroxidase, M/s Dakopatts, Denmark) followed by 1:100 dilution of streptavidin-biotin- peroxidase complex at room temperature for 30 min each. The antibody was localized with substrate chromogen solution of 3,3’-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, USA). Tissue sections were counterstained with Harris haematoxylin, dehydrated with ethanol and mounted under a coverslip.
In each case, the entire section was systemically examined on high-power field (x400) microscope for p53 immunoreactivity. The level of immunoreactivity was expressed as the percentage of stained cancer cells (0% to 100%). Section with nuclear staining in less than 10% of the tumor cells were regarded as negative. Positive p53 nuclear staining was categorized into the 3 following patterns: (+), only a few scattered positively stained cells (10%–25% of all tumor cells);
(++), localized aggression of positively stained cells (more than 25% to 70% of all tumor cells); and (+++), diffused aggression of positively stained cells (more than 70% of all tumor cells). Overexpression of the p53 protein was defined when (++) or (+++) staining pattern were observed. Negative control sections were processed immunohistochemically without the primary antibody and positive control sections were from a breast cancer known to express high level of p53.
Statistical analysis
Statistical analysis was performed with the SPSS 16.0 softwarepackage (SPSS Inc., Chicago, USA) to correlate the results of p53 staining to the pathologic features of tumors. According to the cut-off values, the results of p53 analyses were used as dichotomized (categorical) variable. The χ2 test and Fisher’s exact test were used to test differences in categorical values for significance.
Student’s t-test was used to test continuous values. A p value of ≤0.05 was considered statistically significant, and all reported values represent two-sided tests.
RESULTS
Patients and tumor characteristics
Out of 128 collected cases, 25 had not shown any sign of gastric cancer in H&E staining thus excluded and rest were included for further study. Patients included, 78 men and 25 women, age ranging from 25 to 71 years (mean age of 56 years) (Table 1). From 103 selected cases, 37 were from anterior, 23 were from middle and 43 were from distal part of the stomach. Intestinal type gastric cancer was more common in older (>50 yr)patients (69.38%, 34 out of 49). In females, diffused
FIGURE 1.
Immunohis- tochemical staining of p53 protein in gas- tric cancer using monoclonal antibody. p53 protein expres- sion is seen primarily on cell nucleus in (a) (original magni- fication × 200) and (b) (original magnification
× 400).
S. No Sample P53 overexpressiona Cell differentiationb Histologyc Staged
1 C4 (++) M I II
2 C5 (++) P D II
3 C6 (+++) W I IB
4 C14 (+++) P D II
5 C22 (++) M I II
6 C27 (++) P D IV
7 C28 (++) P D IB
8 C34 (+++) P D IIIA
9 C35 (++) P D IIIA
10 C38 (++) M I IB
11 C39 (++) M I II
12 C40 (+++) P D IIIB
13 C43 (++) P D IIIA
14 C44 (++) M I IB
15 C45 (+++) P D II
16 C46 (+++) P D IIIB
17 C47 (++) W I IA
18 C48 (++) M I IB
19 C49 (++) M I II
20 C50 (++) P D IIIB
21 C53 (++) W I IA
22 C54 (+++) P D IIIB
23 C55 (++) M I IB
24 K2 (+++) P D IIIA
25 K7 (++) M I II
26 K8 (++) W I IB
27 K9 (++) W I IA
28 K11 (++) M I II
29 K15 (++) P D IIIB
30 K16 (++) W I IB
31 K18 (+++) P D IIIA
32 K19 (++) M I II
33 K20 (++) M I IB
34 D4 (++) W I IB
35 D7 (++) M I II
36 D12 (+++) M I II
37 D13 (+++) M I II
ap53 nuclear staining pattern: (-),<10% stained cells; (+), 10–25% stained cells; (++), 26–70% stained cells; and (+++) >70% stained cells; bcell dif- ferentiation: W: well, M: moderately, P: poorly; cHistological pattern, I: Intestinal, D: Diffused; dCancer stages according to AJCC.
TABLE 2. p53 overexpression in histologically positive gastric cancer patients.
p53 overexpression n p value Variables Negative Positive
Total number 66 37 103
Age (years) 0.254
<40 11 2 13
40–65 43 27 70
>65 12 8 20
Sex - - 0.004
Male 44 34 78
Female 22 3 25
Cell differentiation 0.077
Well 7 4 11
Moderately 24 15 39
Poorly 38 15 53
Histology 0.011
Intestinal 23 22 45
Diffused 34 15 49
Undiff/Mixed 9 - 9
Location 0.528
Cardiac 15 6 21
Fundus 11 5 16
Body 16 7 23
Antrum 24 19 43
Stage 0.469
I 19 13 32
II 25 13 38
III 17 10 27
IV 5 1 6
TABLE 3. Statistical comparison between clinicopathological factors and p53 expression in 103 Indian patients with resected gastric cancer.
type histology were predominant (60%, 15 out of 25) as compared to intestinal type (25%, 6 out of 25) whereas no such inclination were seen in males (Table 1).
Immunohistochemical staining of p53
The p53 nuclear staining analysis was performed on 103 selected patients under study and results were calculated as percentage of labeled nuclei in each case. A distinct nuclear, dark brown colored, immunoreactions for p53 was recorded as positive, the nuclear staining pattern was diffuse with little variation (Figure 1).
The staining of p53 was mainly nuclear, and p53 was occasionally observed in the cytoplasm. In the present series, the p53 overexpression (>25% of stained nuclei) was found in 37 cases (35.92%) and 66 cases (64.07%) were categorized p53 negative as shown in Table 2.
Correlation between p53 expression and clinicopathological characteristics
Clinicopathologic features of the registered patients are reported in Table 1 and correlation between p53 expression and clinicopathological variables are shown in Table 3. No significant correlations were found between p53 expression and the age, cellular differentiation, tumor location or stage. In contrast, there was a significant correlation between p53 status and the sex (p = 0.004). In present study, we did not
classify tumor according to WHO guidelines into tubular, papillary, mucinous, signet and unclassified type but preferred the Lauren’s classification because of its simple representation. We found a significant correlation of histological subtype (p = 0.011) with p53 overexpression in gastric cancer tissue. Purpose of the study was also to identify the association of diet, consumption of beverage, tobacco and alcohol use, literacy, occupational hazards, and the living environment with gastric cancer and p53 overexpression but we could not find any statistically significant correlation (Table 4).
DISCUSSION
Mutations of the p53 gene are the most common genetic alteration, known to occur in a wide range of human cancers (11). Under normal conditions wild type p53 protein is rapidly degraded and is therefore present only at very low levels within the cell. The acquisition of a mutant genotype is thought to increase the half-life of the mutant protein and this leads to its accumulation within the cell (14).The accumulated protein is detected by immunohistochemical techniques and proposed as an indirect method of screening tumors for mutation within the p53 gene. We tried to find out the correlation of p53 overexpression with clinicopathological as well as etiological factors.
In our study, the p53 staining was positive more often in intestinal type than in diffused type of gastric cancer (p
= 0.011). This is in consistent with two previous studies (15)but in conflicts with the other previous reports (16).
These differences might be explained by differences in patient’s materials or differences in interpretation of histological finding. We found P53 expression is more amplified in the poorly differentiated type (n = 15, 40.54%) than in the well differentiated type (n = 4, 10.81%) as reported by Isozaki et al. (17). Earlier studies suggest that a p53 impaired function represent an early event in the carcinogenetic process of gastric cancer and our available data of comparison between different stages of gastric cancer and p53 overexpression is in its accordance (18). These findings support the hypothesis that abnormalities of tumor suppressor genes may be involved in the pathogenesis of gastric cancer.
The cause of cancer of the stomach is unknown;
however, a series of study has shown the association of etiological and dietary factor with gastric cancer like diet, alcohol, pickled foods, dry fish, and poppy seeds, salty or fried foods, irregular meals, red meat and cigarette (19-22).Hukka smoking, consumption of Brassica vegetables, red chillies and salted tea have been reported to be associated with high incidence of stomach and esophageal cancer among Kashmiri population (23).Gajalakshmi et al. has shown country made alcohol and cigarette as independent risk factor for gastric cancer, alcohol is generally consumed in the form of country-made liquor (extracted from palm tree, 30–40% ethanol) whereas tobacco is consumed as bidi (a type of local cigarette made by wrapping 0.2–0.3 g of tobacco in dried temburni leaf (Diospyros melanoxylon)) or/and tobacco chewing among low socioeconomic strata in India (24).We could not find any such risk association with alcohol or tobacco consumption.
In the present study, these established etiological risk factors did not emerged as significantly associated with p53 overexpression in stomach cancer. We found a very high association between non-vegetarian diet and gastric cancer; 95.14% of non-vegetarians and 4.86% of pure vegetarians, who had never taken a meat product in their diet, had gastric cancer. Consumption of non-vegetarian food and its association with gastric cancer in India has not been reported previously. The risk of gastric cancer is dramatically decreased in populations whose diet includes a high intake of fruits and vegetables, which may be partly attributable to the consumption of antioxidant micronutrients. Our finding
Variables p53 overexpression n p value Negative Positive
Diet 0.086
Vegetarian 5 – 5
Non-vegetarian 61 37 98
Salted tea 0.297
Yes 32 14 46
No 34 23 57
Dry fish 0.49
Yes 31 20 51
No 35 17 52
Poppy 0.703
Yes 26 16 42
No 40 21 51
Fried meat/rice 0.882
Yes 24 14 38
No 42 23 65
Pickle 0.755
Yes 36 19 55
No 30 18 48
Tobacco chewing 0.128
Yes 7 8 15
No 59 29 88
Smoking 0.465
Yes 22 15 37
No 44 22 66
Alcohol 0.211
Yes 11 10 21
No 55 27 82
Education 0.112
Literate 41 17 58
Illiterate 25 20 45
Blood Group 0.482
O 10 8 18
A 32 13 45
B 15 8 23
AB 9 8 17
Geography 0.823
West Bengal 37 23 60
Jammu & Kashmir 20 10 30
New Delhi 9 4 13
Area 0.614
Urban 46 24 70
Rural 13 20 33
of prevalence of blood group A (n = 45, 43.68%) among gastric cancer patient is in accordance with researchers who studied correlation between ABO blood type and gastric cancer (25).More cases were reported from the urban population (n = 70, 67.96%) than the rural population (n = 33, 32.04%). India has many distinct geographical areas and the dietary nature varies drastically from region to region. Frequent intake of dry fish has been conclusively shown to be a high risk factor for stomach cancer in West Bengal (26), and our present findings in West Bengal are in agreement with this. Our findings add to the evidence that diet plays some role in stomach cancer risk and suggest the need for further evaluation of risks associated with carbohydrates and starchy foods as well as the mechanisms involved.
In conclusion, the present study was able to demonstrate the significant association of p53 immunoreactivity in gastric cancer with Lauren subtype and gender. The phenomenon of p53 overexpression was correlated with the tumor progression like differentiation, stage, grade, etc. and our findings support the hypothesis that abnormalities of tumor suppressor genes may be involved in the pathogenesis of gastric cancer. We found IHC analysis as easy and rapid method for evaluation of p53 protein expression that can potentially be used as biomarker for simple and early diagnosis of gastric cancer. We could not get significant association of dietary factor with gastric cancer as reported by many researchers; therefore, we suggest evaluating association at bigger data set.
TABLE 4. Statistical comparison of p53 overexpression with etiological factor of gastric cancer.
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ACKNOWLEDGMENTS
We would like to acknowledge the Department of Bioscience, Jamia Millia Islamia, New Delhi (India);
Center of Excellence in Genomic Medicine Research and Deanship of Scientific Research, King Abdulaziz University, Jeddah (KSA) for providing necessary support and research facilities. We would like to thank Dr. Sahni P, Dr. Roy A, Dr. Zargar SA and Dr. Saxena S for
providing surgical specimens and clinicopathological facilities. We also thank Dr. Begum S, Dr. Alam MT, Dr.
Asad S, Alam R, and Barha G for statistical analysis and technical support. We are also thankful to King Abdullah City for Science and Technology, Saudi Arabia (Strategic Grant No. 10-BIO1073- 03 and 10-BIO1258-03), for funding and partial support.
