Four gastric cancer cell lines (MKN-7, MKN-45, MKN-74 and KATO-III) were cultured in RPMI-1640 (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetal calf serum (Mitsubishi Kasei, Tokyo, Japan), as well as 100 units/ml each of penicillin and streptomycin at 37°C in a humidified atmosphere containing 5% CO₂, as described previously (24,25). These cell lines were used for reverse transcription-polymerase chain reaction (RT-PCR) assay.

Blood specimens were obtained preoperatively from 93 patients (64 men and 29 women; age range, 35–87 years; average age, 68 years) with gastric cancer who underwent curative gastrectomy with lymph node dissection at Kagoshima University Hospital (Kagoshima, Japan) between 2003 and 2005. None of the patients had received endoscopic mucosal resection, palliative resection, preoperative chemotherapy, and/or radiotherapy in the present study. Furthermore, patients who had synchronous or metachronous cancer in other organs were excluded. Patients were classified and staged on the basis of criteria of the tumor-node-metastasis (TNM) classification of gastric carcinoma established by the International Union Against Cancer (UICC) (26). Normal peripheral blood lymphocytes (PBLs) isolated from 22 healthy volunteers were used as a control group. After discharge, all patients were followed up every 3–6 months by blood tumor marker studies (CEA and CA19-9), radiography, ultrasonography and computed tomography at Kagoshima University Hospital. The median follow-up period after surgery was 25 months (range, 1–74 months).

To investigate STC2 protein expression in gastric cancer, 30 paraffin-embedded archival tissue (PEAT) specimens of resected primary gastric tumors from patients enrolled in this study were used for immunohistochemical analysis.

All specimens were collected from patients after informed consent was obtained in accordance with the institutional guidelines of our hospital.

Blood specimens (5 ml) from each patient were preoperatively collected in tubes containing sodium citrate, and then blood cells were separated in lymphocyte separation buffer (Gentra Systems, Inc., Minneapolis, MN, USA). Total RNA was extracted from the cell lines and blood specimens using Isogen (Nippon Gene, Toyama, Japan). Total RNA was isolated and purified using phenol-chloroform extraction as previously described (24,25). The concentration and purity of the total RNA were determined using a GeneQuant Pro UV/Vis spectrophotometer (Amersham Pharmacia Biotech, Cambridge, UK).

Primer and probe sequences of STC2 and glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) were designed for RT-PCR assays of each marker. The forward primers, fluorescence resonance energy transfer probe sequence, and reverse primers for STC2 and GAPDH were as follows: STC2 forward, 5′-GACTTGCTGCTGCACGAAC-3′; probe, 5′-FAM-ACGTGGACCTCGTGAACTTGCTG-TAM RA-1-3′; reverse, 5′-TGCTCACACTGAACCTGCAC-3′ and GAPDH forward, 5′-GGGTGTGAACCATGAGAAGT-3′; probe, 5′-FAM-CAGCAATGCCTCCTGCACCACCAA-TA MRA-1-3′; reverse, 5′-GACTGTGGTCATGAGTCCT-3′. The RT-PCR products for STC2 and GAPDH were resolved as 107- and 136- base pair fragments, respectively. The integrity of the RNA was confirmed by RT-PCR assay using GAPDH.

All total RNA samples were reverse-transcribed using the Advantage RT-for-PCR kit (Clontech Laboratories, Inc., Palo Alto, CA, USA) as previously described (24,25). Quantitative RT-PCR (qRT-PCR) proceeded using the LightCycler System (Roche Diagnostics, Mannheim, Germany). The reaction mixtures contained cDNA transcribed from 250 ng of RNA using each primer, probe, MgCl₂, and LightCycler FastStart DNA Master hybridization probes (Roche Diagnostics). The amplification profile comprised precycling at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 10 sec, annealing for 20 sec (60°C for STC2, and 55°C for GAPDH), and extension at 72°C for 10 sec. Plasmids for each marker were synthesized using pT7Blue-2 T-Vector (Novagen, Madison, WI, USA) according to the manufacturer's instructions. Standard curves for each assay were generated using the threshold cycles of 6 serial dilutions of plasmid templates (10⁶–10¹ copies). The mRNA copy number was determined using LightCycler software (Roche Diagnostics). Each assay was repeated in duplicate with positive (cell line), negative (H₂O), and reagent (without cDNA) controls to evaluate the quality of the qRT-PCR assay. Absolute copy numbers in qRT-PCR assays were computed on the basis of standard curves of plasmid templates. Copy numbers of STC2 mRNA were normalized by those of GAPDH mRNA (relative STC2 mRNA copies; absolute STC2 mRNA copies/absolute GAPDH mRNA copies).

Serial dilutions (10⁴, 10³, 10², 10¹ and 0) of MKN-74 tumor cells mixed with 1×10⁷ PBLs, which were isolated from a healthy volunteer without STC2 mRNA expression, were used for determining the sensitivity of the qRT-PCR analysis. This in vitro assay was repeated 3 times to verify its reproducibility.

The PEAT sections (3 μm) of surgical primary gastric tumors were incubated on slides at 50°C overnight, deparaffinized with xylene and then rehydrated with a graded series of ethanol. The sections were autoclaved in citrate buffer (0.01 mol/l, pH 6.0) at 120°C for 10 min to activate the antigen. After cooling at room temperature, endogenous peroxidase was blocked using a peroxidase blocking reagent (DakoCytomation, Carpinteria, CA, USA) for 10 min. Non-specific binding was blocked at room temperature for 30 min with Protein Block Serum-Free (DakoCytomation). The sections were incubated at room temperature for 60 min with an anti-human STC2 polyclonal antibody (Proteintech Group, Inc., Chicago, IL, USA) diluted 1:200 in Dako antibody diluent with background reducing components (DakoCytomation). After three 5-min washes in phosphate-buffered saline (PBS), the reaction for STC2 was developed by the ABC method (Vectastain ABC kit; Vector Laboratories) and visualized using diaminobenzidine tetrahydrochloride (DakoCytomation). Negative controls were treated with PBS without the primary antibody under the same conditions. On the basis of immunostainable intensity, STC2 immunoreactivity was classified into 3 groups: negative, weak, and strong immunoreactions.

Differences in STC2 mRNA expression between gastric cancer cell lines and PBLs from healthy volunteers, and between PBLs from patients with gastric cancer and healthy volunteers, were evaluated by the Wilcoxon rank-sum test. The relationship between the status of STC2 expression and categorical clinicopathological factors was assessed by the Chi-square and Fisher's exact tests. Survival curves were generated using the Kaplan-Meier method, and differences in survival were examined using the log-rank test. Prognostic factors were assessed by univariate and multivariate analyses (Cox proportional hazard regression model). All statistical calculations were performed using SAS statistical software (SAS Institute Inc., Cary, NC, USA). A P-value of <0.05 was considered to indicate a statistically significant result.

STC2 mRNA expression in 4 gastric cancer cell lines, blood specimens from 93 patients with gastric cancer, and from 22 healthy volunteers without cancers was assessed by qRT-PCR.

The range of relative STC2 mRNA copies was 2.49×10⁻⁶ to 2.75×10⁻² in gastric cancer cell lines, 0 to 9.4 in blood from patients, and 0 to 1.35×10⁻⁷ in normal PBLs from healthy volunteers. The mean relative numbers of STC2 mRNA copies (± SD) were 1.37×10⁻²±1.55×10⁻² in gastric cancer cell lines, 1.03×10⁻¹±9.75×10⁻¹ in blood from patients, and 2.02×10⁻⁸±4.43×10⁻⁸ in normal PBLs (Fig. 1). Accordingly, the relative numbers of STC2 mRNA copies were significantly higher in the gastric cancer cell lines and in blood from patients than in normal PBLs (P=0.0002 and P=0.01, respectively). STC2 mRNA expression was identified in 43 (46.2%) of the 93 patients with gastric cancer.

This spiking study was planned to investigate the relationship between STC2 mRNA expression and the number of tumor cells in an in vitro assay.

STC2 expression was identified in MKN-74 cells at a density of 10¹ tumor cells/1×10⁷ PBLs, and the relative numbers of STC2 mRNA copies gradually decreased as the numbers of tumor cells within normal PBLs decreased (Fig. 2).

All patients were classified into 2 groups based on the status of STC2 expression (positive, n=43; negative, n=50) to evaluate the relationship between STC2 expression and clinicopathological features.

STC2 expression was significantly correlated with age, depth of tumor invasion, lymph node metastasis, stage and venous invasion (P=0.023, P=0.045, P=0.035, P=0.007 and P=0.027, respectively; Table I).

The 5-year survival rates were 58.4 and 80.9% in patients with STC2-positive and -negative expression, respectively (Fig. 3). Five-year survival rates were significantly lower in patients with STC2-positive expression than in those with STC2-negative expression (P=0.014; Fig. 3).

Univariate analysis demonstrated that age, depth of tumor invasion, lymph node metastasis, distant metastasis, lymphatic invasion, venous invasion and STC2 expression were significantly correlated with postoperative survival (P=0.020, P=0.003, P=0.002, P=0.046, P=0.030, P=0.041 and P=0.012, respectively; Table II). Multivariate analysis demonstrated that age, lymph node metastasis and distant metastasis were independent prognostic factors (P=0.002, P=0.024 and P=0.047, respectively; Table II). Consequently, STC2 expression was not an independent prognostic factor in the multivariate analysis (P=0.433).

To confirm STC2 expression in primary tumor sites, immunohistochemical evaluation was carried out on 30 surgical PEAT specimens of primary gastric tumors. Immunohistochemical analysis showed that STC2 protein expression was identified in the membrane and/or cytoplasm of gastric tumor cells (Fig. 4). Furthermore, primary gastric tumors had various immunoreactions for STC2 (Fig. 4A–C). Finally, strong immunoreaction was identified in 46.7% (7/15) and 26.7% (4/15) of patients with STC2-positive and -negative mRNA expression, respectively.