(5-7). Some studies indicated a significant relationship between cytotoxin-associated gene A (CagA) seropositivity, which indicates an infection of CagA-positive ...
ANTICANCER RESEARCH 28: 1277-1284 (2008)
Prognostic Significance of the Local Expression of Interleukin-12 in Patients with Advanced Gastric Cancer NOBUO NAGASHIMA1,2, YOSHIFUMI NAKAYAMA1, YUZURU INOUE1, JUN NAGATA1, KENTARO MATSUMOTO1, NORITAKA MINAGAWA1, TAKEFUMI KATSUKI1, KAZUNORI SHIBAO1, KEIJI HIRATA1, TATSUHIKO SAKO1,3, TETSUO HAMADA4 and NAOKI NAGATA1 1Department
of Surgery 1, University of Occupational and Environmental Health, Kita-Kyushu City; 2Department of Surgery, Kurate Municipal Hospital, Kurate-machi; Departments of 3Surgery and 4Pathology, Kyushu Rosai Hospital, Kita-Kyushu City, Japan
Abstract.
Background: Interleukin (IL)-12 is a heterodimeric cytokine that exhibits potent antitumor and antimetastatic activities. Very few studies have so far investigated the local expression of IL-12 in tumor specimens of gastric cancer. The purpose of this study was to investigate the immunohistochemical expression of IL-12 in patients with gastric cancer. Patients and Methods: IL-12 was immunohistochemically stained using monoclonal antihuman IL-12 antibody (1-1A4) in surgical specimens of 117 gastric cancer patients. The IL-12-positive cell density was calculated. The relationships among the IL-12-positive cell density, clinicopathological factors and 5-year survival rate were evaluated. Results: Among the patients (n=117), the 5year survival rate after surgery was not statistically different between the patients with high and low IL-12 positive celldensity. However, in the patients with advanced gastric cancer (n=85), those with a high IL-12-positive cell density showed a significantly better prognosis in comparison with those with a low IL-12-positive cell density (p=0.0104). A multivariate analysis indicated that the IL-12-positive cell density and TNM stage are significant prognostic factors. Conclusion: IL-12-positive cell density may be a significant independent prognostic factor in surgical specimens of advanced gastric cancer. Although the mortality rate of gastric cancer in Japan has improved in the last decade (1, 2), it is still the second highest cause of cancer-related deaths (3). Japan is one of
Correspondence to: Y. Nakayama, Department of Surgery 1, University of Occupational and Environmental Health, 1-1 iseigaoka, Yahata-nishi-ku, Kita-kyushyu 807-8555, Japan. Tel: +81 93 691 7441, Fax: +81 93 603 2361 Key Words: Interleukin-12, gastric cancer, immunohistochemistry, prognosis.
0250-7005/2008 $2.00+.40
the countries with the highest mortality and incident rate of gastric cancer in the world (4). Recently, many studies have led to the conclusion that infection with Helicobacter pylori is a major cause for the development of distal gastric cancer (5-7). Some studies indicated a significant relationship between cytotoxin-associated gene A (CagA) seropositivity, which indicates an infection of CagA-positive H. pylori, and gastric cancer (8-10). In Japan, the high mortality rate of the distal gastric cancer might be attributed to the very high rates of H. pylori infection, which include the high prevalence of CagA-positive H. pylori infection (11). IL-12 is a 70-75 kDa heterodimeric cytokine composed of two N-glycosylated polypeptide chains of 40 kDa and 35 kDa linked by a single disulfide bond (12-14). The main producers of IL-12 are monocytes, macrophages, dendritic cells and B-cells, and its receptor is detected on activated Tor NK-cells (15-17). IL-12 has demonstrated several antitumor effects, primarily by inducing the Th-1 cellular immune response (18). IL-12 induces NK-cells, T-cells, as well as macrophages and dendritic cells to produce INF-Á, enhances the lytic activity of NK-cells, alloreactive lymphocyte responses, and specific cytotoxic T-cell responses (19-22). The systemic expression of IL-12 in cancer patients has been measured and evaluated using several methods including serum analysis using ELISA (23-25), peripheral blood mononuclear cell (PBMC) analysis by ELISA in response to lipopolysaccharide (LPS) stimulation (26-28), and IL-12 mRNA level analysis by RT-PCR (29-30). The serum IL-12 levels in patients with T4 disease or distant metastasis were significantly lower than those in patients with less severe lesions (31). The production of IL-12 in PBMC stimulated with Staphylococcus aureus Cowan-1 decreased significantly with advancing disease and was lowest in patients with distant metastasis and cachexia (26). These studies indicated that the systemic expression of IL12 is reduced in patients with more advanced gastric cancer.
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ANTICANCER RESEARCH 28: 1277-1284 (2008) On the other hand, few reports have so far investigated the local expression of IL-12 in cancer specimens by immunohistochemical staining (32, 33). The significance of IL-12-expressing cells in the tumor microenvironment has not been evaluated in detail. Therefore, the present study investigated the relationship of clinicopathological factors, prognosis and the density of IL12-positive cells at the tumor sites in patients diagnosed with primary gastric carcinoma.
Patients and Methods Patients. Between 1980 and 1984, 117 patients with primary gastric cancer who underwent surgery at the Department of Surgery 1, University Hospital of Occupational and Environmental Health, Japan, were recruited to this study. The clinical data of these patients is summarized in Table I. Informed consent was obtained from all patients prior to the study. No patients had received chemotherapy or radiotherapy before surgery. The clinicopathological findings were determined according to UICC tumor-node-metastasis (TNM) classifications (34). Antibody. Regarding the immunohistochemical staining of IL-12, anti-human IL-12p40 monoclonal antibody (1-1A4, SEROTEC, UK) was used. This antibody recognizes the p40 subunit of human IL-12 and binds to both the free subunit and the p35/p40 heterodimer. Immunohistochemical staining of IL-12. Immunohistochemical staining (IHC) of endogenous IL-12 was performed on formalinfixed 2-Ìm sections of tissues embedded in paraffin. The 2-Ìm sections were deparaffinized with xylene and rehydrated in a series of ethanol. IL-12 staining was carried out using the Ventana DAB Universal Kit and an automated stainer (NexES IHC, Ventana Medical Systems, Yokohama, Japan) according to the manufacturer’s instructions, using a mouse monoclonal anti-human IL-12 antibody at a 1:100 dilution. Staining evaluation. Each section was usually scanned at low magnification (x100) to identify the area of the tumor with the highest density of IL-12-positive cells ‘hot spot’ at the invasive front of the tumor. IL-12-positive cells were then counted at x400 magnification using an ocular grid by a researcher without knowledge of the clinical outcome or prognosis. The mean counts of three hot spots/specimen were calculated and used in this study. Clinicopathological assessment. The tumors were staged by two pathologists, who had no prior knowledge of the results of the assays, according to UICC TNM classifications (34). Clinicopathological factors such as age, gender, tumor size, nodal involvement, depth of invasion, vessel invasion, histopathological type, peritoneal dissemination, hepatic metastasis and staging were analyzed for association with IL-12-positive cell density. Statistical analysis. The data shown represent the mean±SD and were statistically analyzed using Student’s t-test and the regression theory, as appropriate. For the survival analysis, the Kaplan-Meier method was applied and differences were tested using the Log-rank
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Table I. Characteristics of all patients. No. of patients Gender (M/F) Age (years) (mean±S.D.) Lymph node metastasis (_/+) Hepatic metastasis (_/+) Peritoneal dissemination (_/+) Depth of invasion T1 T2a T2b T3 T4 Tumor size (cm)(mean±S.D.) Lymphatic invasion (_/+) Venous invasion (_/+) Histopathological type Differntiated Undifferentiated TNM stage IA IB II IIIA IIIB IV
117 79/38 60.0±12.4 50/67 109/8 102/15 32 13 42 27 3 5.4±3.0 26/91 50/67 59 58 30 21 13 22 5 26
test. Univariate and multivariate analyses of survival were conducted using the Cox proportional hazards model. Statistical significance was established at the p
