Targeted Proteins Reveal Cathepsin D as a Novel Biomarker in Differentiating Hepatocellular Carcinoma from Cirrhosis and Other Liver Cancers

Objective: Hepatocellular carcinoma (HCC) represents a global health concern, particularly in Southeast Asia where hepatitis B virus (HBV) infection is common. In this study, we applied tissue-based proteomics to identify novel serological proteins for HCC and validated their performance in serum specimens. Methods: In a discovery set, liver tissue specimens of HBV-related HCC, intrahepatic cholangiocarcinoma (iCCA) and colorectal cancer with liver metastasis (CRLM) were analyzed using mass spectrometry (LTQ-Orbitrap-XL). A subset of proteins that showed highly expressed in HCC were then confirmed by Western blotting. Additionally, clinical significance of selected candidate proteins was tested in serum samples of 80 patients with HBV-related HCC, 50 patients with HBV-related liver cirrhosis and 30 healthy controls. Results: Based on LTQ-Orbitrap-XL mass spectrometer, various differentially expressed proteins (DEPs) between tumor and adjacent non-tumor tissues were identified. These included 77 DEPs for HCC, 77 DEPs for iCCA and 55 DEPs for CRLM. Among selected candidate proteins, annexin A2 and cathepsin D were confirmed to be overexpressed in HCC tissue by Western blot analysis. In a validate cohort, serum cathepsin D level, but not annexin A2, was significantly higher in HCC compared with the non-HCC groups. Serum cathepsin D level was also positively correlated with tumor size and tumor stage. Additionally, the combined assay of serum cathepsin D and alpha-fetoprotein had a high sensitivity in detecting early HCC (83%) and intermediate/advanced HCC (96%). Moreover, patients with low serum cathepsin D (<305 ng/mL) displayed significantly better overall survival than those whose serum levels were high (≥305 ng/mL). Conclusions: Proteomics and subsequent validation revealed cathepsin D as a novel biomarker for HCC. Apart from its diagnostic role, serum cathepsin D might also serve as a prognostic biomarker of HCC. Additional large-scale studies are needed to verify our finding

Transcriptomic Analyses Reveal Long Non-Coding RNA in Peripheral Blood Mononuclear Cells as a Novel Biomarker for Diagnosis and Prognosis of Hepatocellular Carcinoma

Novel biomarkers are highly required for the diagnosis and predicting prognosis of hepatocellular carcinoma (HCC). In this study, we investigated the profiles of long non-coding RNAs (lncRNAs) obtained from the peripheral blood mononuclear cells (PBMCs) of patients with HCC and PBMCs from a co-culture model using transcriptomic analysis. The differentially expressed lncRNAs (DElncRNAs) were then characterized and integrated as cancer-induced lncRNAs. Among them, three up-regulating DElncRNAs including MIR4435-2HG, SNHG9 and lnc-LCP2-1 and one down-regulating, lnc-POLD3-2, were identified. The functional analysis showed that these enriched lncRNAs were mainly associated with carcinogenesis and immune responses. Following further validation in PBMCs samples (100 HBV-related HCC, 100 chronic hepatitis B and 100 healthy controls), MIR4435-2HG, lnc-POLD3-2 and their combination were revealed to be sensitive biomarkers in discriminating HCC from non-HCC (AUROC = 0.78, 0.80, and 0.87, respectively), particularly among individuals with normal serum alpha-fetoprotein levels. Additionally, high circulating SNHG9 expression was shown to be an independent prognostic factor of overall survival in patients with HCC. These results indicate that determining these lncRNAs in PBMCs could serve as novel diagnostic and prognostic biomarkers for HBV-related HCC

Diagnostic and prognostic roles of circulating miRNA-223-3p in hepatitis B virus-related hepatocellular carcinoma.

BackgroundCirculating microRNAs (miRNAs) have been shown to dysregulate in many cancer types including hepatocellular carcinoma (HCC). The purpose of this study was to examine the potential diagnostic or prognostic roles of circulating miRNAs in patients with hepatitis B virus (HBV)-related HCC.MethodsPaired cancerous and adjacent non-cancerous liver tissue specimens of patients with HBV-related HCC were used as a discovery set for screening 800 miRNAs by a Nanostring quantitative assay. Differentially expressed miRNAs were then examined by SYBR green quantitative RT-PCR in a validation cohort of serum samples obtained from 70 patients with HBV-related HCC, 70 HBV patients without HCC and 50 healthy controls.ResultsThe discovery set identified miR-223-3p, miR-199a-5p and miR-451a significantly lower expressed in cancerous tissues compared with non-cancerous tissues. In the validated cohort, circulating miR-223-3p levels were significantly lower in the HCC group compared with the other groups. The combined use of serum alpha-fetoprotein and miR-223-3p displayed high sensitivity for detecting early HCC (85%) and intermediate/advanced stage HCC (100%). Additionally, serum miR-223-3p had a negative correlation with tumor size and BCLC stage. On multivariate analysis, serum miR-223-3p was identified as an independent prognostic factor of overall survival in patients with HCC. In contrast, circulating miRNA-199a-5p and miR-451a did not show any clinical benefit for the diagnosis and prognostic prediction of HCC.ConclusionsOur results demonstrated that miR-223-3p was differentially expressed in cancerous compared with paired adjacent non-cancerous tissues. In addition, circulating miRNA-223-3p could represent a novel diagnostic and prognostic marker for patients with HBV-related HCC

Cell-Free DNA Analysis by Whole-Exome Sequencing for Hepatocellular Carcinoma: A Pilot Study in Thailand

Cell-free DNA (cfDNA) has been used as a non-invasive biomarker for detecting cancer-specific mutations. However, the mutational profile of cfDNA in Thai patients with hepatocellular carcinoma (HCC) has not been investigated. Here, we demonstrated the utility of using whole-exome sequencing (WES) of cfDNA to define the somatic mutation profiles of HCC in Thai patients. The comprehensive profile of cfDNA was determined with WES to identify variants in matched cfDNA and germline DNA from 30 HCC patients in Thailand who underwent nonoperative therapies. The level of cfDNA was higher in HCC patients compared with chronic hepatitis patients (p-value &lt; 0.001). Single nucleotide variants were present in somatic genes in cfDNA, including in ZNF814 (27%), HRNR (20%), ZNF492 (20%), ADAMTS12 (17%), FLG (17%), OBSCN (17%), TP53 (17%), and TTN (17%). These same mutations were matched to HCC mutation data from The Cancer Genome Atlas (TCGA) and a previous Thai HCC study. The co-occurrence of HRNR and TTN mutations in cfDNA was associated with shorter overall survival in HCC patients (hazard ratio = 1.60, p-value = 0.0196). These findings indicate that the mutational profile of cfDNA accurately reflected that of HCC tissue and suggest that cfDNA could serve as a useful biomarker for diagnosis and prognosis in Thai HCC patients. In addition, we demonstrated the use of the pocket-sized sequencer of Oxford Nanopore Technology to detect copy-number variants in HCC tissues that could be applied for onsite clinical detection/monitoring of HCC