Background
Esophageal squamous cell carcinoma (ESCC), the major histopathological form of esophageal cancer, is one of the most lethal malignancies of the digestive tract and is the fourth most frequent cause of cancer deaths in China [
1]. Despite the improvements in surgical techniques and adjuvant chemoradiation for patients with ESCC, the five-year survival rate of patients with advanced ESCC is still poor [
2]. This poor survival rate is largely due to the lack of serological markers for early diagnosis and prediction of disease progression; patients are frequently diagnosed with ESCC when they have already reached an advanced stage of disease [
3]. There is thus a growing need to identify useful biological markers for early, non-invasive diagnosis of ESCC and for monitoring tumor progression [
4].
In addition to the traditional tumor markers CEA, SCCA and CYFRA21-1, autoantibodies against tumor-associated antigens were recently reported in sera from patients with ESCC. Similar to the traditional tumor markers, these autoantibodies were shown to be useful molecular markers for ESCC. Some patients with ESCC mount an immunological reaction against several tumor-associated antigens, including p53 [
5‐
7], myomegalin [
8] and TRIM21 [
9]. Recently, a proteomics-based approach identified several autoantibodies in sera of patients with ESCC, such as anti-heat shock protein 70 [
10] and anti-peroxiredoxin VI [
11]. The presence of these autoantibodies in sera has been reported as a useful marker for early diagnosis or for prediction of disease progression in patients with ESCC.
Most recently, we identified CDC25B autoantibodies in sera from patients with ESCC using a proteomics-based technique[
12]. Three CDC25B phosphatases exist in higher eukaryotes, CDC25A, CDC25B and CDC25C[
13]. CDC25B has been shown to play an important role in tumorigenesis [
14]. First, CDC25B can transform fibroblast cells lacking functional retinoblastoma protein or harboring mutated Ras protein[
15]. Second, CDC25B activates the mitotic kinase CDK1/cyclin B complex in the cytoplasm to stimulate cell cycle progression [
16]. Furthermore, overexpression of CDC25B has been observed in a variety of human cancers, including colon cancer[
17], medullary thyroid carcinoma [
18], breast cancer [
19], non-Hodgkin's lymphomas[
20], non-small cell lung cancer [
21] and ESCC[
22‐
25]. We previously reported that aberrant expression of CDC25B in ESCC tumor cells can induce CDC25B autoantibodies in sera of ESCC patients, and antibodies against CDC25B were detected in sera of 36.3% of patients with ESCC, but not in sera of healthy controls, by reverse capture ELISA [
12]. Our findings suggest that CDC25B autoantibodies are a novel serum marker for ESCC.
Although higher levels of anti-CDC25B antibodies were found in the sera of patients with ESCC than in the sera of healthy controls, the relationship between tumor burden, tumor staging and antibody levels remains unknown. In addition, the potential utility of anti-CDC25B antibodies for diagnosis of ESCC has not been clearly addressed. In this study, we established a reverse capture ELISA to detect anti-CDC25B antibodies in sera from patients with ESCC and evaluated the clinical values of CDC25B autoantibodies for diagnosis of ESCC and prediction of tumor progression.
Methods
Patients and sera
Sera were collected from 134 patients with primary ESCC at the time of diagnosis before tumor resection at the Cancer Center of Sun Yat-sen University between January 2003 and December 2004. Ninety-three patients were male and 41 patients were female. The patients ranged in age from 38 to 81 years (mean, 58.5 years), and none of them had received radiation therapy or chemotherapy before surgery. Sera from 134 healthy volunteers (91 males and 43 females with ages ranging from 40 to 70 years (mean, 61 years)) were collected and used as controls. Prior to the use of these sera, informed consent was obtained from patients and experiments were approved by the Institute Research Ethics Committee. After collection, sera were immediately aliquoted and stored at -80°C until use.
Cell lines
The ESCC cell lines Eca-109, TE-1, and Kyse140 (Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) were grown in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 μg/L streptomycin, and 100 μg/L penicillin in a humidified incubator containing 5% CO
2 at 37°C. The immortalized esophageal cell line NE-3 was obtained from Dr. Jin (the University of Hong Kong, P. R. China)[
26] and cultured in Keratinocyte-SFM (Invitrogen, Carlsbad, CA).
Western blot analysis
Western blots were performed as described previously [
27]. The membranes were stained with a 1:1000 dilution of an anti-CDC25B antibody (Cell Signaling Technology, Danvers, MA) or with a 1:2000 dilution of a mouse monoclonal anti-α-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). A non-tumorous tissue protein was obtained from a patient with ESCC who underwent surgical esophageal tissue resection at the Cancer Center of Sun Yat-sen University (Guangzhou, P. R. China) during 2009 and used as a negative control.
Preparation of Antigen Protein
Antigen protein was extracted from the ESCC cell lines and prepared as reported previously [
28]. Briefly, after washing the cells three times with phosphate-buffered saline (PBS), the cells were collected and incubated at a concentration of 10
7 cells/ml in a lysis buffer composed of Tris base (10 mmol/L), NaCl (150 mmol/L), Triton-X (0.1%) and a proteinase inhibitor cocktail, placed on ice, vortexed every 10 min for 1 h, and centrifuged at 10,000 × g for 20 min at 4°C. The supernatant was then collected as an antigen protein sample and stored at -80°C until use. The final protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Fremont, CA).
Reverse capture ELISA for Detection of CDC25B Autoantibodies
A 96-well plate (Costar) was coated overnight with purified anti-CDC25B monoclonal antibody (100 ng/well in 50 mM bicarbonate buffer (pH 9.0), Cell Signaling Technology, Danvers, MA) at 4°C. Wells were then blocked for 2 h at 37°C with 3% bovine serum albumin (BSA) in PBS. The antigen protein sample was diluted in PBS (pH 7.0) to final concentrations of 20 mg/ml, 10 mg/ml and 5 mg/ml, added to blocked wells (100 μl/well) and incubated overnight at 4°C. Wells were then washed three times with PBST (0.1% (v/v) Tween 20 in PBS), and the 100 μl serum samples (1:200 dilution with PBST) were incubated in the wells for 2 h at 37°C. Rabbit anti-human CDC25B polyclonal antibody (1:10,000 dilution in PBST, Abcam) was used as a positive control, and 3% BSA served as a negative control. After washing the wells four times with PBST, each well was incubated with a 1:10,000 dilution of 100 μl goat anti-human or anti-rabbit IgG-HRP conjugate (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 37°C. The wells were then washed with PBST and incubated with TMB developing reagent for 5 min in the dark. The reactions were stopped with 0.5 mol/L H2SO4 and the absorbance of each well was measured at 450 nm using a Multiskan Spectrum plate reader (Thermo LabSystems). Sera from ESCC patients and healthy volunteers were tested simultaneously, and each sample was assayed twice in duplicate wells.
CEA, SCC and CYFRA21-1 Assay
Serum CEA and CYFRA21-1 were assessed by an electrochemiluminescence immunoassay using E170 analyzer (Roche Diagnostics Gmbh, Roche, USA). Serum SCC-Ag was measured by a microparticle enzyme immunoassay (ABBOTT Diagnostics, Abbott, USA).
Statistical Analysis
All statistical analyses were performed using the SPSS 16.0 software package. The cut-off value for seropositivity of CDC25B-Abs was identified by the ROC curve. Pearson's chi-square test or Fisher's exact test was employed to assess the association between CDC25B seropositivity and clinicopathologic characteristics. The statistical difference in CDC25B-Abs levels between patients with tumors and healthy control subjects was evaluated using the Mann-Whitney U test. Survival curves were estimated by Kaplan-Meier plots and log-rank tests. Cox proportional hazard regression analysis was used to estimate the hazard ratios of independent factors for survival. P < 0.05 in all case was considered statistically significant.
Discussion
The identification of tumor antigens that elicit an immune response is important for clinical applications; tumor antigens may used for early diagnosis, prognosis, and immunotherapy against the disease[
29]. In this study, we show that CDC25B-Abs in sera from ESCC patients were more sensitive than CEA, SCC-Ag and CYFRA21-1 for diagnosis of ESCC. Moreover, serum levels of CDC25B-Abs were correlated with the clinicopathologic characteristics present in patients with advanced ESCC.
CEA, SCC-Ag and CYFRA21-1 have been used as tumor markers for diagnosis of ESCC [
30]. However, reliance on the three tumor markers for the detection of ESCC has not been satisfactory, especially because of the poor sensitivity of these tumor markers for ESCC[
31]. In line with previous studies, our current study showed that the sensitivity of CEA, SCC-Ag or CYFRA21-1 for detection of ESCC was less than 35%[
32‐
34]. To circumvent the problem of low sensitivity, we and others have begun to evaluate the use of autoantibodies against tumor antigens to detect ESCC. Ralhan has shown that anti-p53 antibodies were found in 60% sera from patients with ESCC[
5], and Shimada has reported that anti-p53 antibodies were found in 40% sera from patients with ESCC and surveillance of serum p53-Abs was superior to CEA, SCC-Ag and CYFRA21-1 [
6]. Autoantibody against Prx VI was found in sera from 50% of patients with ESCC[
11]. Serum anti-myomegalin antibodies were present in 47% of patients with ESCC [
8]. Our previous study showed that 36.3% of ESCC patients with autoantibody responses to CDC25B [
12]. These results suggest that autoantibodies increase the sensitivity of detection of ESCC and might be useful tumor markers for ESCC diagnosis.
In the current study, CDC25B autoantibodies were detected in sera of ESCC patients by reverse capture ELISA. This technology is based on capturing specific antigens from tumor cell lysates with antibodies, allowing the antigens to be immobilized in their native configuration [
35‐
37]. Due to optimization of the reverse capture ELISA in current study, the sensitivity of this assay is higher than in our previous report (36.3%), but its specificity is lower than that reported in our previous study (100%) [
12]. The rate of CDC25B-Abs seropositivity in patients with ESCC was significantly higher than the seropositivity rates of tumor markers SCC-Ag, CEA and CYFRA21-1. Moreover, the combination of CDC25B-Abs and conventional tumor markers, CEA, SCC-Ag, and CYFRA21-1 significantly increased the sensitivity of detection of ESCC. Our data suggest that CDC25B-Abs could be a potential biomarker for ESCC diagnosis.
In addition, our results demonstrate that CDC25B autoantibodies were more prevalent in sera from patients with advanced ESCC than in sera from patients with early stage disease (
P < 0.001) and that in the patients with clinical stage III-IV and N1 subgroup, CDC25B-Abs seronegative patients survived longer than CDC25B-Abs seropositive patients. This observation may be explained by the higher incidence of CDC25B overexpression in advanced ESCC than in early stage tumors[
22,
25]. CDC25B protein expression increased as tumors progressed; none of the healthy control subjects expressed CDC25B, while one-fourth of the dysplasia subjects and one-half of the patients with invasive cancer expressed CDC25B[
25]. Moreover, overexpression of CDC25B was also more frequently found in patients with deep tumor invasion and lymph node metastasis than in patients with early stage disease [
22,
38]. Overexpression of CDC25B in advanced ESCC may thus lead to high production of CDC25B-Abs in patients with advanced tumors. These results suggest that detection of serum CDC25B-Abs is a useful non-invasive marker for identifying advanced ESCC patients with poor prognosis.
In summary, the levels of CDC25B-Abs in sera from ESCC patients were significantly higher than those in sera from healthy subjects. Detection of CDC25B-Abs in combination with CEA, SCC-Ag, CYFRA21-1 results in significantly increased sensitivity of detection, with 64.2% of ESCC patients testing positive for at least one of these markers. Moreover, our study has demonstrated the prognostic significance of serum CDC25B-Abs in ESCC and the clinical implications of CDC25B-Abs seropositivity on lymph node metastasis and advanced stage ESCC. High levels of CDC25B autoantibodies in sera were significantly associated with poor survival in advanced ESCC. CDC25B autoantibodies are thus a useful prognostic predictor for advanced ESCC.
Acknowledgements
This study was supported by grants from the National Natural Science Foundation of China (30630068, 30872931, and 30972762) and the Ministry of Science and Technology of China (No. 2007AA02Z477, 2006DAI02A11, and 2006AA02Z4B4).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MSZ is responsible for the study design. JD and BHZ performed the experiments and drafted the manuscript. LHX participated in the data analysis and Western blots. All authors read and approved the final manuscript.