Introduction

Current guidelines recommended that patients with clinical stage II/III gastric adenocarcinoma should be treated with subtotal or total gastrectomy followed by adjuvant chemoradiotherapy. After R0 resection, patients with T1-2, N0 tumors may be observed and fluoropyrimidine-based adjuvant chemoradiotherapy is recommended in some of the selected patients with T2, N0 tumors with high-risk features such as poorly differentiated, lymphovascular and neural invasion [1].

The stage is the most important prognostic factor in gastric cancer. However, gastric cancer is not a uniform disease, and therefore, prognosis varies even among patients in the same stage. For this reason, in order to better identify the biological subset, more classification parameters need to be defined in addition to TNM and classic histopathological characteristics. The expressions of epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR) and cErbB-2 (Her-2/Neu) have been found associated with poor prognosis in patients with gastric cancer [2, 3]. On the contrary, the absence of p21 expression has been associated with poor prognosis in patients with gastric cancers [4–6].

We aimed to investigate the expression and mutation/polymorphism of p21 and Her-2 proteins and also relationship between that genes status and histopathological factors and prognosis in patients with pathologically staged T3-4, N0-3(+) gastric adenocarcinoma after subtotal or total gastrectomy, with the goal of identifying factors related to predicting outcome.

Materials and methods

Patient selection

For this retrospective analysis, subjects were selected from 382 patients with gastric cancer treated at the Trakya University Medical Faculty Hospital in Edirne, Turkey, from January 2000 to December 2008. For the present analysis, the patient selection criteria were as the following: (1) without any preoperative therapy; (2) undergone total or subtotal gastrectomy; (3) pathologically confirmed T3-4, N0-3(+) gastric adenocarcinoma; (4) no evidence of distant metastases. After the medical and pathologic records were carefully reviewed, 44 patients with locally advanced gastric adenocarcinoma were selected.

Immunohistochemical staining for biomarkers

All information on biomarker staining was extracted from the pathologic records. All dissected tumor specimens were routinely fixed in buffered formalin and embedded in paraffin. Sections of 5-mm thickness were stained with hematoxylin and eosin for histologic diagnosis and then labeled with two primary antibodies: anti-P21 (Clone NCC-RAS-001, code M0367, DAKO; dilution 1:100) and anti-Her 2 (Clone: PN2A, code M7269, DAKO; dilution 1:400). For antigen retrieval, sections were treated with 10 mM citrate at pH 6.0 in a 750-W microwave oven for three 5-min cycles. Then, the sections were immunostained with horseradish peroxidase (HRP) polymer (DAKO) in accordance with manufacturer’s specifications. Diaminobenzidine was used to develop the stains and hematoxylin for the counterstaining. Negative controls consisted of substituting normal mouse serum for the primary antibodies. Expression of two markers’ was quantified by using a visual grading system based on staining intensity on a scale from 0 to 3.

DNA extraction and polymerase chain reaction (PCR–RFLP and PCR-SSCP) analysis

Genomic DNA was isolated from paraffin-embedded tissues using the High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland) in compliance with the manufacturer’s protocol. All PCR amplifications were performed in a total volume of 50 μL containing 100 ng of extracted DNA, 20 pmol/L of each forward and reverse primer, and 25 μL of HotStarTaq Master Mix [containing 2.5 units of HotStarTaq DNA polymerase, 1× PCR buffer with 1.5 mmol/L MgCl2, and 200 μmol/L of each dNTP (Qiagen, Hilden-Germany)].

SNP in Her-2 transmembrane segment involving codon 655 was analyzed by PCR–RFLP. The 148-bp transmembrane segment of Her-2 was PCR-amplified using the gene-specific primers, that is, forward (Her-2/F) 5′-AGAGCGC-CAGCCCTCTGACGTCCAT-3′, and reverse (Her-2/R) 5′-TCCGTTTCCTGCAGCAGTCTCCGCA-3′. Amplification was performed with initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C, annealing at 62 °C, and extension at 72 °C for 1 min each and a final extension at 72 °C for 10 min. The PCR product was digested with 5 units of BsmAI restriction enzyme (New England Biolabs, Ipswich, MA-USA) at 55 °C for 2 h and run on 3 % agarose gel, stained with Et-Br and visualized under UV light. BsmAI digestion gave fragments of 116 and 32 bp for the Val (GTC) allele and a single 148-bp fragment for the IIe (ATC) allele.

SSCP analysis was performed by PCR amplification of exon 3 of the p21 gene. Exon 3 was amplified with oligonucleotides 5′-GCCCCCCACTGTCTTCCT and 5′-GCGCTTCCAGGACTGCAGG. Amplification was consisted in 10 min, denaturation step at 95 °C followed by 37 cycles of 30 s at 95 °C, 30 s at 62 °C, and 30 s at 72 °C. Totally, 20 μl solution [containing 10 μl of each PCR product was diluted in 10 μl of denaturing loading buffer (98 % formamide; 0.05 % bromphenol blue; and 0.05 % xylene cyanol FF)] was electrophoresed on 8 % non-denaturing polyacrylamide gels. Gels were run in 1× Tris–borate-EDTA (TBE), respectively, at 200 V for 7–15 h. After electrophoresis, silver staining was performed by a staining kit (Promega, USA).

All SSCP analyses were repeated twice each under electrophoresis conditions. Negative assay controls consisted of water and extraction buffer blanks for PCR.

Definition of treatment failure

All treatment failures were verified through review of the medical records. Local-regional failure was defined as recurrence within pelvis, including the tumor bed, regional lymph nodes and anastomosis. Distant failure was indicated as disease recurrence detected in the liver, lung, brain and other organs.

Statistical analyses

The data used for this analysis included patient age and sex, cellular differentiation, the presence or absence of lymphatic, vascular or perineural invasion, the number of dissected lymph nodes, and whether adjuvant therapy had been administered. Two potential predictive molecular markers (p21 and Her-2), detected by immunohistochemical staining polymerase chain reaction of surgical specimens, were also evaluated as potential prognostic factors. Characteristics were described in terms of frequency for the categorical variables, means and standard deviations for continuous data, and medians for non-normally distributed continuous data. Survival time was calculated from the date of surgery to the date of event or the last follow-up.

Events were defined: local-regional failure, distant metastasis and death. Survival curves were estimated by using the Kaplan–Meier method and compared with log-rank test in the univariate analysis. Cox proportional hazards regression was used for multivariate modeling and for examining the prognostic significance of the variables identified in the models. p values of less than 0.05 were taken to indicate statistically significant differences.

Results

Patient characteristics

Patient characteristics are presented in Table 1. Thirty-four patients were men and 10 patients were women; the median age was 63 years (range, 26–75). Nineteen patients (43.2 %) were diffuse histology and sixteen patients (36.4 %) were intestinal histology. Thirty-six patients (81.6 %) were T3, and in thirty patients (68.2 %), lymph node involvement was positive. Surgical margin was positive in six patients (13.6 %).

Table 1 Patient characteristics
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Thirty-four patients (77.3 %) were treated with chemoradiotherapy, and 10 patients (14.7 %) were treated with only chemotherapy or radiotherapy. Main chemotherapy regimen was 5-FU plus folinic acid.

The results of immunohistochemical staining and polymerase chain reaction

Her-2 expression was negative in forty patients (90.9 %), and p21 expression was negative in seventeen patients (38.6 %). II type Her-2 gene polymorphism was positive in only one patient (2.3 %). p21 gene mutation was positive in 9 (20.5 %) patients (Table 2).

Table 2 Immunohistochemical staining and polymerase chain reaction results
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Correlation of molecular markers and clinicopathological factors

For all clinicopathological factors, there was statistically significant correlation between negative p21 expression and diffuse-type histology, whole gastric invasion, vascular/neural invasion and undifferentiated histology. There was significant correlation between p21 gene mutation and diffuse type, and undifferentiated histology (Table 3). We could not find any correlation between any clinicopathological factors and Her-2 polymorphism/expression.

Table 3 Correlation between p21 expression/mutation and clinicopathological factors
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Molecular markers and survival

The median follow-up interval was 23.23 months (range, 3.83–83.20 months). Univariate analysis showed that total gastrectomy, whole gastric invasion, positive vascular and neural invasion, negative p21 expression and recurrent disease were negative effects on overall survival (p = 0.012, 0.026, 0.047, 0.005, 0.038 and 0.001, respectively). Cox multivariate analysis revealed only two factors to be associated with overall survival: negative p21 expression and recurrent disease (p = 0.01 and 0.002). The median survival rate of patients with negative expression was significantly poorer than that of patients with positive expression of p21 (17 vs. 27 months, p = 0.01, cox regression).

The 3-year disease-free survival (DFS) and overall survival rates for all patients were 18.8 and 30.45 %. The median overall survival was 26.52 months (range, 5.23–83.60 months), and median DFS was 19.45 months (range, 1.17–83.60 months). During the follow-up period, 4 patients developed recurrent disease and fourteen patients developed with distant metastasis.

We could not find any relation between DFS and any parameter.

Discussion

The function of p21 protein was investigated in cell cultures, and it was showed that it provides DNA repair or apoptosis of cell by stopping at the G1 phase checkpoint in irradiated cells [7–9]. In addition, direct inhibition of cell proliferation in tumor cells and healthy fibroblasts was showed in different studies [10, 11]. In these studies, this effect of p21 protein has been advocated tumor suppressor p53 gene-dependent effect. In two different studies, p53-independent effect of p21 protein was investigated in cell culture and inducing effect of p21 gene was showed after DNA damage in cases of suppressed p53 gene [12, 13].

In the healthy gastric mucosa while epithelial cells are progressing in the cell cycle, epithelial cells are going to the surface of gastric mucous and at the end of this period epithelial cells have been acquired much better differentiation, which cover the surface of the stomach. These cells in the stomach mucosa which are covering the surface have been shown p21 positivity, and as a result of this, it has been reported that p21 positive cells are well differentiated [14]. Despite being positive staining in all normal stomach tissue samples, Xie et al. [15] detected positive p21 staining rate only 39.8 % in gastric cancer tissue samples, and it was significantly higher in patients with well-differentiated histology. Ogawa et al. [16] reported a statistically significant relationship between p21 expression and the well-differentiated histology. In our study, p21 expression was significantly higher in patients with well-differentiated histology.

Statistically significant relationship between p21 expression and histopathological parameters, such as tumor invasion, lymph node metastasis, lymphatic and venous invasion, has been reported in different studies [17, 18]. Ogawa et al. [16] reported a statistically significant reduction on the expression of p21 in patients with undifferentiated histology, deep tumor invasion, presence of venous and lymphatic invasion. Similarly, Kouraklis et al. [6] reported statistically significant reduction in the expression of p21 in patients with depth of tumor invasion, presence of venous and lymphatic invasion, and liver metastases. In our study, diffuse and undifferentiated histology, whole gastric involvement, the presence of vascular and neural invasion were found significantly higher in patients with negative expression of p21.

Ogawa et al. [16] reported significantly lower recurrence rate in patients with positive p21 expression, and they reported a longer overall survival in these patients. Gomyo et al. [19] reported significantly better overall survival rates in patients with the presence of p21 expression. Kouraklis et al. [6] reported a higher overall survival in patients with positive p21 expression. In contrast to these, Müller et al. [20] failed to demonstrate any relationship between the p21 expression and overall survival. In our study, there was no relation between p21 expression and recurrence, but there was statistically improved overall survival in the presence of p21 expression.

In previous studies which are investigating the role of p21 in gastric cancer, it has been focused on especially p21 expression. There is only one study which investigates the role of p21 gene mutation in gastric cancer in the literature. In this study, Park et al. [21] investigated the presence of p21 gene mutation in gastric cancer tissue samples of 20 patients, but they failed to detect any mutation on exon 2 and exon 3. In our study, we studied p21 mutation on exon 3, and p21 gene mutation was identified in 9 (20.5 %) patients. In addition, p21 gene mutation was significantly higher in diffuse-type and undifferentiated patients. Our results which show this relation are similar with result of p21 expression studies.

Her-2 overexpression rate is 8–28 % in gastric cancer studies. Chariyalertsak et al. [22] reported Her-2 overexpression rate is in patient with early stage, advanced stage, recurrent and inoperable cases, respectively, 6.9, 15.9 and 28.6 %. In our study, Her-2 expression rate was 9.1 %.

The results of studies which are investigating the relationship between prognostic factors and Her-2 expression in gastric cancer patients show differences. There are some studies that show significantly relation between Her-2 expression and papillary histology, liver metastases, well differentiation, serosa invasion, lymph node metastasis and advanced stage according to the depth of invasion [23–25]. In addition, in our study, there was no significant correlation between Her-2 expression and histopathological parameters, and there was no significant relationship detected between Her-2 expression and overall survival, and DFS.

Satiroglu-Tufan et al. [26] reported Her-2 gene polymorphism rate in patients with gastric cancer (IIe/Val) is 20 %, and they reported significant relation with stage IV disease. In our study, Her-2 gene polymorphism was detected, VV phenotype, in only 1 patient. Significant relationship could not be identified between histopathological parameters and overall survival.

Conclusion

The negative expression of p21 correlates with disease survival and may be a poor prognostic factor in patients with resected gastric cancer treated with adjuvant chemotherapy. For clearly assessment of effect of p21 and Her-2 expression and mutation/polymorphism on overall survival, more studies which contain more patients are needed.