Bei Zhang

An Analysis of Immunoreactive Signatures in Early Stage Hepatocellular Carcinoma

Background Hepatocellular carcinoma (HCC) is prevalent worldwide and early diagnosis of HCC is critical for effective treatment and optimal prognosis. Methods Serum was screened first by immunoproteomic analysis for HCC-related tumor associated antigens (TAAs). Selected TAAs were clinically evaluated retrospectively in patients with HCC, liver cirrhosis, chronic hepatitis and healthy controls. Levels of autoantibody to the selected TAAs were measured by protein microarrays containing protein antigens of the candidate TAAs. Analyses were done by using receiver operating characteristics (ROC) to calculate diagnostic accuracy. Findings Twenty-two candidate TAAs were assessed by protein microarray analysis in 914 participants with serum α-fetoprotein (AFP) available. Twelve candidate TAAs were statistically different in signal intensity between HCC and controls. Among them, CENPF, HSP60 and IMP-2 showed AUC (area under the curve) values of 0.826, 0.764 and 0.796 respectively for early HCC. The highest prevalence of autoantibody positivity was observed in HCC cases with BCLC tumor stage A, well-differentiated histology and Child-Pugh grade C. Specifically, 73.6% or 79.3% cases of early HCC with negative AFP were positive for autoantibody to CENPF or HSP60. Interpretation Tumor-associated autoimmune reactions may be triggered by early stage HCCs. Measurement of serum autoantibody to TAAs may be complementary to AFP measurements and improve diagnosis of early HCC.

Novel role of apatinib as a multi-target RTK inhibitor in the direct suppression of hepatocellular carcinoma cells

Although apatinib has been demonstrated with potential antitumor activity in multiple solid tumors, the underlying mechanism of apatinib for the treatment of hepatocellular carcinoma (HCC) remains unclear. In the present study, we explored if there are any direct suppression effects of apatinib on HCC cells and its relevant targets. We investigated the effect of apatinib on viability of five HCC cell lines and an intrahepatic cholangiocarcinoma cell line, and colony formation, apoptosis and migration of representative HCC cells in vitro; and HCC progression in a xenograft mouse model. Using a phospho-receptor tyrosine kinase pathway array with 49 different tyrosine kinases, we screened and verified the tyrosine kinase targets involved in apatinib response. Apatinib treatment significantly inhibited HCC cell viability, proliferation, colony formation, and migration, and enhanced cell apoptosis in a concentration-dependent manner (p < 0.05). Furthermore, apatinib showed a favorable anti-tumor growth effect (71% of inhibition ratio, p < 0.05) in an established human HCC xenograft mice model with good safety. RTK pathway arrays and western blots analysis demonstrated that apatinib significantly downregulated the phosphorylation levels of several tyrosine kinase receptors, particularly PDGFR-α and IGF-IR, and inhibited Akt phosphorylation. These data suggest that the apatinib may have a direct anti-HCC effect as a direct multi-target RTK inhibitor of HCC cells and a promising potentiality in HCC clinical therapies.

Screening and clinical evaluation of dominant peptides of centromere protein F antigen for early diagnosis of hepatocellular carcinoma

Tumor-associated antigens, such as centromere protein F (CENP‑F), have been recognized as potential serological biomarkers for early diagnosis of hepatocellular carcinoma (HCC); however, the exact regions corresponding to the dominant peptides of CENP‑F antigen remain to be explored. We aimed to screen and evaluate potential dominant peptides of CENP‑F for early diagnosis of HCC. Dominant peptides of CENP‑F were predicted by BioSun version 3.0, and the corresponding recombinant proteins were prepared. Enzyme‑linked immunosorbent assays were conducted for initial screening of dominant peptides, and selected dominant peptides were subjected to further clinical evaluation. Eight dominant peptides of CENP‑F antigens were predicted at amino acids (a.a) 121‑220, 335‑416, 1100‑1265, 1670‑1791, 1759‑2093, 2075‑2210, 2485‑2592, and 2808‑2960. Initial screening of the predicted peptides in samples of 47 HCC cases showed the highest diagnostic value for 121‑220 a.a and 1670‑1791 a.a peptides with area under the curve (AUC) values of 0.795 [95% confidence interval (CI), 0.706‑0.884] and 0.809 (95% CI, 0.721‑0.896), sensitivity of 58.3 and 85.4%, and specificity of 93.9 and 65.3%, respectively. Further evaluation of the two peptides in 405 samples comprised of 153 HCC, 126 liver cirrhosis and 126 healthy controls, presenting an AUC of 0.743 (95% CI, 0.674‑0.812) for 121‑220 a.a peptide in detecting early‑stage HCCs. Specifically, the 121‑220 a.a peptide showed a complementary effect in combination with α‑fetoprotein (AFP) for the detection of early‑stage HCC with increased AUC value of 0.840 (95% CI, 0.781‑0.899), and sensitivity of 81.4% and specificity of 72.2%. In conclusion, our study identified the 121‑220 a.a dominant peptide as the region of CENP‑F antigen with the highest immunogenicity and demonstrated its value in combination with AFP for diagnosis of early-stage HCC.

Non-HFE mutations in haemochromatosis in China: combination of heterozygous mutations involving HJV signal peptide variants

Introduction Hereditary haemochromatosis (HH) caused by a homozygous p.C282Y mutation in haemochromatosis (HFE) gene has been well documented. However, less is known about the causative non-HFE mutation. We aimed to assess mutation patterns of haemochromatosis-related genes in Chinese patients with primary iron overload. Methods Patients were preanalysed for mutations in the classic HH-related genes: HFE, HJV, HAMP, TFR2 and SLC40A1. Whole exome sequencing was conducted for cases with variants in HJV signal peptide region. Representative variants were analysed for biological function. Results None of the cases analysed harboured the HFE p.C282Y; however, 21 of 22 primary iron-overload cases harboured at least one non-synonymous variant in the non-HFE genes. Specifically, p.E3D or p.Q6H variants in the HJV signal peptide region were identified in nine cases (40.9%). In two of three probands with the HJV p.E3D, exome sequencing identified accompanying variants in BMP/SMAD pathway genes, including TMPRSS6 p.T331M and BMP4 p.R269Q, and interestingly, SUGP2 p.R639Q was identified in all the three cases. Pedigree analysis showed a similar pattern of combination of heterozygous mutations in cases with HJV p.E3D or p.Q6H, with SUGP2 p.R639Q or HJV p.C321X being common mutation. In vitro siRNA interference of SUGP2 showed a novel role of downregulating the BMP/SMAD pathway. Site-directed mutagenesis of HJV p.Q6H/p.C321X in cell lines resulted in loss of membrane localisation of mutant HJV, and downregulation of p-SMAD1/5 and HAMP. Conclusion Compound heterozygous mutations of HJV or combined heterozygous mutations of BMP/SMAD pathway genes, marked by HJV variants in the signal peptide region, may represent a novel aetiological factor for HH.

Direct comparison of five serum biomarkers in early diagnosis of hepatocellular carcinoma

Background Although a number of serum biomarkers for detection of hepatocellular carcinoma (HCC) have been explored, their exact diagnostic value remains unclear. We aimed to conduct a direct comparison of five representative serum biomarkers for detecting HCC and to derive multi-marker prediction algorithms. Patients and methods In total, 846 patients were recruited from three hospitals in China, including 202 HCC patients, 226 liver cirrhosis patients, 215 chronic hepatitis B virus-infected patients, and 203 healthy volunteers. Serum levels of alpha-fetoprotein (AFP), lens culinaris agglutinin-reactive AFP (AFP-L3), des-gamma-carboxyprothrombin (DCP), squamous cell carcinoma antigen, and centromere protein F autoantibody were measured by ELISA. The diagnostic performances of individual biomarkers and multi-marker combinations were evaluated by receiver operating characteristics analysis. The bootstrapping method was adopted to adjust for potential overfitting of all diagnostic indicators. Results DCP exhibited the best diagnostic performance, with areas under the curve (AUC) for detecting HCC of 0.82 (95% CI 0.64–0.80) and sensitivity of 65.2% (95% CI 63.3–82.1%) at 90% specificity. Of note, DCP showed similar diagnostic efficacy for detecting AFP-positive and AFP-negative HCC. After a comprehensive search for multi-marker combinations, a two-marker prediction algorithm including AFP and DCP was constructed and yielded an AUC of 0.87 (95% CI 0.68–0.84) for detecting HCC. In addition, the combination showed good ability in discriminating early-stage HCC and decompensated liver cirrhosis, with an AUC of 0.81 (95% CI 0.75–0.86). Conclusion DCP could be a complementary biomarker in the early diagnosis of HCC. The constructed multi-marker prediction algorithms could contribute toward distinguishing HCC from non-malignant chronic liver diseases.

Molecular alterations of the NF2 gene in hepatocellular carcinoma and intrahepatic cholangiocarcinoma

Neurofibromatosis type 2 with mutations in the neurofibromin 2 (NF2) gene, encoding the Merlin protein, is an autosomal dominant disorder characterized by enhanced cancer predisposition, particularly tumors of the central nervous system. Recent animal studies indicate that disruption of NF2/Merlin function in oval cells, which are hepatic progenitor cells, may lead to the development of primary liver cancers including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC); however, its role in human primary liver cancer remains unclear. In the present study, we explored the role of NF2/Merlin in human primary liver cancers. Tumor tissues (n=144) were used for the screening of NF2 mutation, while whole blood samples from 219 HCC and 194 healthy control cases were used for analysis of single nucleotide polymorphisms (SNPs) in liver cancer. The expression and amplification of NF2/Merlin and its downstream gene in the Hippo pathway, Yes-associated protein (YAP), were also analyzed. Missense NF2 mutations were identified in 2 of 106 (1.9%) HCCs and 2 of 38 (5.3%) ICCs. Allele frequency of NF2 IVS4-39 A/A was significantly higher in the HCCs than that in the healthy controls. Noteworthy, NF2/Merlin showed a dual role as a tumorigenic gene and tumor-suppressor gene; Merlin was expressed at higher levels in tumors than in adjacent non-tumor tissues of HCC; while the rate of Merlin upregulation was significantly lower in poorly differentiated ICCs. In addition, a significant negative correlation between Merlin and YAP expression was observed in ICC. In conclusion, we provide initial evidence of human primary liver cancers characterized by molecular alterations of NF2/Merlin and the involvement of the Hippo pathway in the pathogenesis of human liver cancer.

Development and evaluation of an unlabeled probe high‐resolution melting assay for detection of ATP7B mutations in Wilson's disease

Wilsons disease (WD) is a rare autosomal recessive disorder characterized by the deposition of copper mainly in the liver or nerve system that leads to their dysfunction. Mutations in the gene encoding ATPase, Cu+ transporting, beta polypeptide (ATP7B) are causative for WD. The aim of this study was to develop a rapid and convenient assay for detection of the three most common causative ATP7B mutations, p.R778L, p.P992L, and p.V1106I.

“Haemochromatotic” characteristics of the human BEL‐7402 cell line

Hereditary haemochromatosis (HH) is an inherited disease characterized by excessive absorption and accumulation of iron in various organs (Bacon et al, 2011), and is related to mutations in the HFE, HFE2, HAMP, TFR2 and SLC40A1 genes (Pietrangelo, 2015). Mutations in HFE2 associated with HH results in reduced phosphorylation of Smad1/5/8 and hepcidin (HAMP) down-regulation (Babitt et al, 2006). As liver cells are the most affected cells in HH, immortalized liver cell lines including hepatoma cell lines are important tools to investigate the mechanism and pathogenesis of HH. Here, we showed that a widely used hepatoma cell line, BEL7402, carries a HFE2 mutation, resulting in impaired