New molecular staging with G-factor supplements TNM classification in gastric cancer: a multicenter collaborative research by the Japan Society for Gastroenterological Carcinogenesis G-Project committee

This study was conducted after obtaining approval from the society’s ethics committee at the annual meeting in 2007, and then requesting approval from the ethics committee of each of the four institutions, Department of Surgical Oncology, Osaka City University graduate School of Medicine (Osaka, Japan); Department of Gastroenterological Surgery, Kanazawa University (Kanazawa, Japan); Department of Surgery and Science, Graduate School of Medicine, Kyushu University (Fukuoka, Japan); and Department of Gastroenterological Surgery, Saga University, Faculty of Medicine (Saga, Japan), that supplied resected specimens. Each institution provided samples according to an implementation planning report. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki, 1975. Recent reports suggest that there was a difference of at least 20 % in 5-year survival when patients were stratified according to the expression of useful prognostic factors [4‐11]. To detect as much difference in survival in a retrospective analysis, approximately 100 cases who had recurrence and 100 cases who were recurrence-free after a sufficient follow up were deemed necessary.

The aforementioned four institutions ultimately registered 210 cases of gastric cancer who underwent curative R0 resection by gastrectomy with more than D1 lymph node dissection (D1+ to D2). Of the 210 cases, 104 were Stage II and 106 were Stage III according to the Japanese Classification of Gastric Carcinoma (the 13th edition). In addition, 104 cases were confirmed to have postoperative recurrence or death within 5 years, whereas 106 cases were confirmed to have been recurrence-free for 5–10 years. Upon gaining approval from the ethics committees of each of the aforementioned institutions, tissue samples were obtained, along with clinicopathologic data such as age, gender, occupation, operative procedure, degree of penetration into the wall (pT), lymph node metastasis (pN), final stage, ly and v factors, histological type, presence or absence of adjuvant therapy and regime, recurrence type, postoperative disease-free survival (DFS) and postoperative overall survival (OS). The data were subsequently analyzed by the Department of Oncology at the Institute of Geriatrics and Medical Science, Osaka City University, Graduate School of Medicine.

The G project committee was elected, and as a preliminary step, the members conducted a PubMed literature search of articles published between 1990 and 2005 using the key words “gastric cancer” and “independent prognostic factor.” A total of 822 articles on gastric cancer were extracted and reviewed (Table 1). Of the 50 molecules identified, the molecules or other factors frequently highlighted in both univariate and multivariate analyses as prognostic factors were epidermal growth factor (EGF) (35 papers), p53 (21), vascular endothelial growth factor (VEGF) (18), microsatellite instability (12), TGF-β (6), urokinase-type plasminogen activator (u-PA) (5) and E-cadherin (5). Based on these results and considering the known functions of the molecules, the committee selected p53 and VEGF (VEGF-A and VEGF-C) as candidate molecules to be nominated as G-factor. More recent articles were reviewed in the meantime, and the committee decided to add the following five molecules as candidates, based on their emerging roles in cancer biology reported after 2005: matrix metalloproteinase-7 (MMP-7, a member of the metalloprotease family) [12], human epidermal growth factor receptor 2 (HER2) [13], Regenerating islet-derived family, member 4 (Reg IV) [14, 15], olfactomedin 4 (GW112, an anti-apoptotic factor) [16], and Claudin-18 (the tight junction molecule) [17].

The surgical specimens were formalin-fixed and paraffin-embedded, and sent to the Department of Molecular Pathology at Hiroshima University (Hiroshima, Japan). Immunohistochemical staining was performed at this facility using eight primary antibodies against the eight candidate factors. The Envision+ staining kit (Dako Corp., Glostrup, Denmark) was used for immunostaining. Paraffin-embedded specimens were sectioned at 4 μm, hydrophilized, and microwaved for 30 min in pH 6.0 citric acid buffer or autoclaved in ethylenediaminetetraacetic acid (EDTA) buffer to activate the antigen. Intrinsic peroxidase was deactivated by incubation with 3 % H₂O₂ for 10 min. After rinsing, blocking was performed using sheep serum and reacting with each primary antibody. Anti-p53 primary antibody was cloned name DO-7 (Dako); VEGF-A, polyclonal antibody (Santa Cruz); VEGF-C, polyclonal antibody (American Research Products); Reg IV, polyclonal antibody (see Ref. [14]); GW112, N212 (see Ref. [15]); Claudin-18, polyclonal antibody (Invitrogen); MMP-7, 141-7B2 (Daiichi Fine Chemicals). All primary antibodies were diluted 1:50 and incubated at room temperature for 1 h, washed with phosphate buffered saline (PBS), and incubated with a secondary antibody at room temperature for 1 h. The Envision+ Rabbit Peroxidase Detection System (Dako Corp.) contained the primary rabbit antibodies VEGF-A, VEGF-C, Reg IV, and Claudin-18, whereas the Envision+ Mouse Peroxidase Detection System (Dako Corp.) was used to detect p53, olfactomedin 4, and MMP-7. After washing with PBS, the samples were incubated in diaminobenzidine (DAB) solution for 10 min, stained, washed with PBS, and counter-stained with hematoxylin. Expression levels of p53, VEGF-A, VEGF-C, MMP-7, HER2, Reg IV, olfactomedin 4 and Claudin-18, were analyzed by immunostaining using one representative specimen from the center of the tumor. The stained area was scored by the percentage of immunopositive cells as an index of the expression of each molecule. Cases that were not at all stained were scored as 0, cases with < 10 % of stained tumor cells were 1+, cases with 10–50 % of stained tumor cells were 2+, while cases with > 50 % of stained tumor cells were 3+. Evaluation of immunostaining was conducted independently by two pathologists, and any discrepancies in assessment were discussed and reassessed by microscopy. Scores of 2+ and 3+ were determined as positive for immunostaining.

Each representative positive expression in histological image for gastric cancer is depicted in Fig. 1. The positive staining rate of each molecule was 41.4 % for p53, 55.2 % for VEGF-A, 73.3 % for VEGF-C, 25.7 % for Reg IV, 66.2 % for olfactomedin 4, 63.4 % for Claudin-18, 46.7 % for MMP-7, and 13.0 % for HER2, respectively.

Examination of the all 210 gastric cancer cases revealed a significant correlation between the postoperative recurrence and pT stage (p = 0.008), pN stage (p = 0.078), final stage (p < 0.001), ly factor (p = 0.002), and v factor (p < 0.001), but not with surgical procedure or the type of adjuvant chemotherapy received. As for the prognostic significance of candidate molecular factors, no significant correlation was observed between the immunostaining expression of eight aforementioned factors and postoperative recurrence (Table 2).

In terms of OS for all gastric cancer cases (Stage II and III), cases with positive p53 expression had significantly worse prognosis (p = 0.02) when compared with p53-negative cases. Cases positive for MMP7 tended to have worse prognosis in comparison with the negative cases, although not significantly so (p = 0.09) (Fig. 2a). In terms of DFS, p53-positive and MMP7-positive cases had significantly worse prognosis (p = 0.006 and p = 0.04, respectively) (Fig. 2b). No significant difference in prognosis was observed between patients positive and negative for four other factors (VEGF-A, Reg IV, olfactomedin 4, and Claudin-18) (data not shown).

Figure 3 reveals prognostic evaluation (OS and DFS) in Stage II gastric cancer cases (n = 104). MMP7-positive cases suffered from significantly worse prognosis both in terms of OS (p = 0.027) and DFS (p = 0.014). In addition, VEGF-C positive cases had more favorable prognosis (p = 0.044 for OS and p = 0.048 for DFS) compared with VEGF-C negative cases. The similar analysis of OS and DFS revealed no significant difference in survival among Stage III patients (n = 106) for any of the eight factors.

According to the aforementioned results, we selected p53 and MMP-7 as G-factor that might serve our purpose. Consequently, we analyzed the association between the combination of p53 and MMP-7 expression and prognosis. All gastric cancer patients in the current series were stratified into three groups as follows, based on the expression of these molecules; G0 group (negative for both molecules, n = 69), G1 group (positive for either of the molecules, n = 97), and G2 group (positive for both of the molecules, n = 44). In all of the 210 gastric cancer cases, G2 cases demonstrated significantly higher recurrence rate (59 %) compared to 38 % in G0 cases (p = 0.047). In stage-by-stage analyses, the recurrence rate of G2 cases (50 %) tended to be higher in comparison with that of G0 and G1 cases for Stage II (25 and 20 %, respectively, p = 0.069), but not for Stage III (p = 0.414).

In the univariate analysis, G2 cases were associated with a shorter OS (hazard ratio 1.83, 95 % CI 1.15–2.90; p = 0.01) and DFS (hazard ratio 2.15, 95 % CI 1.26–3.68; p = 0.005) in comparison with G0 and G1 cases. The multivariate Cox’ regression analysis revealed that the G2 group was not an independent prognostic factor for OS (hazard ratio 1.59, 95 % CI 0.99–2.55; p = 0.052), but was independently associated with a shorter DFS (hazard ratio 1.90, 95 % CI 1.10–3.30; p = 0.022) (Table 3).

Survival analysis revealed that long term outcome of G0 cases was significantly better than that of G1 cases (p = 0.024 for OS and 0.019 for DFS) and G2 cases (p = 0.001 for OS and DFS) in all cases analyzed. In addition, G2 was particularly strong as marker of poor prognosis among Stage II cancer (p = 0.004 for comparison of OS with G0 and 0.029 for comparison with G1) (Fig. 4).