Phosphorylated Smad2 in Advanced Stage Gastric Carcinoma

We examined surgical samples from patients at the Osaka City University Hospital, Osaka, Japan. A total of 135 patients who had undergone resection of primary gastric tumours and were confirmed histologically to have advanced gastric cancer, were enrolled in this study. "Advanced cancer" indicates cancer invasion of the muscularis propria or serosa. None of the patients had undergone preoperative radiation or chemotherapy. Pathological diagnoses and classifications followed the Japanese Classification of Gastric Carcinoma [13]. Hepatic metastasis and peritoneal metastasis were examined at laparotomy. Peritoneal lavage cytology of the abdominal cavity was performed at laparotomy, and exfoliated cancer cells were microscopically examined. Depth of tumour invasion, differentiation, lymph node metastasis, venous invasion and lymphatic invasion were based on microscopic examination of materials obtained by surgical resection. The histological classification was based on the predominant pattern of tumour. The histological subtypes were: papillary adenocarcinoma, well-differentiated tubular adenocarcinoma and moderately-differentiated tubular adenocarcinoma, regarded as intestinal-type. The subtypes were: solid poorly-differentiated adenocarcinoma, non-solid poorly-differentiated adenocarcinoma, signet-ring cell carcinoma and mucinous carcinoma, regarded as diffuse-type. Lymph node metastasis was decided on the regional lymph nodes metastasis. The regional lymph nodes of the stomach are classified into three stations numbered as described in the Japanese classification [13] that depending upon the location of the primary tumour. As the number of increase, it indicated that spread to distant lymph nodes. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki (1975). This study was approved by the Osaka City University ethics committee. Informed consent was obtained from all patients prior to entry.

All the H&E-stained slides of the surgical specimens were reviewed, and the representative section of the tumour that included the site of deepest invasion was selected for the immunohistochemical study.

A rabbit polyclonal anti-human P-Smad2 antibody (Chemicon International, Themecula, CA. 1:2000) was used to detect p-Smad2. The methods for immunohistochemical staining of p-Smad2 have been described in the manufacturer's instructions. In brief, the slides were deparaffinized, and were heated for 20 min at 105°C by autoclave in Target Retrieval Solution (Dako, Carpinteria, CA). Sections were then incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. The specimens were incubated with p-Smad2 antibody (1:2,000) overnight at 4°C. The sections were incubated with biotinylated goat anti-rabbit immunoglobulin G for 30 min, followed by three washes with PBS. The slides were treated with streptavidin-peroxidase reagent, and were incubated in PBS diaminobenzidine and 1% hydrogen peroxide v/v, followed by counterstaining with Mayer's hematoxylin. We previously reported the expression level of p-Smad2 in gastric cancer cell lines [14]. The omission of the primary antibody served as negative controls.

P-Smad2 staining was evaluated at the invasion front of gastric cancers. Both hematoxylin and eosin staining were used as a reference slide to select cancer areas at the invading front. The existence of cancer cells was continuously examined from the serosa to the mucosa under a microscope. The invading front was determined at the lesion where cancer cells were first found from serosal side. P-Smad2 staining was evident in the nuclei of cancer cells. The percentage of p-Smad2 immunopositive cells was determined by counting 3 areas randomly chosen in the tissue in a total of 300 nuclei of cancer cells. Evaluation was made by two double-blinded independent observers who were unaware of clinical data and histologic diagnoses. The percentage of nucleus stained cells for p-Smad2 was also determined. There were 15 (11%) cases of a discrepant evaluation between the two independent observers. In those cases, we rechecked and discussed and the final score was obtained by consensus.

Staining was scored using the Allred scoring system as described previously [15]. For semi-quantitative analysis of p-Smad2 immuno-reactivity, the intensity of staining and percentage of tumour stained cells were evaluated in representative high-power (×400) and low-power fields (×200) using optical microscopy: intensity was scored 0-3 (0 = no immunoreactivity, 1 = weak, 2 = moderate, and 3 = intense immunoreactivity) and proportion was scored 0-5 (0 = 0%; 1 = 1-20%; 2 = 21-40%; 3 = 41-60%; 4 = 61-80%; 5 = 81-100%). The two scores were then added to obtain the final result of 0-8. The median score of all cases was 6.07. Then the cut-off point was determined 6; p-Smad2 expression was considered high expression when scores were ≥7 and low expression when scores were <7.

Comparative analyses of the data were performed using the Chi-square test or Fisher's exact test. The survival durations were calculated using the Kaplan-Meier method and analyzed by the log-rank test to compare the cumulative survival durations in the patient groups. Cox proportional-hazards regression was used to compute univariate and multivariate hazards ratios for the study parameters. A p-value of less than 0.05 was defined as being statistically significant. SPSS 10.0 software (SPSS Japan, Tokyo, Japan) was used for the analyses.

Phospho-Smad2 was mainly immunolocalized in the nuclei of cancer cells (Figure 1A). The intensity of staining and percentage of stained tumour cells were variable. Regarding intensity, score 0 was found in no cases (0%), score 1 in 55 cases (41%), score 2 in 33 cases (24%), and score 3 in 47 (35%) cases. Upon evaluation of positive cell percentages, score 0 was found in no cases (0%), score 1 in 2 cases (1%), score 2 in 11 cases (8%), score 3 in 16 cases (12%), score 4 in 43 cases (32%), and score 5 in 63 cases (47%). The total Allred scores varied from 2 to 8. The high expression level of p-Smad2 was found in 63 (47%) of 135 gastric carcinomas. The relationships between p-Smad2 expression and clinicopathological features of the tumours are shown in Table 1. P-Smad2 expression level was significantly high in diffuse type carcinoma (p = 0.011) and significantly correlated with peritoneal metastasis (p = 0.017), lymph node metastasis by Japanese classification [13] or UICC classification [16] (p = 0.047, p = 0.004) and peritoneal cytology (p = 0.026). "Peritoneal cytology" means peritoneal lavage cytology at laparotomy as a standard method for the detection of free tumor cells and a useful predictor of peritoneal recurrence in gastric cancer. In 12 cases, peritoneal cytology was not performed. Then the total number of peritoneal cytology was 123 cases. There was no statistically significant association between the phospho-Smad2 expression and either hepatic metastasis, venous invasion or lymphatic invasion. The number of metastatic lymph nodes in high-expression group (median = 12.3) was significantly (p = 0.002) higher than that in low-expression group (median = 5.77), while no difference of the number of affected lymph nodes was shown between low-p-Smad2 groups (median = 41.1) and high-p-Smad2 groups (median = 44.4).

In this series of advanced gastric cancer, survival of phospho-Smad2 high level expression patients was significantly poorer than that of phospho-Smad2 low level expression patients (p = 0.035, Figure 1B). In univariate analysis, high phospho-Smad2 expression (p = 0.048), morphological type 4 (p < 0.001), diffuse-type tumours (p < 0.001), lymph node metastasis (p < 0.001), peritoneal metastasis (p < 0.001), peritoneal free cancer cells (p < 0.001), lymphatic invasion (p = 0.006) and clinical stage IV (p < 0.001) were significantly associated to poor patient survival (Table 2). In multivariate analysis, morphologic type 4, diffuse-type tumours and clinical stage IV were statistically independent prognostic factors, but phospho-Smad2 was not (Table 3).