|Year : 2017 | Volume
| Issue : 5 | Page : 813-816
Change of circulating antibodies against CD25-derived peptide antigen in hepatocellular carcinoma
Jiaxin Wang, Yangchun Xu, Huan Zhao, Xuan Zhang
Jilin Provincial Key Laboratory on Molecular and Chemical Genetics, The Second Hospital of Jilin University, Changchun, China
|Date of Web Publication||13-Dec-2017|
The Second Hospital Jilin University, 218 Ziqiang Street, Changchun 130041
Source of Support: None, Conflict of Interest: None
Aims: Several studies have shown altered levels of plasma anti-CD25 antibodies in patients with malignancy in lung, esophagus and breast. The present study was thus designed to test whether circulating anti-CD25 antibody levels were changed in hepatocellular carcinoma (HCC).
Methods: An enzyme-linked immunosorbent assay (ELISA) was developed in-house to detect plasma IgG antibodies to CD25-derived linear peptide antigens in 122 patients with HCC and 121 control subjects.
Results: Student's t-test showed that plasma anti-CD25 IgG levels were significantly higher in HCC patients than control subjects (t = 4.96, P < 0.001), in which male patients mainly contributed to the increased IgG levels (t = 5.11, P < 0.001). Further analysis showed that plasma anti-CD25 IgG levels were dependent on the stages of HCC although there was no significant correlation between plasma anti-CD25 IgG levels and BCLC stages (r = 0.145, P = 0.110, N = 122); a significant increase in anti-CD25 IgG levels was observed in HCC patients with stages B (t = 4.43, P < 0.001) and C+D (t = 4.89, P < 0.001) as compared with control subjects. Receiver operating characteristic (ROC) curve analysis showed that the area under the ROC curve (AUC) was 0.66 (SE = 0.035, 95% CI 0.60-0.73). The sensitivity of anti-CD25 IgG assay was 12.3% against a specificity of 99.2%.
Conclusions: The present study suggests that circulating anti-CD25 IgG antibodies may have prognostic rather than early diagnostic values for HCC.
Keywords: Autoantibody, CD25, hepatocellular carcinoma, tumor immunity
|How to cite this article:|
Wang J, Xu Y, Zhao H, Zhang X. Change of circulating antibodies against CD25-derived peptide antigen in hepatocellular carcinoma. J Can Res Ther 2017;13:813-6
| > Introduction|| |
Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and is ranked second in global cancer-related mortality. The current research into HCC is mainly focusing on how to reduce its high incidence in the population and to improve its poor prognosis. While alpha-fetoprotein (AFP) levels in blood have been used for clinical diagnosis of HCC, but such a test lacks specificity and sensitivity. A number of studies suggest that circulating antibodies against tumor-associated antigens (TAAs) are often positive in patients with malignant tumors.,
What mechanism is involved in triggering the secretion of antibodies to TAAs in patients with cancer remains unknown, but regulatory T-lymphocytes (Treg), a key subset of CD4+ T cells, may contribute to the regulation of tumor-related immune responses. Treg cells are a kind of important immune modulators in the immune system and are likely to play a major role in the development of HCC.,,
Interleukin-2 receptor alpha chain (IL2RA), also known as CD25, is a protein encoded by the IL2RA gene. It is a transmembrane protein present in activated lymphocytes, especially Treg cells. An increase in CD25+ Treg cell number has been reported in individuals with various cancers., Possibly, CD25 plays a role in the process of tumor immunity. A few recent studies performed in a Chinese population found that circulating IgG antibodies against CD25-derived peptide antigens were significantly increased in patients with some solid cancers such as esophageal squamous cell carcinoma, breast cancer, and nonsmall cell lung cancer. Accordingly, the present work was undertaken to examine whether circulating anti-CD25 IgG antibodies was also associated with HCC in the Chinese population.
| > Materials and Methods|| |
This study recruited 122 patients, who were diagnosed as having HCC, by the First Hospital of Jilin University, Changchun, China. Of these 122 HCC patients aged 54.5 ± 9.7 years, 104 were males and 18 were females. Their diagnosis was made mainly based on image examination and AFP levels in the circulation. HCC staging was made with the Barcelona Clinic Liver Cancer (BCLC) staging system, and these 122 patients with HCC were classified into three subgroups based on their stages, Group 1 (Stage 0 + A), Group 2 (Stage B), and Group 3 (Stage C + D). Plasma samples were taken before any anticancer treatment. A total of 121 healthy controls (99 males and 22 females) aged 54.9 ± 8.9 years were also recruited from local communities for this study. Clinical interview and image examination were applied to rule out those controls who had history of any malignancies as well as history of a severe form of autoimmune conditions, including autoimmune thyroid disease, pernicious anemia, Type 1 diabetes, celiac disease, ankylosing spondylitis, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease.
All the individuals were of Chinese Han origin, and all gave informed written consent to participate in this study. This work was approved by the Ethics Committee of the Second Hospital of Jilin University, Changchun, China and conformed to the requirements of the Declaration of Helsinki.
Enzyme-linked immunosorbent assay (ELISA) was developed in-house with the synthetic peptide antigen (H-IYHFVVGQMVYYQCVQGYRALHRGPAESVE-OH) as reported in previous studies,,, and the test of plasma anti-CD25 IgG levels was performed based on a previous study. This peptide sequence is present in the extracellular domain of human CD25 protein (accession NP_000408). Briefly, the synthetic peptide was dissolved in 67% acetic acid to obtain a concentration of 5 mg/ml as stock solution and then diluted with coating buffer (0.1 M phosphate buffer containing 0.15 M NaCl and 10 mM ethylenediaminetetraacetic acid, pH 7.2) to working solution of 20 μg/ml. Maleimide-activated plates (Thermo Scientific, Shanghai, China) were coated based on the manufacturer's instruction.
The antigen-coated plates were washed twice with 200 μl wash buffer that was phosphate-buffered saline (PBS) (P4417, Sigma-Aldrich, Shanghai, China) containing 0.05% Tween 20; 50 μl plasma sample diluted 1:200 in assay buffer that was PBS containing 0.5% bovine serum albumin was added to each sample well; 50 μl assay buffer was added to each negative control (NC) well and 50 μl positive control (PC) sample was added to each PC well. Following incubation at room temperature for 1.5 h, the plate was washed three times with 200 μl wash buffer, and 50 μl peroxidase-conjugated goat antihuman IgG antibody (ab98567, Abcam, Guangzhou, China) diluted 1:50,000 in assay buffer was added to each well. After incubation at room temperature for an hour, color development was initiated by adding 50 μl stabilized chromogen (SB02, Life Technologies, Beijing, China) and terminated after 20 min by adding 25 μl stop solution (SS) (SS04, Life Technologies). The measurement of optical density (OD) was completed on a microplate reader within 10 min at 450 nm with a reference wavelength of 620 nm. All the samples were tested in duplicate, and the specific binding ratio (SBR) was used to represent the relative levels of plasma IgG antibodies. Calculation of SBR is as follows:
SBR = (ODSample–ODNC)/(ODPC–ODNC)
To minimize an intra-assay deviation, the ratio of the difference between duplicated OD values of each sample to their sum was used to assess the precision for the in-house ELISA antibody test. If the ratio was found to be >10%, the test of this sample was treated as being invalid and not used for data analysis.
The mean ± standard deviation in SBR was used to present data. IBM SPSS Statistics 21.0 (SPSS Inc, Chicago, USA) was used to perform the Student's t-test (two-tailed) to compare the difference in plasma anti-CD25 IgG levels between the patient group and the control group, to carry out Pearson's correlation analysis to examine the correlation between plasma anti-CD25 IgG levels and BCLC stages and to perform receiver operating characteristic (ROC) curve analysis to work out the area under the ROC curve (AUC) with 95% confidence interval (CI) and the sensitivity of anti-CD25 IgG assay against a specificity of >99%.
| > Results|| |
As shown in [Table 1], the Student's t- test showed that plasma anti-CD25 IgG levels were significantly higher in HCC patients than controls (t = 4.96, P < 0.001) and that male patients appeared to mainly contribute to the increased IgG levels (t = 5.11, P < 0.001). Further analysis showed that plasma anti-CD25 IgG levels were dependent on HCC stages [Table 2] although there was no significant correlation between plasma anti-CD25 IgG levels and BCLC stages (r = 0.145, P = 0.110, n = 122); a significant increase in anti-CD25 IgG levels was observed in HCC patients with Stage B (t = 4.43, P < 0.001) and Stage C + D (t = 4.89, P < 0.001) as compared with controls [Table 2].
|Table 1: Comparison of anti-CD25 IgG levels between hepatocellular carcinoma patients and controls|
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|Table 2: Circulating anti-CD25 IgG levels in hepatocellular carcinoma patients at different stages|
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ROC curve analysis showed that overall AUC was 0.67 (standard error = 0.035, 95% CI 0.60–0.73), in which Group 1 (Stage 0 + A) had the lowest AUC and Group 3 (Stage C + D) had the highest AUC [Table 3] and [Figure 1]. The sensitivity of anti-CD25 IgG assay was 12.3% against a specificity of 99.2%.
|Table 3: Receiver operating characteristic analysis of circulating IgG autoantibodies to CD25 in hepatocellular carcinoma|
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|Figure 1: Receiver operating characteristic curve analysis of circulating anti-CD25 IgG levels in hepatocellular carcinoma patients and controls|
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| > Discussion|| |
The diagnosis of HCC mainly relies on imaging detection, blood AFP tests, and liver biopsy. However, the survival time after the onset of symptoms is generally <1 year. Although technology for early detection has been improved, there is still a need to develop novel approaches for the management of advanced HCC. Qiu et al. have reported an increase in plasma levels of interleukin-35 as an independent prognostic indicator in HCC and indicated that such an event could take place as a compensatory mechanism or protective immune response during cancer development. In this study, we demonstrated that plasma anti-CD25 IgG levels were significantly increased only in patients at Stages B, C, and D, but not in early stage [Table 2]; the detection of circulating anti-CD25 IgG levels may just have a prognostic value for malignant disease.
CD25 is mainly expressed in CD4+ Treg cells, which has been found to be important for the surface of the logo.,, The increased number of Treg cells surrounding tumor tissues and in splenic tissues is associated with the low immune function, tumor recurrence, and metastasis.,,, Treg cells represent a unique lineage of T cells with a critical role in maintaining immune homeostasis and are very potent suppressors of the immune response. It has recently been shown that a slight decrease in percentage of Treg cells (1.3%) could result in the development of autoimmune diseases. In this study, we found that circulating anti-CD25 IgG levels were significantly increased only in patients at intermediate and late stages but not in early stages. This change may be due to an increase in activated Treg cells. The increased expression of CD25 molecules may stimulate the secretion of anti-CD25 IgG in patients with a late stage HCC.
Current BCLC staging system plays an important role in predicting the prognosis of HCC and deciding a plan for treatment of the disease. However, patients at the same stage with the same therapeutic regimen show heterogeneous outcomes. Therefore, introduction of novel biomarkers may be useful for developing precision treatment through accurate prediction of the prognosis of HCC.
There are a couple of limitations in this study. First, the sample size used for antibody testing in the female group was rather small. Therefore, this initial work needs to be replicated independently with a larger sample set. Second, we used only synthetic peptides as antigens to develop the in-house ELISA; it may be useful to develop an ELISA antibody test with recombinant CD25 protein to compare the differences in sensitivity and specificity between two ELISA approaches.
We thank all the patients and controls for their participation in this study.
Financial support and sponsorship
This work was supported by Hailanshen Biomedical and Technology Ltd, Shenzhen, China.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]