Mei Liu

Autoantibodies to tumor-associated antigens as biomarkers in human hepatocellular carcinoma (HCC)

Tumor-associated antigens (TAAs) recognized by cellular and/or humoral effectors of the immune system are attractive targets for diagnostic and therapeutic approaches to human cancer. Different approaches can be used to comprehensively characterize and validate the identified TAA/anti-TAA systems, which are potential biomarkers in cancer immunodiagnosis. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The high fatality rate of HCC within one year after its detection might be partly attributed to a lack of diagnostic methods that enable the early detection. Our previous studies have shown that novel autoantibodies can appear which are not detected prior to pre-malignant conditions during transition from chronic liver disease to HCC. The hypothesis we advance is the transition to malignancy can be associated with autoantibody response to certain cellular proteins that might have some role in tumorigenesis. We propose that the information that the cancer patient’s immune system is conveying in the form of autoantibodies to tumor-associated antigens (TAAs) should be utilized to a greater extent in identifying early signs of tumorigenesis. In this review, we will focus on the important features of TAA and the possibility that autoantibodies to TAAs can be used as biomarkers in immunodiagnosis and prognosis of HCC.

Using immunomic approach to enhance tumor-associated autoantibody detection in diagnosis of hepatocellular carcinoma.

To explore the possibility of using a mini-array of multiple tumor-associated antigens (TAAs) as an approach to the diagnosis of hepatocellular carcinoma (HCC), 14 TAAs were selected to examine autoantibodies in sera from patients with chronic hepatitis, liver cirrhosis and HCC by immunoassays. Antibody frequency to any individual TAA in HCC varied from 6.6% to 21.1%. With the successive addition of TAAs to the panel of TAAs, there was a stepwise increase of positive antibody reactions. The sensitivity and specificity of 14 TAAs for immunodiagnosis of HCC was 69.7% and 83.0%, respectively. This TAA mini-array also identified 43.8% of HCC patients who had normal alpha-fetoprotein (AFP) levels in serum. In summary, this study further supports the hypothesis that a customized TAA array used for detecting anti-TAA autoantibodies can constitute a promising and powerful tool for immunodiagnosis of HCC and may be especially useful in patients with normal AFP levels.

Using immunoproteomics to identify tumor-associated antigens (TAAs) as biomarkers in cancer immunodiagnosis

Since intracellular proteins involved in carcinogenesis have been shown to provoke autoantibody responses, autoantibodies can be used as probes in immunoproteomics to isolate, identify, and characterize potential tumor-associated antigens (TAAs). Once a TAA is identified, several approaches will be used to comprehensively characterize and validate the identified TAA/anti-TAA systems that are potential biomarkers in certain types of cancer. Our ultimate goal is to establish rigorous criteria for designation of an autoantibody to a TAA as a cancer biomarker, examine candidate TAAs for sensitivity and specificity of anti-TAA antibody response, and further develop customized TAA arrays that can be used to enhance anti-TAA antibody detection in cancer. This review will mainly focus on the recent advances in our studies using immunoproteomic approach to identify and characterize TAAs as biomarkers in cancer.

Peroxiredoxin 1 is a tumor-associated antigen in esophageal squamous cell carcinoma

Peroxiredoxin 1 (Prdx1) is an antioxidant and plays an important role in H2O2-mediated cell signaling. We previously found that the expression level of Prdx1 was elevated in esophagus squamous cell carcinoma (ESCC) tissue using a proteomics approach. Since overexpressed protein can induce an autoimmune response, to further examine whether serum from ESCC patients exhibits immunoreactivity against Prdx1, autoantibody responses to Prdx1 were evaluated by ELISA, western blotting and indirect immunofluorescence assay in sera from patients with ESCC and normal individuals. Immunohistochemical study with tissue array slides and western blot analysis with cancer cell lines were also performed to analyze the protein expression profiles of Prdx1 in ESCC tissues and cancer cell lines. The results demonstrated that the positive rate of autoantibody against Prdx1 in ESCC sera was 13.2% (9/68), whereas this rate was 0% (0/89) in normal individuals. Data also showed that expression of Prdx1 was significantly increased in ESCC tissues when compared to expression in paired adjacent normal tissues (P<0.05). The data indicate that Prdx1 may contribute to malignant transformation of the esophagus, and may be used as a biomarker in the immunodiagnosis of ESCC.

Autoantibody response to a novel tumor-associated antigen p90/CIP2A in breast cancer immunodiagnosis

There is an urgent need to identify relevant tumor markers showing high sensitivity and specificity for early immunodiagnosis of breast cancer. Autoantibodies directed against tumor-associated antigens (TAAs) have been shown to be relevant tumor markers. The purpose of this study was to evaluate whether autoantibodies to a tumor-associated antigen p90/CIP2A can be used as diagnostic markers in breast cancer. In this study, autoantibody responses to p90/CIP2A were evaluated by enzyme-linked immunosorbent assay (ELISA), western blotting, and indirect immunofluorescence assay in sera from patients with breast cancer and normal human individuals. The results have demonstrated that p90/CIP2A can induce a relatively higher frequency of autoantibody response in breast cancer (19.1xa0%) compared to the sera of normal individuals (2.3xa0%). The frequency of p90/CIP2A expression in breast cancer tissues was significantly higher than that in adjacent normal tissues (Pu2009<u20090.01). Our preliminary results suggest that autoantibodies against p90/CIP2A may be a useful serum biomarker for early stage breast cancer screening and diagnosis.

A cancer-related protein 14-3-3ζ is a potential tumor-associated antigen in immunodiagnosis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related deaths worldwide. Serum alpha-fetoprotein (AFP) is the conventional biomarker currently used in clinical diagnosis of this malignancy. However, AFP is not reliable for early diagnosis, and especially the sensitivity and specificity of AFP in HCC diagnosis are not optimal. Early detection of HCC is an important issue because of the very poor prognosis and usually no more than 6xa0months survival after diagnosis. Therefore, there is a need for the development of more sensitive and specific methods that can supplement AFP in the early detection of this cancer. In this study, autoantibody responses to 14-3-3ζ in HCC were evaluated by enzyme-linked immunosorbent assay (ELISA), western blot, and indirect immunofluorescence assay. Immunohistochemistry (IHC) with tissue array slides was also performed to analyze protein expression of 14-3-3ζ in HCC and control tissues. The prevalence of autoantibodies against 14-3-3ζ was 16.7xa0% (28/168) in HCC, which was significantly higher than that in liver cirrhosis (LC), chronic hepatitis (CH), and normal human sera (NHS) (Pu2009<u20090.01). The average titer of autoantibodies against 14-3-3ζ in HCC sera was higher compared to that in LC, CH, and NHS (Pu2009<u20090.01). In the further study, anti-14-3-3ζ antibodies have been detected in the sera from several HCC patients with serial bleeding samples. A stronger reactive band with 14-3-3ζ in western blot can be seen in sera at 9xa0months before the clinical diagnosis of HCC. Our preliminary data indicate that anti-14-3-3ζ autoantibodies may be potential biomarkers for early-stage HCC screening and diagnosis.

Humoral autoimmune response to nucleophosmin in the immunodiagnosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a worldwide prevalent cancer with an exremely poor prognosis. Detection of serum α-fetoprotein (AFP) and liver imaging techniques are the conventional methods used clinically for the identification of this malignancy. However, these techniques are not reliable for early diagnosis, and particularly the sensitivity and specificity of AFP in HCC diagnosis are not optimal. Therefore, there is an urgent need for the development of more sensitive and specific methods that can improve AFP quantification in the early detection of HCC. In the present study, autoantibody responses to nucleophosmin (NPM1) in HCC patients were evaluated by enzyme-linked immunosorbent assay (ELISA), western blotting and indirect immunofluorescence. Immunohistochemistry (IHC) with tissue array slides was also performed to analyze protein expression of NPM1 in HCC and control tissues. The prevalence of autoantibodies against NPM1 was 22.4% (17/76) in HCC, which was significantly higher than that in sera from patients with liver cirrhosis (LC), chronic hepatitis (CH) and systemic lupus erythematosus (SLE) (P<0.01). The average titer of autoantibodies against NPM1 in HCC sera was higher compared to that in LC, CH, SLE and normal human sera (NHS) (P<0.01). In addition, anti-NMP1 autoantibodies were detected in sera from several HCC patients with serial bleeding samples. A stronger reactive band corresponding to NMP1 was visualized in the western blot analyses, utilizing sera from patients 3–6 months before the clinical diagnosis of HCC. Our data indicate that NPM1 and the anti-NPM1 system may have potential as an early-stage biomarker for HCC screening and diagnosis.

Overexpression of HCC1/CAPERα may play a role in lung cancer carcinogenesis

HCC1/CAPERα is considered to be a novel human tumor-associated antigen, and the tumor-specific immunity of HCC1/CAPERα has been reported in several types of cancer. However, there was very limited evidence indicating its function in tumorigenesis. In the present study, to elucidate the roles and underlying molecular mechanism of HCC1/CAPERα in lung cancer, we examined the expression of HCC1/CAPERα in human non-small cell lung cancer (NSCLC) cell line and NSCLC tissue microarray (TMA). Immunohistochemistry with TMA was performed to detect HCC1/CAPERα expression in NSCLC and adjacent lung tissues. NSCLC cell line constitutively transfected by pcDNA3.1-HCC1/CAPERα, and empty pcDNA3.1 vector were used. These cells were analyzed by Western blot, MTT, immunofluorescence, wound healing assay, and transwell assays. It was found that HCC1/CAPERα was mainly localized in the nucleus of the lung cancer cells and overexpression of HCC1/CAPERα may promote lung cancer cells proliferation and increase cells migration. The frequency of HCC1/CAPERα expression in NSCLC tissues was significantly higher than that in adjacent and normal tissues (Pu2009<u20090.01). Our data suggest that overexpression of HCC1/CAPERα may increase the proliferation and migration of NSCLC cells, and HCC1/CAPERα could be a promising biomarker for lung cancer.

Autoantibody Response to Murine Double Minute 2 Protein in Immunodiagnosis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. Although new therapeutic strategies have been continuously developed and applied to clinical treatment for HCC, the prognosis is still very poor. Thus, early detection of HCC may enhance effective and curative management. In this study, autoantibody responses to MDM2 protein in HCC patients serum were evaluated by enzyme-linked immunosorbent assay (ELISA) and part sera were evaluated by Western blotting and indirect immunofluorescence assay. Immunohistochemistry (IHC) over tissue array slides was also performed to analyze protein expression of MDM2 in HCC and control tissues. The prevalence of autoantibodies against MDM2 was significantly higher than that in liver cirrhosis (LC), chronic hepatitis (CH), and normal human sera (NHS). The average titer of autoantibodies against MDM2 in HCC serum was higher compared to that in LC, CH, and NHS. A high titer of autoantibodies against MDM2 in ELISA could be observed in the serum in 6 to 9 months before the clinical diagnosis of HCC in the serum of several HCC patients with serial bleeding samples. Our preliminary data indicate that MDM2 and anti-MDM2 system may be a potential biomarker for early stage HCC screening and immunodiagnosis.

Abstract 885: Using immunomic approach to enhance tumor-associated autoantibody detection in diagnosis of hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnMany studies demonstrated that the immune system can recognize the antigenic changes in cancer cells, and further develop autoantibodies against these antigens which have been called tumor-associated antigens (TAAs). During transition from chronic liver disease to hepatocellular carcinoma (HCC), autoantibodies can appear which are not detected in the pre-malignant conditions. In order to explore the possibility and usefulness of using a mini-array of multiple TAAs as an approach in the diagnosis of HCC, a total of 14 TAAs, including IMP1, IMP2/p62, IMP3/Koc, p53, Survivin, p16, CAPERα, CIP2A/p90, RalA, NPM1, MDM2 and 14-3-3 ζ, c-Myc and CyclinB1, were selected for expression of full-length recombinant proteins. Antibodies against 14 TAAs in 30 sera from patients with chronic hepatitis, 30 sera from patients with liver cirrhosis and 76 sera from patients with HCC as well as sera from 16 HCC patients with serial bleeding samples were detected by enzyme-linked immunosorbent assay (ELISA). Positive results were also confirmed by Western blotting. Antibody frequency to any individual TAA in HCC varied from 6.6% to 21.1%. With the successive addition of TAAs to the panel of TAAs, there was a stepwise increase of positive antibody reactions. According to the evaluation indexes for accuracy of screening test, the sensitivity and specificity in using a panel of 14 TAAs for immunodiagnosis of HCC was 69.7% and 83.0%, respectively. The interesting finding was that these anti-TAA autoantibodies could be detected at least one year before the diagnosis of HCC, suggesting that using a penal of 14 TAAs for enhancing autoantibody detection would be a valuable approach in the early diagnosis of HCC. The mini-array of 14 TAAs can identify 43.8% HCC patients who shows serum alpha-fetoprotein (AFP) negative. This study deals with the concept of “cancer immunomics” which allows a global analysis of the auto-antibodies against antigens in a neoplasm. It also determinates that malignant transition to HCC is associated with increased autoantibody responses to certain cellular proteins. In summary, the data from this study further support the hypothesis that a customized TAA array can constitute promising and powerful tool for enhancing the serological anti-TAA antibody detection of HCC, especially in patients with serum AFP negative.nnCitation Format: Liping Dai, Mei Liu, Qing Zhu, Xinxin Liu, Yurong Chai, Pengfei Ren, Kaijuan Wang, Chunhua Song, Peng Wang, Mingan Wang, Eng M. Tan, Jianying Zhang. Using immunomic approach to enhance tumor-associated autoantibody detection in diagnosis of hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 885. doi:10.1158/1538-7445.AM2014-885