Identification of Hepatocellular-Carcinoma-Associated Antigens and Autoantibodies by Serological Proteome Analysis Combined with Protein Microarray

Abstract

To comprehensively study autoantibodies in patients with hepatocellular carcinoma (HCC), we used an approach-based serology and proteomics technologies. Total proteins extracted from HepG2 cells and HepG2.2.15 cells were separated by two-dimensional gel electrophoresis (2DE) and then transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently incubated with sera from HCC patients or from normal controls. As a result, 13 HCC-associated antigens were identified. Antigenicity of eight proteins was further confirmed using recombinant proteins by Western blotting (WB) and protein microarray. The results of antigen microarray analysis showed strong signals of keratin 8 and lamin A/C in chronic hepatitis controls; therefore, the autoantibodies to keratin 8 and lamin A/C may not be HCC-specific. These two antigens were removed from subsequent analyses. The frequencies of positive reactions to DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, eukaryotic translation elongation factor 2 (eEF2), apoptosis-inducing factor (AIF), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), prostatic binding protein, and triosephosphate isomerase (TIM) were significantly higher in HCC than in chronic hepatitis and normal individuals. Positive reactions to DEAD box polypeptide 3, eEF2, AIF, and prostatic binding protein were significantly more frequent in HCC than in any other cancer. The sensitivity of any individual antigen in HCC at stage I ranged from 50 to 85%. When the combinations of six antigens were analyzed, the sensitivity increased to 90%. We conclude that the detection of autoantibodies against the six antigens may have value on early diagnosis of HCC.

Proteomic Analysis of Differentially Expressed Proteins in Peripheral Cholangiocarcinoma

“…Other proteins that have also been shown to be elevated in hepatocellular carcinoma include fibrinogen gamma chain precursor [30], glutathione S transferase [31], glycerol-3-phosphate dehydrogenase [32], histone-binding protein RBBP4 [33] and nucleophosmin [34]. Lamin-A/C was elevated in sera of patients with a hepatocellular carcinoma [35]. Other proteins have been shown to be associated with different tumor types such as L-lactate dehydrogenase B chain which is elevated in lung cancer [36], niban which is overexpressed in renal tumors [37], and non-syndromic hearing impairment protein 5 which shows altered expression in colorectal cancer [38].…”

Autoantibodies against tumor-associated antigens for detection of hepatocellular carcinoma

“…However, tumors > 5 cm in diameter are present more frequently in patients with anti-eEF2 autoantibodies compared with those with small tumors (> 5 cm in diameter) (P < 0.05) [35] . The rates of detection of autoantibodies against AIF and hnRNP A2 in patients with HCC without regional lymph node metastasis were significantly higher compared with those with regional lymph node metastasis (P < 0.05) [35] . There is a significant difference in size of tumors of patients with HCC cases that correlates with prevalence of autoantibodies against hnRNP L-67-88, with the average tumor size of 5.84 ± 4.23 cm in patients with detectable autoantibodies whereas 3.70 ± 2.07 cm in patients without detectable autoantibodies [36] .…”

“…There is evidence, however, showing that there are no statistically significant differences in patients with HCC in the prevalence of autoantibodies against DEAD box 3, eEF2, AIF, hnRNP A2, PBP, and TIM and patients' characteristics of sex, histological grade, or TNM classification [35] . However, tumors > 5 cm in diameter are present more frequently in patients with anti-eEF2 autoantibodies compared with those with small tumors (> 5 cm in diameter) (P < 0.05) [35] . The rates of detection of autoantibodies against AIF and hnRNP A2 in patients with HCC without regional lymph node metastasis were significantly higher compared with those with regional lymph node metastasis (P < 0.05) [35] .…”

Serologic Autoantibodies as Diagnostic Cancer Biomarkers—A Review

“…SERPA and protein microarray studies have found autoantibodies to proteins, including EEF2, heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked (DDX3X), apoptosis inducing factor (AIF), prostatic binding protein (PBP), and TIP to be significantly higher in patients with hepatocellular carcinoma than in healthy individuals or patients with chronic hepatitis. The sensitivity of any of the four markers: DDX3X, PBP, EEF2, and AIF was found to be 50% to 85% and increased to 90% when analyzed as a biomarker panel (115).…”