Impaired or reduced CD3 zeta chain (CD3-ζ) expression in T cells has been identified in various cancers and may be associated with an ineffective immune response. The clinical significance of CD3-ζ chain expression in tumor-infiltrating lymphocytes (TILs) in gastric carcinoma remains unclear.
The authors immunohistochemically investigated CD3-ζ expression in TILs in 185 patients who had undergone curative gastrectomy. CD3-ζ/CD3 epsilon (CD3-ϵ) ratios were calculated. Patients were divided into two groups: a normal CD3-ζ group (n = 121) and a reduced CD3-ζ group (n = 64). Patients with a zeta per epsilon ratio of greater than 66% were placed in the normal CD3-ζ group.
Patients in the normal CD3-ζ group had fewer lymph node metastasis (P < 0.01) and a shallower depth of invasion (P < 0.05) than those in the reduced CD3-ζ group. The 5-year survival rate was 72% in the normal CD3-ζ group, which was significantly better than that in the reduced CD3-ζ group (55%; P < 0.01). When stratified according to clinical stage, the prognostic value was significantly different only in Stage IV patients. Multivariate analysis showed that CD3-ζ expression was an independent prognostic factor (P = 0.03) next to depth of invasion and lymph node involvement.
Many researchers have demonstrated a significant correlation between tumor-infiltrating lymphocytes (TILs) and clinical outcome in cancer patients.1–4 These findings suggest that tumor cells are eliminated by the host immune system via tumor antigen specific T cells.5 However, cytotoxic lymphocytes around tumor sites do not always exhibit antitumor activity and most are ineffective against autologous tumor cells. Impaired or reduced CD3 zeta chain (CD3-ζ expression is considered to alter or hamper cytoplasmic signaling in activating T cells.6–8 Experimentally reduced or impaired CD3-ζ expression in peripheral T cells first was identified in tumor-bearing mice9 and was found in TILs in malignant lymphoma,10 renal cell carcinoma,11 and in peripheral blood lymphocytes in cervical,12 colon,13 and stomach carcinomas.14 It therefore is believed that CD3-ζ impairment in various types of cancer patients leads to an ineffectiveness in immune response to autologous tumor. Recent studies have shown that antioxidant compounds9 or adoptive immunotherapy15 could normalize or preserve CD3-ζ expression in lymphocytes. This suggests that cytokine stimulation may recover or normalize impaired or reduced CD3-ζ expression, which may improve the immunologic response to tumor cells. In the current study, we retrospectively investigated lymphocytic CD3-ζ expression immunohistochemically and estimated the clinical implication of CD3-ζ expression in gastric carcinoma.
MATERIALS AND METHODS
A total of 185 patients with gastric carcinoma who had undergone curative gastrectomy for gastric carcinoma at Kagoshima University Hospital from 1985 to 1997 were enrolled in the current study. The mean age of this patient population was 63.3 years (range, 30–87; median, 56.5 years), and the male/female ratio was 2.5 (133:52). No patients had received chemotherapy preoperatively (Table 1). Clinical data and pathologic evaluation were assessed according to the General Rules for the Japanese Gastric Cancer Study and Pathology.16
Table 1. Patient Profile
No. of patients
U: upper third of the stomach; M: middle third of the stomach, L: lower third of the stomach.
Median Age (yrs)
CD3- ζ expression
Resected specimens were longitudinally sliced into 4-mm-thick sections, fixed in 10% formalin solution and embedded in paraffin. Representative sections including the deepest lesion were prepared and stained with hematoxylin and eosin for pathologic diagnosis.
Monoclonal Antibodies and Immunohistochemistry
The anti-CD3-ζ monoclonal antibody (sc-1239; Santa Cruz Biochemistry, Santa Cruz, CA), recognizing the cytoplasmic epitope of CD3-ζ and anti-CD3-ϵ polyclonal antibody (A0452; DAKO, Denmark) were used for detection of CD3-ζ or CD3-ϵ expression.17 Paraffin embedded specimens, including the most invasive tumors were cut into 4-μm slices and used for immunohistochemical staining. After endogenous peroxidase quenching, antigen was recovered by boiling sections at 120 °C and 3.4 atm for 10 minutes in 1% citrate buffer. Primary antibodies were applied at dilutions of 1:200 for CD3-ζ and 1:400 for CD3-ϵ and reacted overnight at 4 °C. A biotinylated secondary antibody was applied. Complexed streptavidin-biotinylated peroxidase was added at a dilution of 1:200, and antigen expression was visualized with diaminobenzidine.
Evaluation of CD3-ζ and CD3-ϵ expression in TILs
Intratumoral CD3-ζ or CD3-ϵ positive lymphocytes were counted in a total of 10 high-power fields (HPF ×400) in the same areas on consecutive slides and averaged. The CD3-ζ/CD3-ϵ ratio was calculated and evaluated according to Gruiji's classification.6 A total of 100 tumor-infiltrating CD3-ϵ positive cells were observed in randomly selected HPFs (×400), and CD3-ζ positive cells were counted in the same areas on consecutive slides. The CD3-ζ/CD3-ϵ ratio was again calculated. Patients having more than 66% (2 of 3) of CD3-ζ/CD3-ϵ were placed in the CD3-ζ normal group, whereas patients with less than 66% of CD3-ζ/CD3-ϵ were placed in the CD3-ζ reduced group.
The analysis of significant differences in categoric variables was performed using the chi-square test. Postoperative survival curves were calculated by the Kaplan–Meier method and analyzed with the generalized Wilcoxson test. Clinical factors were evaluated as independent prognostic factors by multivariate analysis using the Cox proportional hazard model. All P values are based on two-sided testing, and a significant difference was defined as less than 0.05.
Distribution of CD3 Zeta Positive Lymphocytes in Cancerous or Normal Gastric Tissues
CD3-ϵ positive and CD3-ζ positive cells infiltrating the tumor nest were visualized by immunohistochemical staining (Figs. 1 and 2). The population density of CD3-ζ and CD3–ϵ were evaluated in the same area on consecutive slides. The expression of CD3-ϵ and CD3-ζ in TILs was easily evaluated in the lymph follicle near the tumor (Figs.1a,b). In this example, the CD3-ζ CD3-ϵ was 0.88, indicating normal CD3-ζ expression. Alternatively, in Figure 2, the CD3-ζ CD3-ϵ was calculated to be 0.40, and the patient was placed in the CD3-ζ reduced or impaired group.
The CD3-ζ/CD3-ϵ ratio in cancerous stroma varied from 0 to 1.0 (average, 0.86) in this population. According to Gruiji's classification, the 185 patients examined were divided into 121 (63%) patients with normal CD3-ζ expression and 64 (37%) with CD3-ζ reduced or impaired expression (Table 1).
Correlation between Zeta Chain Expression and Clinical Factors
Thirty-six patients (73%) with reduced CD3-ζ expression were positive for lymph node involvement, which was significantly higher than the 47 patients (38%) with CD3-ζ normal expression (P < 0.05). Patients with reduced CD3-ζ expression also showed deeper tumor invasion than patients with CD3-ζ normal expression (P < 0.01). Accordingly, patients with normal CD3-ζ expression were in significantly earlier clinical stages of disease than those with reduced CD3-ζ expression (P < 0.05). Lymphatic invasion, histologic pattern and tumor location did not correlate with CD3-ζ expression (Table 2).
Table 2. Clinicopathologic Features of Patients with or without CD3-ζ Chain Expression
U: upper third of the stomach; M: middle third of the stomach, L: lower third of the stomach; NS: not significant.
Clinicopathologic data were described according to the General Rules for the Japanese Gastric Cancer Study and Pathology.
Depth of invasion
Lymph node involvement
Relation between CD3 Zeta Expression and Surgical Outcome
The 5-year survival rate of this patient population was significantly better in patients with normal CD3-ζ expression than in those with reduced CD3-ζ expression (P < 0.01; Fig. 3). When stratified according to clinical stage, Stage IV patients with reduced CD3-ζ expression showed significantly poorer prognoses than patients with normal CD3-ζ expression (P < 0.05; Fig. 4). Univariate analysis showed age, depth of invasion, tumor size, lymph node metastasis, and CD3-ζ expression all were related to prognosis. According to multivariate analysis by gender, age, histology, tumor dimension, depth of invasion, lymph node involvement, and CD3-ζ expression, lymph node involvement (hazard ratio [HR], 1.9; P < 0.001), tumor depth (HR, 1.6; P = 0.004), tumor size (HR, 1.6; P < 0.01), and CD3-ζ expression (HR, 1.42; P = 0.03) were all found to be independent prognostic factors (Table 3).
Table 3. Univariate and Multivariate Analysis of Clinicopathologic Factors
Univariate analysis (P value)
Lymph node involvement
Depth of invasion
CD3 zeta expression
The concept that T cells recognize tumor specific antigen and lyse autologous tumor is broadly accepted by clinical researchers.5, 17–19 Although immunotherapy using tumor antigen specific cytotoxic T lymphocytes has been clinically effective against refractory cancers such as malignant melanoma,20 it was not significantly effective in gastrointestinal carcinoma patients. It is reported that cancer cells directly or indirectly affect CD3-ζ expression in T cells and may interfere in their normal response to autologous tumor.14, 21 Specifically, macrophages induced by cancer cells were considered to decrease or alter the function of CD3-ζ in peripheral or TILs.22, 23 CD3-ζ abnormalities were found in both intratumoral lymphocytes and peripheral blood lymphocytes in patients with various types of malignancies. In the current study, we found that 37% of gastric carcinoma patients demonstrated a reduced CD3-ζ expression, which is similar to levels reported in other types of cancers.6, 17
Our retrospective analysis showed that reduced CD3-ζ expression negatively correlated with lymph node involvement, depth of invasion, and clinical stage of disease, which is in accordance with other studies.17 These findings suggest that the reduced response of T cells to tumor growth provided a favorable environment for tumor extension. CD3-ζ expression was correlated with patient survival in gastric carcinoma, which also agrees with reports on other types of tumors.17, 24
Only observed in patients with Stage IV disease, 5-year survival rates were significantly decreased in patients with reduced CD3-ζ expression than in those with normal CD3-ζ expression. Furthermore, CD3-ζ was found to be an independent prognostic factors in this patient population, which indicates that the presence or absence of CD3-ζ may influence surgical outcome directly. Accordingly, CD3-ζ expression in TILs may be an appropriate prognostic marker in gastric carcinomas. CD3-ζ expression levels obtained from biopsy specimens may provide information on preoperative immunologic status or may suggest the use of immunotherapy in gastric carcinoma patients. Once patients with reduced CD3-ζ expression are identified, treatment with CD3-ζ stimulatory cytokines could be performed in an attempt to normalize CD3-ζ expression in T cells, restoring their antitumor effect.15, 25, 26 Retrieval of CD3-ζ function using immunoactivating cytokines may be effective in Stage IV patients with reduced CD3-ζ expression.