Cervical carcinoma is the third most common malignancy among women, and the estimated incidence is 529,800 new cases worldwide.1 Well-established clinicopathological prognostic markers are International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis, and tumor size.2 However, it is estimated that approximately 35% of patients with advanced cervical cancer will have recurrence after therapy.3 To improve the survival rate in this heterogeneous group of patients, further stratification based on new specific prognostic molecular biomarkers is crucial in women with cervical carcinoma. This classification might help clinicians make appropriate therapeutic decisions for patients at high risk of death or recurrence.
Cluster of differentiation (CD) 24 is a mucin-like cell surface protein and is expressed mainly on various developing cells including pre-B-cells, certain neural cells, and epithelial cells.4,5 CD24 plays a role in cell-to-cell interaction and regulation of proliferation and adhesion.6 Previous studies show that CD24 is important in growth and survival of cancer cells. In vivo, the potential role of CD24 on human tumor cells is as a ligand of P-selectin, an adhesion receptor on thrombin-activated endothelial cells and platelets.7 The CD24/P-selectin binding pathway could be important in the interaction of tumor cells with platelets or endothelial cells in vivo and has significance in tumor metastasis.8 Furthermore, Baumann et al9 provided evidence that CD24-expressing cancer cells increased tumor cell proliferation and acquired enhanced spreading, motility, and invasive properties.
Recent studies have demonstrated that the CD24 protein is frequently overexpressed in several human carcinomas, whereas it is rarely detectable in normal tissues.10 Furthermore, overexpression of CD24 seems to correlate with the poor prognosis of several malignancies including colorectal, endometrial, breast, liver, and ovarian tumors.11–15 However, there is very limited information on the prognostic significance of CD24 in patients with cervical carcinoma.
The aim of our study was to determine the expression of CD24 in cervical carcinomas and assess correlations between CD24 expression and clinicopathological parameters. In addition, we evaluated whether the differential expression of CD24 is associated with overall survival in cervical carcinoma.
MATERIALS AND METHODS
A total of 144 patients diagnosed with FIGO stage I to IV cervical carcinoma were studied from 1992 through 1999 at Shin Kong Wu Ho-Su Memorial Hospital (Taipei, Taiwan). Of the 144 patients with cervical carcinoma, 90 were classified in stage I, 32 in stage II, and 22 in stage III or IV. Of these, 95 patients were treated with radical hysterectomy and pelvic lymphadenectomy. After radical hysterectomy, 31 patients were chosen for postoperative radiation therapy because of positive pelvic lymph nodes and/or parametrial invasion or positive surgical margins. These patients were the subjects of various histopathological parameter reviews. All slides were reclassified and graded by 1 pathologist according to World Health Organization criteria; there were 130 squamous cell carcinomas, 11 adenocarcinomas, and three adenosquamous cell carcinomas. Informed consent to use the samples for diagnostic and research purposes was obtained according to the procedures established at our institution. Clinicopathological variables including age, stage of disease, histological cell type, histological grade, lymph node status, tumor size, and survival were abstracted from the medical records of each patient.
The median age of patients at diagnosis was 48.5 years (range, 28–83 years). Clinical follow-up was available for all patients. The mean follow-up period was 115.7 months from the date of surgery or radiotherapy, with a median of 147.0 months (range, 2–202.5 months).
Tissue Microarrays and Immunohistochemical Analysis
Paraffin-embedded tumor tissues were retrieved, and tissue microarray slides containing duplicate 2.00-mm cores from each tumor were constructed (Fig. 1A). Immunostaining of the CD24 protein was performed using the avidin-biotin immunoperoxidase method (Super Sensitive Immunodetection System, QP900-9L, BioGenex, San Romon, CA) and the Optimax Plus Automated Cell Staining System (BioGenex). Sections (5 µm) of tissue microarray blocks were prepared, air dried at room temperature overnight, incubated at 55°C for 1 hour, deparaffinized in xylene, and finally rehydrated using graded alcohol concentrations. Antigen retrieval was performed by boiling for 10 minutes in 0.1 M citrate buffer (pH 6) in a microwave oven (600 W), and then endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase for 10 minutes. The tissue sections were then incubated with a monoclonal antibody against human CD24 (Ab-2, clone 24C02/SN3b, Lab Vision, Fremont, CA) at a dilution of 1:100 for 30 minutes. Next, the tissue sections were incubated with a biotinylated secondary antibody, and staining was achieved by the avidin-biotin peroxidase complex technique with visualization using diaminobenzidine as the chromogen; the sections were also counterstained with hematoxylin. Negative controls were prepared by replacing the primary antibody with normal serum. Paraffin-embedded tissue sections taken from patients with gastric cancer, which is known to stain positively for the CD24 antibody, were used as the positive controls. One known positive and one negative CD24 slide were used as controls in each assay.
The results of immunostaining were recorded independently by 2 investigators. All areas of tumor cells within each section were analyzed, and a positive reaction was defined as CD24 expressed in the cytoplasm of tumor cells. Results of immunoreactivity were quantified with a weighted score described in our previous study.16 First, the percentage of the total number of tumor cells staining positively was categorized and awarded a score of 0 to 4 as follows: 0, less than 5%; 1, 5% to 25%; 2, 26% to 50%; 3, 51% to 75%; and 4, greater than 75%. The intensity of cytoplasmic immunostaining was scored on a 3-point scale: 1, weak; 2, moderate; and 3, intense. A weighted score for each tumor specimen was produced by multiplying the percentage score by the intensity score. Any discrepancies in staining interpretations were resolved by shared observation over a multiheaded scope until a consensus was reached. A series of cutoff values in 1 increment of the weighted score were tested for continuous variables, and those giving the best P values were adopted to plot Kaplan-Meier curves and compare prognostic differences by log rank tests in this study. Based on the trends of survival cures, score of 4 points or greater was considered overexpression for CD24.
The SPSS-Win 17.0 program package (SPSS Inc, Chicago, IL) was used in a personal computer for the basic statistical calculations. The relationships between CD24 expression and clinicopathological parameters were examined using contingency tables, which were further analyzed using the χ2 or Fisher exact test. Survival was censored if the patients were still alive or had died of causes other than carcinoma. Survival curves were constructed using the Kaplan-Meier method, and the curves were compared using the log rank test.
Cox proportional hazards model was used to identify prognostic factors and to assess relative risks of dying. A value of P < 0.05 was considered statistically significant, and all P values were 2 sided.
CD24 Protein Expression
In normal cervical tissue, CD24 was not expressed in squamous epithelium. Table 1 shows the scoring of CD24 expression. Of the 144 invasive carcinomas analyzed, 48 (33.3%) showed no CD24 expression, 46 (31.9%) showed weak CD24 expression (weighted score < 4), and 50 (34.7%) showed strong CD24 expression (weighted score ≥ 4) (Fig. 1B–D). The median weighted score of CD24 expression in the invasive carcinoma tumors was 1.0. For statistical assessment, cases categorized as having a weighted score of 4 or greater were considered the CD24 overexpression group, and this group was compared with the underexpression group (weighted score < 4).
CD24 Overexpression in Association with Clinicopathological Parameters
The relationship between the expression of CD24 protein and clinicopathological features is shown in Table 2. Overexpression of CD24 was significantly associated with histological cell type (P = 0.042). However, CD24 overexpression was not associated with other clinicopathological parameters, including FIGO stage, histological grade, lymph node metastasis, and tumor size.
CD24 Overexpression Predicts Poor Prognosis
At the end of the follow-up, 50 patients (34.7%) had died of cervical carcinoma. The mean survival of the patients was 141.1 months (95% confidence interval [CI], 127.1–155.0), and the 5-year survival rate was 65.3%. Using the Kaplan-Meier method, patients with strong CD24 expression tended to have poorer survival compared with that of patients with CD24 underexpression (5-year survival rate, 52.0% vs 72.3%; log rank P = 0.014) (Fig. 2). There was a mean survival of 114.5 months in patients whose tumors displayed CD24 overexpression and 154.1 months in those with reduced CD24 expression.
In univariate analysis based on Cox regression model, CD24 overexpression was found to be a strong prognostic factor of poor overall survival (relative risk, 1.979; 95% CI, 1.135–3.449; P = 0.016). In addition, advanced FIGO stage, nodal metastasis, parametrial invasion, lymph-vascular space invasion, large tumor size, deep stromal invasion, and vaginal involvement were associated with shorter overall survival.
In multivariate analysis, when the data were stratified with respect to clinical FIGO stage, CD24 expression remained statistically significant. Cox proportional hazards analysis revealed that CD24 overexpression and clinical stage were independent prognostic factors for overall survival (Tables 3). Compared with the relative risk (relative risk, 1) of death for the CD24 underexpression group, the relative risk of death for the CD24 overexpression group was 1.814 (95% CI, 1.019–3.226; P = 0.043). Therefore, the group of patients with CD24 overexpression was 81% more likely to die of cervical cancer than the group with CD24 underexpression.
In this study, we have confirmed that CD24 is expressed in a subset of cervical carcinomas. Furthermore, our results demonstrated that CD24 overexpression is associated with poor outcome in patients with cervical carcinomas.
Up-regulation of CD24 has been considered one of the most important factors in the tumorigenesis of a variety of carcinomas. In addition, accumulating evidence indicates that CD24 overexpression is an important biomarker for progression and reduced overall survival time in many carcinomas.11–15 Our data demonstrated that 37.4% of invasive cervical carcinomas displayed strong CD24 expression. This finding has been previously reported by Kwon et al,17 who revealed that expression of CD24 was significantly higher in squamous cell carcinomas (78.1%) than in the squamous epithelial mucosa of the cervix. In their series of studies of cervical carcinoma, a positive correlation was found between CD24 expression and parametrial invasion and pelvic lymph node metastasis. They also disclosed that CD24 positivity was significantly associated with decreased distant metastasis-free survival and overall survival in squamous cervical cancer, even in those with adjuvant treatment after surgery.18
Consistent with the results obtained from Sung et al,18 the most interesting finding of our study was the strong correlation between CD24 expression and the clinical outcome. To the best of our knowledge, this is the second study investigating CD24 expression in a large tumor sample of cervical carcinoma and its association with survival. We found CD24 expression to be an independent predictor of shortened patient survival by univariate and multivariate analyses in cervical carcinoma. These data support the concept that CD24 expression can be used to stratify patients with a higher risk of disease aggressiveness. Moreover, in our current study, loss of CD24 expression was found to be a significant prognostic factor for overall survival using log rank and multivariate survival analyses. Thus, immunohistochemical analysis of CD24 status in routine formalin-fixed, paraffin-embedded specimens may be an indicator of tumor behavior.
In this study, despite the significant correlation with overall survival, CD24 expression was not associated with FIGO stage, histological grade, tumor size, and lymph node status. Although there seems to be a discrepancy between these findings, it is reasonable to speculate that CD24-positive tumor cells could attach more easily to the endothelium lining the vascular wall and thus enter into the circulation. Hematogenous metastasis is a fatal step in the progression of cervical cancer, and we feel that this is the most likely explanation of the decreased survival times of strongly CD24-expressing tumors.
Although the molecular basis for overexpression of CD24 in cervical squamous carcinoma remains unclear, frequent expression of CD24 in cervical neoplasia suggests that this protein is involved in the carcinogenesis of these lesions.
Numerous studies have linked CD24 expression with tumorigenesis and tumor progression. Based on in vitro studies, CD24 seems to be involved in tumor cell proliferation,19 invasion, and metastasis.20 This is advocated by animal models, in which CD24 expression has been related to promote tumor cell invasiveness and metastasis in vivo.9 Furthermore, Overdevest et al21 showed that CD24 was important for metastatic lung outgrowth in bladder cancer and treatment with anti-CD24 monoclonal antibodies was efficacious in reducing clinically detectable lung metastases and prolonging survival in mice. Similarly, in a human xenograft model, down-regulation of CD24 markedly retarded tumorigenicity of human cancer cell lines in mice treated with monoclonal antibodies.22 Identifying selective CD24 inhibitors as a therapeutic approach would provide new insights into the treatment of patients with cervical carcinoma. Another interesting finding of our analysis was the absence of CD24 expression in adenocarcinoma. This observation may be explained by different underlying biological mechanisms of the tumorigenesis and progression in this tumor. Our study had a small number of patients, and this finding warrants further larger studies to clarify.
In summary, we have demonstrated that CD24 overexpression is a significant predictor of survival. On the basis of our preliminary results, CD24 overexpression may define a distinct subgroup of patients with cervical cancer with a worse prognosis and might aid the clinician in the selection of appropriate therapy for individual patients. Although this has to be substantiated in a large number of patients, cancer therapy targeted against CD24 may be a promising strategy for the treatment of patients with cervical carcinoma.
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