Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 5  |  Page : 813-816

Change of circulating antibodies against CD25-derived peptide antigen in hepatocellular carcinoma


Jilin Provincial Key Laboratory on Molecular and Chemical Genetics, The Second Hospital of Jilin University, Changchun, China

Date of Web Publication13-Dec-2017

Correspondence Address:
Xuan Zhang
The Second Hospital Jilin University, 218 Ziqiang Street, Changchun 130041
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_823_17

Rights and Permissions
 > Abstract 


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

How to cite this URL:
Wang J, Xu Y, Zhao H, Zhang X. Change of circulating antibodies against CD25-derived peptide antigen in hepatocellular carcinoma. J Can Res Ther [serial online] 2017 [cited 2019 Dec 11];13:813-6. Available from: http://www.cancerjournal.net/text.asp?2017/13/5/813/220485




 > Introduction Top


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.[1] While alpha-fetoprotein (AFP) levels in blood have been used for clinical diagnosis of HCC, but such a test lacks specificity and sensitivity.[2] A number of studies suggest that circulating antibodies against tumor-associated antigens (TAAs) are often positive in patients with malignant tumors.[3],[4]

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.[5],[6],[7]

Interleukin-2 receptor alpha chain (IL2RA), also known as CD25, is a protein encoded by the IL2RA gene.[8] It is a transmembrane protein present in activated lymphocytes, especially Treg cells.[9] An increase in CD25+ Treg cell number has been reported in individuals with various cancers.[10],[11] 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,[12] breast cancer,[13] and nonsmall cell lung cancer.[14] 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 Top


Subjects

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,[15] 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.

Antibody testing

Enzyme-linked immunosorbent assay (ELISA) was developed in-house with the synthetic peptide antigen (H-IYHFVVGQMVYYQCVQGYRALHRGPAESVE-OH) as reported in previous studies,[12],[13],[14] and the test of plasma anti-CD25 IgG levels was performed based on a previous study.[16] 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.

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 Top


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

Click here to view
Table 2: Circulating anti-CD25 IgG levels in hepatocellular carcinoma patients at different stages

Click here to view


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

Click here to view
Figure 1: Receiver operating characteristic curve analysis of circulating anti-CD25 IgG levels in hepatocellular carcinoma patients and controls

Click here to view



 > Discussion Top


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.[17] 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.[18] 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.[19],[20],[21] The increased number of Treg cells surrounding tumor tissues and in splenic tissues is associated with the low immune function, tumor recurrence, and metastasis.[22],[23],[24],[25] 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.[26] 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.[15] 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.

Acknowledgments

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.



 
 > References Top

1.
Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology 2005;42:1208-36.  Back to cited text no. 1
    
2.
Farinati F, Marino D, De Giorgio M, Baldan A, Cantarini M, Cursaro C, et al. Diagnostic and prognostic role of alpha-fetoprotein in hepatocellular carcinoma: Both or neither? Am J Gastroenterol 2006;101:524-32.  Back to cited text no. 2
[PUBMED]    
3.
Chapman C, Muray A, Chalrabarti J, Thorpe A, Woolston C, Sahin U, et al. Autoantibodies in breast cancer: Their use as an aid to early diagnosis. Ann Oncol 2007;18:868-73.  Back to cited text no. 3
    
4.
Tan HT, Low J, Lim SG, Chung MC. Serum autoantibodies as biomarkers for early cancer detection. FEBS J 2009;276:6880-904.  Back to cited text no. 4
[PUBMED]    
5.
Shara S, Khosla R, David P, Rastogi A, Vyas A, Singh D, et al. CD4+CD25+ CD127 (low) regulatory T cells play predominant anti-tumor suppressive role in hepatitis B virus-associated hepatocellular carcinoma. Front Immunol 2015;6:49.  Back to cited text no. 5
    
6.
Zhou Y, Wang B, Wu J, Zhang C, Zhou Y, Yang X, et al. Association of preoperative EpCAM circulating tumor cells and peripheral Treg cell levels with early recurrence of hepatocellular carcinoma following radical hepatic resection. BMC Cancer 2016;16:506.  Back to cited text no. 6
[PUBMED]    
7.
He X, Qu F, Zhou F, Zhou X, Chen Y, Guo X, et al. High leukocyte mtDNA content contributes to poor prognosis through ROS-mediated immunosuppression in hepatocellular carcinoma patients. Oncotarget 2016;7:22834-45.  Back to cited text no. 7
    
8.
Leonard WJ, Donlon TA, Lebo RV, Greene WC. Localization of the gene encoding the human interleukin-2 receptor on chromosome 10. Science 1985;228:1547-9.  Back to cited text no. 8
[PUBMED]    
9.
Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995;155:1151-64.  Back to cited text no. 9
[PUBMED]    
10.
Li CH, Kuo WH, Chang WC, Huang SC, Chang KJ, Sheu BC. Activation of regulatory T cells instigates functional down-regulation of cytotoxic T lymphocytes in human breast cancer. Immunol Res 2011;51:71-9.  Back to cited text no. 10
[PUBMED]    
11.
Erfani N, Mehrabadi SM, Ghayumi MA, Haghshenas MR, Mojtahedi Z, Ghaderi A, et al. Increase of regulatory T cells in metastatic stage and CTLA-4 over expression in lymphocytes of patients with non-small cell lung cancer (NSCLC). Lung Cancer 2012;77:306-11.  Back to cited text no. 11
[PUBMED]    
12.
Guan S, Liu B, Zhang C, Lee KH, Sun S, Wei J, et al. Circulating autoantibody to CD25 may be a potential biomarker for early diagnosis of esophageal squamous cell carcinoma. Clin Transl Oncol 2013;15:825-9.  Back to cited text no. 12
    
13.
Liu T, Song YN, Shi QY, Liu Y, Bai XN, Pang D, et al. Study of circulating antibodies against CD25 and FOXP3 in breast cancer. Tumour Biol 2014;35:3779-83.  Back to cited text no. 13
    
14.
Chen C, Wang W, Meng Q, Wu N, Wei J. Further study of circulating IgG antibodies to CD25-derived peptide antigens in non-small cell lung cancer. FEBS Open Bio 2016;6:211-5.  Back to cited text no. 14
[PUBMED]    
15.
Llovet JM, Fuster J, Bruix J; Barcelona-Clínic Liver Cancer Group. The Barcelona approach: Diagnosis, staging, and treatment of hepatocellular carcinoma. Liver Transpl 2004;10 Suppl 1:S115-20.  Back to cited text no. 15
    
16.
Hallford P, St. Clair D, Halley L, Mustard C, Wei J. A study of type-1 diabetes associated autoantibodies in schizophrenia. Schizophr Res 2016;176:186-90.  Back to cited text no. 16
    
17.
Hoofnagle JH. Hepatocellular carcinoma: Summary and recommendations. Gastroenterology 2004;127:S319-23.  Back to cited text no. 17
[PUBMED]    
18.
Qiu X, Wang X, Song Y, Chen L. Plasma level of interleukin-35 as an independent prognostic indicator in hepatocellular carcinoma. Dig Dis Sci 2016;61:3513-21.  Back to cited text no. 18
[PUBMED]    
19.
Piccirillo CA, Shevach EM. Cutting edge: Control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells. J Immunol 2001;167:1137-40.  Back to cited text no. 19
[PUBMED]    
20.
Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell 2008;133:775-87.  Back to cited text no. 20
[PUBMED]    
21.
Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 2003;4:330-6.  Back to cited text no. 21
[PUBMED]    
22.
Pan XD, Mao YQ, Zhu LJ, Li J, Xie Y, Wang L, et al. Changes of regulatory T cells and foxP3 gene expression in the aging process and its relationship with lung tumors in humans and mice. Chin Med J (Engl) 2012;125:2004-11.  Back to cited text no. 22
[PUBMED]    
23.
Lim HW, Hillsamer P, Banham AH, Kim CH. Cutting edge: Direct suppression of B cells by CD4+CD25+ regulatory T cells. J Immunol 2005;175:4180-3.  Back to cited text no. 23
[PUBMED]    
24.
Smyth MJ, Teng MW, Swann J, Kyparissoudis K, Godfrey DI, Hayakawa Y. CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer. J Immunol 2006;176:1582-7.  Back to cited text no. 24
[PUBMED]    
25.
Onishi Y, Fehervari Z, Yamaguchi T, Sakaguchi S. Foxp3+ natural regulatory T cells preferentially form aggregates on dendritic cells in vitro and actively inhibit their maturation. Proc Natl Acad Sci U S A 2008;105:10113-8.  Back to cited text no. 25
[PUBMED]    
26.
Whiteside TL. What are regulatory T cells (Treg) regulating in cancer and why? Semin Cancer Biol 2012;22:327-34.  Back to cited text no. 26
[PUBMED]    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  >Abstract>Introduction>Materials and Me...>Results>Discussion>Article Figures>Article Tables
  In this article
>References

 Article Access Statistics
    Viewed851    
    Printed16    
    Emailed0    
    PDF Downloaded31    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]