Diagnostic Biomarker For Hepatocellular Carcinoma Research Articles

Development of a novel panel based on micro-RNAs (21, 29a, 200 and 335) and alpha-fetoprotein as diagnostic biomarkers for hepatocellular carcinoma associated with hepatitis C infection

Background and study aimsMicroRNAs (miRNAs) play key roles in cancer biology; they are used as potential tools in cancer diagnosis. This study investigated the microRNA expression profile of patients with hepatocellular carcinoma (HCC). Patients and methodsSerum microRNA expression profiles (miRNA-29a, miRNA-200, miRNA-335 and miRNA-21) were analysed in 137 patients with HCC and liver cirrhosis and 49 healthy subjects (used as negative controls) using real-time quantitative reverse transcription polymerase chain reaction. Alpha-fetoprotein (AFP), as a routine tumour marker, was also assessed using enzyme-linked immunosorbent assay. ResultsThe expression levels of miRNA-21, miRNA-335 and miRNA-200 were significantly up-regulated, whereas those of miRNA-29a were remarkably down-regulated in patients with HCC compared with those in healthy subjects. miRNA-200 had the most elevated area under the receiver operating characteristic curve (AUC) among single miRNAs used to predict HCC occurrence (AUC = 0.72). The highest discriminatory power was recorded using a panel based on the combination of four miRNAs, miRNA-200, miRNA-29a, miRNA-21 and miRNA-355, and AFP levels (AUC = 0.92). The four-miRNA panel combined with AFP levels exhibited high accuracy in predicting HCC with small tumour sizes of <2 cm (AUC = 0.90) and ≥2 cm (AUC = 0.93). The combination of the four-miRNA panel and AFP resulted in an AUC value of 0.83 for single lesions, which was lesser than that recorded for ≥2 lesions (AUC = 0.94, 0.95, respectively). ConclusionThe combination of the four-miRNA panel and AFP levels can be used as a sensitive and specific biomarker for HCC.

Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 is a diagnostic and prognostic biomarker for hepatocellular carcinoma.

BACKGROUNDPhosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-Rex1) was reported to be a risk factor in several cancers, including breast cancer, lung cancer, and melanoma, but its expression and role in hepatocellular carcinoma (HCC) have not yet been fully studied.AIMTo explore the expression of P-Rex1 in HCC, and further evaluate its potential application in the diagnosis and prognosis of HCC, especially in hepatitis B virus (HBV)-related patients.METHODSP-Rex1 expression in HCC was evaluated by real-time-quantitative polymerase chain reaction. The expression of P-Rex1 was subjected to correlation analysis with clinical features, such as lymph node invasion, distant metastasis, HBV infection, patient's age and gender. Receiver operating characteristic analysis was used to examine the potential role of P-Rex1 as a diagnostic biomarker in HCC. Kaplan-Meier analysis was used to determine the association between P-Rex1 expression and overall survival, progression-free survival and relapse-free survival. Bioinformatic analysis was used to validate the clinical findings.RESULTSP-Rex1 expression was significantly increased in HCC tumors than adjacent tissues. The expression of P-Rex1 was higher in HCC patients with lymph node invasion, distant metastasis, HBV infection and positive alpha-fetoprotein, respectively. The receiver operating characteristic analysis showed that P-Rex1 was a diagnostic biomarker with a higher area under the curve value, especially in patients with HBV infection. Survival analysis showed that patients with higher P-Rex1 expression had a favorable survival rate, even in early-stage patients.CONCLUSIONP-Rex1 expression was highly increased in HCC, and the expression level of P-Rex1 was positively correlated with tumor progression. P-Rex1 has a higher efficiency in the diagnosis of HBV-related HCC, and could also be used as a favorable prognostic factor for HCC patients.

Hypermethylation of SCAND3 and Myo1g Gene Are Potential Diagnostic Biomarkers for Hepatocellular Carcinoma.

Presently, there is a lack of effective blood-based biomarkers facilitating the diagnosis of hepatocellular carcinoma (HCC). Thus, we aimed to investigate novel methylation markers for HCC diagnosis, and explore relationships between biomarker methylation and clinicopathology of HCC. The methylation status of the SCAN domain containing three (SCAND3) and myosin 1g (Myo1g) genes in HCC cell lines and tissues were detected by digital droplet PCR. The serum SCAND3 and Myo1g methylation levels were analyzed in HCC-afflicted patients and unafflicted controls. The results indicated SCAND3 and Myo1g methylation were abnormally high in the HCC cell lines and tissues. The values of serum SCAND3, Myo1g, and SCAND3 + Myo1g methylation with respect to facilitating the detection, and early detection of HCC were better than for alpha-fetoprotein (AFP) alone. Furthermore, when we combined SCAND3 + Myo1g with AFP, a high sensitivity and specificity resulted. Notably, in the AFP-negative HCC group, the methylation of SCAND3 and Myo1g also showed an excellent diagnostic performance. Besides this, a high serum SCAND3 methylation level was an independent risk factor for predicting portal vein tumor thrombus (PVTT) in HCC patients (OR = 4.746, p = 0.013). Finally, SCAND3 and Myo1g enhanced the HCC diagnostics as noninvasive serum methylation biomarkers, and the SCAND3 methylation status effectively indicated HCC accompanied by PVTT.

Serum-derived three-circRNA signature as a diagnostic biomarker for hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a common tumor characterized by high morbidity and mortality rates. The importance of circRNA in cancer diagnosis has been established. The study aimed to identify differentially-expressed circRNAs (DECs) in human blood exosomes from patients with HCC and to investigate their diagnostic value.MethodsThe circRNA expression profiles of HCC and normal human blood samples were downloaded and processed from the exoRBase database. At the cutoff criteria of a fold change (FC) > 2.0 and P < 0.05, DECs were screened utilizing the limma package in the R software. A receiver operator characteristic curve (ROC) was used to study its diagnostic value. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was performed to confirm the three-circRNAs expression in the blood samples with HCC. Various bioinformatics tools were used to characterize the potential biological pathways induced by circRNAs.ResultsCompared with the normal samples, seven up-regulated and five down-regulated circRNAs were determined in the HCC samples. ROC analyses demonstrated that hsa_circ_0004001, hsa_circ_0004123, hsa_circ_0075792, and a combination of the three biomarkers exhibited higher sensitivity and specificity. The qRT-PCR confirmed that the three circRNAs were upregulated in the blood samples with HCC. Chi squared tests implied that the expression of three circRNAs was positively correlated with the TNM stage and tumor size. The circRNAs participated in VEGF/VEGFR, PI3K/Akt, mTOR, and Wnt signaling pathways by targeting miRNAs.ConclusionsThe study established the existence of seven up-regulated and five down-regulated circRNAs in HCC. Additionally, hsa_circ_0004001, hsa_circ_0004123, hsa_circ_0075792, and a combination of the three were utilized as valuable diagnostic biomarkers in HCC.

Identification of noninvasive diagnostic biomarkers for hepatocellular carcinoma by urinary proteomics

Hepatocellular carcinoma (HCC) ranks fourth in cancer mortality worldwide, and third in China. Hepatitis B virus (HBV) infection is a main risk factor for HCC in China, and the early diagnosis of HCC in high-risk population is very important. However, the commonly used diagnostic biomarker alpha-fetoprotein has limitations in clinical practice. In order to identify reliable and noninvasive HCC urinary biomarkers, a high-throughput proteomics streamline was applied in the analysis of urine samples from 74 HCC and 82 high-risk patients with chronic HBV infected liver diseases. Candidate diagnostic markers were screened by feature selection algorithm, and were combined with random forest or simple voting algorithms in the training dataset. Then the multiple feature models were validated in an independent test dataset. The selected features were further verified by Multiple Reaction Monitoring (MRM) in another independent dataset. By integrating 7 features screened in the discovery phase, random forest model achieved AUC of 0.92 and 0.87 in training and test datasets, respectively, while voting model performed better with AUC of 0.94 and 0.90, respectively. In the MRM dataset, the 7 features were targeted quantified, and voting model integrating the 7 features achieved AUC of 0.95. Our work highlights the potential of noninvasive urinary protein biomarkers in HCC diagnosis with high-risk population, which will be beneficial to HCC auxiliary diagnosis and HCC surveillance. SignificanceA high throughput urinary proteome analysis platform was committed into the discovery of noninvasive HCC biomarkers in high-risk patients with chronic HBV infected liver diseases. The combination of 7 urinary features achieved good performance in distinguishing HCC from high-risk population. The expression of the 7 features was validated by targeted MRM, and the integration of the features also worked well in the MRM dataset. This is the first time that urinary proteomic strategy was applied in discovering HCC biomarkers from high-risk population. This result will be helpful for HCC auxiliary diagnosis and surveillance in a noninvasive way.

Polo-like kinase 1 as a promising diagnostic biomarker and potential therapeutic target for hepatocellular carcinoma.

Hepatocellular carcinoma is a major cause of cancer mortality worldwide. The outcome of hepatocellular carcinoma depends mainly on its early diagnosis. To date, the performance of traditional biomarkers is unsatisfactory. Polo-like kinase 1 is a serine/threonine kinase that plays essential roles in cell cycle progression and deoxyribonucleic acid damage. Moreover, polo-like kinase 1 knockdown decreases the survival of hepatocellular carcinoma cells; therefore, polo-like kinase 1 is an attractive target for anticancer treatments. Nobiletin, a natural polymethoxy flavonoid, exhibits a potential antiproliferative effect against a wide variety of cancers. This study targets to identify a reliable diagnostic biomarker for hepatocellular carcinoma and provide a potential therapeutic target for its treatment. Polo-like kinase 1 levels were analyzed in 44 hepatocellular carcinoma patients, 33 non-hepatocellular carcinoma liver cirrhosis patients and 15 healthy controls using the enzyme-linked immunosorbent assay method. Receiver operating characteristics curve analysis was used to establish a predictive model for polo-like kinase 1 relative to α-fetoprotein in hepatocellular carcinoma diagnosis. Furthermore, in the in vitro study, gene expressions were assessed by quantitative polymerase chain reaction in two human hepatocellular carcinoma cell lines after treatment with doxorubicin and polo-like kinase 1 inhibitor volasertib (Vola) either alone or in combination with nobiletin. Cell viability was also determined using the crystal violet assay.: Serum polo-like kinase 1 levels in hepatocellular carcinoma patients were significantly higher than liver cirrhosis and control groups (p < 0.0001). Polo-like kinase 1 showed a reasonable sensitivity, specificity, positive predictive value, and negative predictive value in hepatocellular carcinoma diagnosis. Moreover, nobiletin improved inhibition of cell growth induced by Vola and doxorubicin. Regarding reverse transcription polymerase chain reaction results, nobiletin suppressed expressions of polo-like kinase 1 and proliferating cell nuclear antigen and elevated expressions of P53, poly (ADPribose) polymerase 1, and caspase-3. Nobiletin/doxorubicin and nobiletin/Vola showed a significant increase in caspase-3 activity indicating cell apoptosis. Polo-like kinase 1 may be a potential biomarker for hepatocellular carcinoma diagnosis and follow-up during treatment with chemotherapies. In addition, nobiletin synergistically potentiates the doxorubicin and Vola-mediated anticancer effect that may be attributed partly to suppression of polo-like kinase 1 and proliferating cell nuclear antigen expression and enhancement of chemotherapy-induced apoptosis.

Uncovering the potential differentially expressed miRNAs as diagnostic biomarkers for hepatocellular carcinoma based on machine learning in The Cancer Genome Atlas database.

The present study aimed to identify novel diagnostic differentially expressed microRNAs (miRNAs/miRs) in order to understand the molecular mechanisms underlying hepatocellular carcinoma. The expression data of miRNA and mRNA were downloaded for differential expression analysis. Optimal diagnostic differentially expressed miRNA biomarkers were identified via a random forest algorithm. Classification models were established to distinguish patients with hepatocellular carcinoma and normal individuals. A regulatory network between optimal diagnostic differentially expressed miRNA and differentially expressed mRNAs was then constructed. The GSE63046 dataset and in vitro experiments were used to validate the expression of the optimal diagnostic differentially expressed miRNAs identified. In addition, diagnostic and prognostic analyses of optimal diagnostic differentially expressed miRNAs were performed. In total, 14 differentially expressed miRNAs (all upregulated) and 2,982 differentially expressed mRNAs (1,989 upregulated and 993 downregulated) were identified. hsa-miR-10b-5p, hsa-miR-10b-3p, hsa-miR-224-5p, hsa-miR-183-5p and hsa-miR-182-5p were considered as the optimal diagnostic biomarkers for hepatocellular carcinoma. The mRNAs targeted by these five miRNAs included secreted frizzled related protein 1 (SFRP1), endothelin receptor type B (EDNRB), nuclear receptor subfamily 4 group A member 3 (NR4A3), four and a half LIM domains 2 (FHL2), NK3 homeobox 1 (NKX3-1), interleukin 6 signal transducer (IL6ST) and forkhead box O1 (FOXO1). ‘Bile acid biosynthesis and cholesterol’ was the most enriched signaling pathways of these target mRNAs. The expression validation of the five miRNAs was consistent with the present bioinformatics analysis. Notably, hsa-miR-10b-5p and hsa-miR-10b-3p had a significant prognosis value for patients with hepatocellular carcinoma. In conclusion, the five differentially expressed miRNAs may be considered as diagnostic biomarkers for patients with hepatocellular carcinoma. In addition, the differential expression levels of the targets of these five mRNAs, including SFRP1, EDNRB, NR4A3, FHL2, NKX3−1, IL6ST and FOXO1, may be involved in hepatocellular carcinoma tumorigenesis.

Cisplatin resistance-associated circRNA_101237 serves as a prognostic biomarker in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-associated mortality worldwide. Despite clinical advances, the survival rate of patients with HCC remains low, as most patients are diagnosed with HCC when they are already at the advanced stage. Certain circular RNAs (circRNAs) are closely associated with the development of liver cancer. In the present study, a circRNA array was performed to screen differentially expressed circRNAs in HCC tissues. The further analysis focused on the newly identified circRNA_101237, the host gene of which, cyclin-dependent kinase 8, is located at chr13:26974589-26975761. CircRNA_101237 was determined to be upregulated in tumor tissue and serum of patients with HCC as compared with that in paracancerous tissues and the serum of healthy controls, respectively. In addition, the expression of circRNA_101237 was associated with tumor size, lymph node metastasis, distant metastasis and TNM stage. Univariate and multivariate analysis indicated that serum circRNA_101237 levels were an independent predictor of survival prognosis in patients with HCC. The overall survival of patients with high expression of circRNA_101237 was reduced compared with that of patients with low expression of circRNA_101237. Of note, cisplatin induced the expression of circRNA_101237 in HCC cells in a dose- and time-dependent manner in vitro, and the levels of circRNA_101237 in the serum of patients with cisplatin-resistant HCC and in cisplatin-resistant Huh7 cells were increased. The present study provided novel insight into the use of circRNA_101237 as a diagnostic biomarker for HCC and a potential therapeutic target.

The development of a Glypican-3-specific binding peptide using in vivo and in vitro two-step phage display screening for the PET imaging of hepatocellular carcinoma.

Glypican-3 (GPC3) is a diagnostic biomarker for hepatocellular carcinoma (HCC). Although numerous designs targeting GPC3 have been reported, the HCC diagnostic agents with specific tumor accumulation and low background, particularly in normal liver tissue, are still in need. Peptides have attracted considerable attention as an imaging probe due to their low immunogenicity, short in vivo circulation time, and acceptable production cost. Herein, a two-step phage display screening approach was performed against GPC3-high expression tumor xenografts in vivo, followed by human recombinant GPC3 protein in vitro. A GPC3-specific binding peptide, named TJ12P2, with the sequence of Ser-Asn-Asp-Arg-Pro-Pro-Asn-Ile-Leu-Gln-Lys-Arg (SNDRPPNILQKR) was identified. The apparent Kd value between TJ12P2 and the GPC3 protein was measured as 158.2 ± 26.25 nM. After 18F labeling, 18F-AlF-NOTA-TJ12P2 was found accumulated in the tumors by positron emission tomography (PET) imaging in two HCC subcutaneous tumor models (HepG2 and SMMC-7721) with high GPC3 expression. Static PET imaging revealed that 18F-AlF-NOTA-TJ12P2 accumulation in the HepG2 and SMMC-7721 tumors reached 1.825 ± 0.296 %ID g-1 and 1.575 ± 0.520 %ID g-1, with tumor-to-muscle ratios of 4.14 ± 0.50 and 4.25 ± 0.25, respectively, at 30 min post-injection (p.i.). Much less accumulation (0.533 ± 0.078 %ID g-1) of the 18F-AlF-NOTA-TJ12P2 was found in the control PC3 tumors with low GPC3 expression. More importantly, no obvious normal liver uptake of TJ12P2 was observed in the abovementioned animal models. As a result, a novel peptide targeting GPC3, TJ12P2, with strong affinity and specificity was identified using a two-step phage display screening technique in the present study. The 18F-AlF-NOTA-TJ12P2 may be a promising PET imaging probe with translational potential for accurate HCC diagnosis.

Methylation of tumour suppressor genes RUNX3, RASSF1A and E-Cadherin in HCV-related liver cirrhosis and hepatocellular carcinoma

ABSTRACTBackground: HCV infection is related to aberrant methylation of several genes. RASSF1A, E-Cadherin and RUNX3 are tumour suppressor genes that may be inactivated by hypermethylation in many tumours including hepatocellular carcinoma (HCC). We hypothesized that methylation is a diagnostic biomarker for HCC in patients with HCV-related liver cirrhosis.Methods: We recruited 207 cases of HCV-related liver cirrhosis, 193 HCC patients and 53 healthy controls. Methylation-specific polymerase chain reaction for detection of circulating hypermethylated RASSF1A, E-Cadherinand RUNX3. Alpha fetoprotein (AFP) was measured by commercial immunoassay.Results: Significant hypermethylation of the three genes was found in the HCC group compared to both cirrhosis and healthy groups (P < 0.001), whereas no significant difference in hypermethylation was found between cirrhosis and healthy groups (P = 0.17, 0.50 and 0.14, respectively). No significant links were found between hypermethylated RASSF1A, E-Cadherin and RUNX3 and stages of Barcelona Clinic of Liver Cancer score (P =0.21, 0.63 and 0.98, respectively). No significant associations were found between AFP value and hypermethylated genes in cirrhosis and HCC groups (P = 0.82) except with E-Cadherin in HCC (P = 0.02). In multiple regression analysis, RASSF1A and E-Cadherin were predictors of HCC within cirrhosis cases, but only E-Cadherin was an independent risk factor for prediction of HCC in cases with low AFP (P = 0.01).Conclusions: The presence of hypermethylated serum RASSF1A, E-Cadherin and RUNX3 is linked to HCC in patients with HCV-related cirrhosis. Only E-Cadherin is an independent risk factor for prediction of HCC with low AFP. These findings may be of diagnostic value.

Identification of novel diagnostic and prognostic biomarkers for hepatocellular carcinoma.

The differential expression of a featured set of genes may serve as a diagnostic biomarker in hepatocellular carcinoma (HCC) patients. The aim of this study was to identify prognostic biomarkers for the diagnosis and survival of HCC based on the analysis of a large cohort of patients. Clinical and RNA‑seq data were obtained from The Cancer Genome Atlas (TCGA) database. A transcriptomics analysis was conducted to detect differentially expressed genes (DEGs). Samples from 53tumors and 20normal tissues of HCC patients were obtained to further analyze the connection between overall survival (OS) and DEG levels. Based on the OS and progression‑free survival (PFS), 4 DEGs (GABRR1, SOX11, COL24A1 and MYLK2) were identified from the TCGA dataset. Using gene ontology (GO) analysis, it was demonstrated that the DEGs were associated with several biological processes, including multicellular organismal and single‑multicellular organism processes, which are involved in the development and migration of HCC. In addition, the four genes were significantly upregulated in tumor tissues. Notably, the mRNA expression of the four genes had a negative association with OS and PFS in HCC patients determined using a Kaplan‑Meir analysis. The four‑gene signature is a potential novel biomarker for the prediction of HCC patient survival.

KCNK levels are prognostic and diagnostic markers for hepatocellular carcinoma.

Two-pore-domain (KCNK, K2P) K+ channels are transmembrane protein complexes that control the flow of ions across biofilms, which underlie many essential cellular functions. Because KCNK family members are known to contribute to tumorigenesis in various types of cancer, we hypothesized that they might be differentially expressed in hepatocellular carcinoma (HCC) cells as compared to healthy tissue and serve as diagnostic or prognostic biomarkers. We tested this hypothesis through bioinformatic analyses of publicly available data for the expression of various KCNK subunits in HCC. We observed reduced expression of KCNK2, KCNK15, and KCNK17 in liver cancer, as well as overexpression of KCNK9, all of which correlated with a better prognosis for HCC patients per survival analyses. Moreover, ROC curves indicated that KCNK2, KCNK9, KCNK15, and KCNK17 levels could be used as a diagnostic biomarker for HCC. Finally, our western blot and qRT-PCR results were consistent with those obtained from bioinformatic analyses. Taken together, these results suggest that KCNK2, KCNK9, KCNK15, and KCNK17 could serve as potential diagnostic and prognostic biomarkers of HCC.

SUCO as a Promising Diagnostic Biomarker of Hepatocellular Carcinoma: Integrated Analysis and Experimental Validation.

BackgroundHepatocellular carcinoma (HCC) is not frequently diagnosed until the late stage due to its concealed symptoms. Therefore, the identification of biomarkers that have effective diagnostic performance and act as potential key therapeutic targets for HCC becomes urgent.Material/MethodsComprehensive analysis of accumulated data downloaded from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) databases was used to obtain more reliable potential diagnostic biomarkers of HCC and to explore related molecular mechanisms. Meta-analysis and summary receiver operating characteristic (SROC) curve analysis were performed to evaluate the differential expression of SUCO gene in HCC and identify the capability of SUCO in distinguishing HCC-tissues from normal liver-tissues.ResultsSUCO was found to be upregulated in HCC-tissues and exhibited a favorable value in diagnosing HCC. Bioinformatics analysis showed that SUCO might play important roles in HCC progression, and was significantly related to cell cycle, cell metabolism, and proliferation.ConclusionsThis study was the first to demonstrate that SUCO was overexpressed in HCC-tissues, and that high expression of SUCO was significantly related to poor overall survival in HCC patients. SUCO might be a potential diagnostic biomarker for HCC patients, which promotes the tumorigenesis and progression of HCC.

Integrin α6 targeted positron emission tomography imaging of hepatocellular carcinoma in mouse models

Integrin α6 emerges to be a diagnostic biomarker for hepatocellular carcinoma (HCC). Here, we translated our previously identified integrin α6 targeted peptide RWY into a positron emission tomography (PET) tracer 18F-RWY for the detection of HCC lesions in following four HCC mouse models including subcutaneous, orthotopic, genetically engineered and chemical induced HCC mice. 18F-RWY produced high PET signals in liver tumor tissues that were reduced by blocking studies using nonradiolabeled RWY peptide. We compared the integrin α6 targeted PET tracer 18F-RWY with the integrin αvβ3-targeted PET tracer 18F-3PRGD2 and the clinical PET tracer 18F-FDG in chemical induced HCC mice. Among 12 HCC identified by enhanced magnetic resonance imaging (MRI) with hepatocellular specific gadoxetate disodium Gd-EOB-DTPA, the sensitivities of 18F-RWY, 18F-3PRGD2 and 18F-FDG were approximately 92%, 73% and 50% while the tumor-to-liver ratios were 4.36 ± 1.41, 1.97 ± 0.43 and 1.63 ± 0.23 respectively. Additionally, PET imaging with the integrin α6 targeted 18F-RWY enabled to visualize small HCC lesions with diameters approximately 0.2 cm that was hard to be distinguished from surround hepatic vascular by enhanced MRI with Gd-EOB-DTPA. These findings potentiate the use of integrin α6 targeted PET tracer 18F-RWY for the detection of HCC.

The diagnostic and prognostic role of RhoA in hepatocellular carcinoma.

The aim of this study was to investigate the expression level of Ras homolog gene family, member A (RhoA) in patients with hepatocellular carcinoma (HCC) and to investigate the prognostic and diagnostic value of RhoA. Data mining from various data bases and wet experiments on samples from Peking Union Medical College Hospital showed that RhoA mRNA and protein expression were significantly higher in the HCC tissues than in the normal tissues. Higher expression at both the mRNA and protein levels was associated with a poorer prognosis. High sensitivity (92.5%) and specificity (90.0%) were observed in the diagnostic model based on protein level rather than mRNA level. RhoA expression was modulated by genetic amplification. The lysosome, pathogenic Escherichia coli infection, purine metabolism and pyrimidine metabolism pathways were mainly enriched in the high RhoA level group, while the hedgehog signaling, linoleic acid metabolism, olfactory transduction and taste transduction pathways were enriched in the low RhoA level group. RhoA is commonly upregulated in HCC tissues, and its expression at both the mRNA and protein levels is associated with poor prognosis. Notably, RhoA protein levels serve as a diagnostic biomarker for HCC.

Dickkopf-1 and Amphiregulin as Novel Biomarkers and Potential Therapeutic Targets in Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is a highly fatal tumor which represents a major health problem worldwide. Due to asymptomatic nature of HCC, most patients present with the progressive stage of disease, so, unfortunately, there are no effective therapies. Existing techniques for HCC surveillance and diagnosis lack the required accuracy. Therefore, searching for new diagnostic and/or therapeutic tools could improve patient survival. This study aimed to estimate the diagnostic role of Dickkopf-1 (DKK1) and amphiregulin (AREG) and to find out their correlation with different clinicopathological parameters in HCC patients. Materials and Methods: Serum levels of DKK1 and AREG in 55 HCC patients, 20 cirrhotic patients, and 15 healthy subjects as control group were measured using the ELISA technique. Results: Both of DKK1 and AREG showed a significant increase in the HCC group compared to cirrhotic and healthy groups. DKK1 at a cutoff point of 8.92 ng/ml showed that the area under the curve (AUC) was 0.826 with 87.3% sensitivity and 82.9% specificity. DKK1 showed a significant correlation with tumor size, liver dysfunction, and poor performance status in HCC patients. AREG at a cutoff point of 8.74 pg/ml showed a sensitivity of 74.5% but low specificity (47.1%). AREG showed a significant correlation with portal vein thrombosis and tumor metastasisin HCC patients. Conclusion: Serum DKK1 could be a diagnostic biomarker for HCC. Both of DKK1 and AREG may play significant roles in tumor progression and may offer promising therapeutic targets in HCC patients.

MiR-155, miR-96 and miR-99a as potential diagnostic and prognostic tools for the clinical management of hepatocellular carcinoma.

Increasing evidence has demonstrated that circulating microRNAs (miRNAs) can be utilized as potential biomarkers for the diagnosis of cancer, as well as a prognostic tool for the management of the disease. Therefore, the present study aimed to evaluate the predictive ability of miRNA (miR)-155, miR-96 and miR-99a for the diagnosis and prognosis of hepatocellular carcinoma (HCC). Tissues were collected from 30 patients with HCC and their matched adjacent normal liver tissues, as well as from serum samples from 30 patients with HCC and 30 healthy controls. Reverse transcription-quantitative PCR was used to measure the expression levels of miR-155, miR-96 and miR-99a. The expression levels of miR-155 and miR-96 were upregulated in the tissues and serum of patients with HCC, whereas miR-99a expression levels were decreased. Receiver operating characteristics (ROC) curve analysis revealed that circulating miR-155, miR-96, miR-99a and a combination of these three miRNAs could serve as diagnostic biomarkers for HCC with areas under the curve (AUC) of 0.84, 0.824, 0.799 and 0.931, respectively. Serum α-fetoprotein (AFP) was detected using electrochemiluminescence immunoassay analyzer. The addition of AFP with the combination of these three miRNAs offered a higher accuracy of HCC diagnosis (AUC, 0.979; sensitivity, 90.0%; specificity, 100.0%). In addition, elevated expression levels of miR-155 and miR-96 were associated with poor survival time of patients with HCC. The panel of miR-155, miR-96, miR-99a and AFP had a higher sensitivity and specificity for the diagnosis of HCC when compared with a single marker. Furthermore, the present data suggested that miR-155 and miR-96 may be potential prognostic markers for the clinical management of patients with HCC.

Abstract 5269: Discovery and validation of plasma acylcarnitines for the early diagnosis of hepatocellular carcinoma

Abstract Introduction: Hepatocellular carcinoma (HCC) is one of the commonest and the leading cause of cancer death globally. The main cause of this high mortality rate is that patients with HCC are usually diagnosed at late stages since they have only subtle clinical presentations at early stages. This late diagnosis is a result of the requirement of high-end imaging modalities, the use of invasive procedures, and the lack of reliable tumour biomarkers. Metabolomics is the study of metabolites in an organism in response to the changes in health status. The purpose of this study was to identify and validate plasma acylcarnitines as potential diagnostic biomarkers of HCC by using techniques in the field of metabolomics. Methods: 228 EDTA-plasma samples of healthy controls, cirrhotic patients, or treatment-naïve patients with HCC, collected from Imperial College Healthcare Tissue Bank, were used as a discovery and training set. Patient factors, including basic patient characteristics, aetiologies of HCC, and Child-Pugh scores, and data relating to tumours, such as the multiplicity, the sizes in diameter, the status of macro-vascular invasion, were collected. A targeted assay was adapted to measure concentrations of carnitine and acylcarnitines with different chain length using the TQ-S tandem quadrupole mass spectrometer. Non-parametric univariate analyses were applied to evaluate the statistical difference between HCC and cirrhosis. Logistic regression was preferred to develop diagnostic models. A Gambian cohort of 165 plasma samples was then used as an external validation. Results: Among sixteen measured carnitine and acylcarnitines, three acylcarnitines, namely acetylcarnitine, succinylcarnitine, and hexanoylcarnitine, statistically increased in HCC patients comparing to cirrhotic patients. A linear model incorporating these three acylcarnitines and alpha-fetoprotein was then developed. The area under the ROC curve of this multivariable regression model was 0.782 (95% C.I., 0.695-0.870) in the training set. When applied to the validation cohort, the sensitivity and specificity of this model at identifying HCC patients from cirrhosis were 82.2% and 67.8%, respectively. Conclusion: This report provides a comprehensive profile of the changes of carnitine and acylcarnitines among HCC, cirrhotic patients and healthy controls, and determines the value of acylcarnitines as diagnostic biomarkers for HCC. Citation Format: Eric Yi-Liang Shen, Abellona U, Simon Taylor-Robinson, Mark Thursz, Elaine Holmes, Jeremy Nicholson. Discovery and validation of plasma acylcarnitines for the early diagnosis of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5269.

Evaluation of Fucosylated Haptoglobin as a Diagnostic Biomarker for Hepatocellular Carcinoma in Egypt

Background: Most Hepatocellular Carcinomas (HCCs) are diagnosed at an advanced stage. Therefore, there is citation-type an urgent need for better methods of detection and surveillance of patients at risk of HCC. Alpha-fetoprotein (AFP) has a suboptimal diagnostic performance for HCC surveillance, so novel and reliable diagnostic biomarkers are required. Objective: The aim of this work is to evaluate fucosylated haptoglobin as a diagnostic biomarker for hepatocellular carcinoma in Egyptian patients. Materials and Methods: This case-control study was carried out on 60 patients classified into 3 groups (20 patients on each); group I (HCC group), group II (Cirrhotic group) and group III (Control group). Diagnosis of liver cirrhosis was done by clinical, biochemical and ultrasound (US), whereas the diagnosis of HCC was done by percutaneous biopsy or radiological (by US and triphasic Computerized Tomography (CT) based on the guidelines of the American-Association for the Study of Liver Diseases. HCC stage was clinically defined according to the Barcelona Clinic Liver Cancer (BCLC) staging system. AFP &amp; fucosylated haptoglobin levels were estimated in all groups. Results: There was a statistically significant positive correlation between serum fucosylated haptoglobin and tumor size in the HCC group. Serum fucosylated haptoglobin (at 116 U/ ml) showed sensitivity 80%, specificity 65%, positive predictive value 53% and negative predictive value 87% with AUC 0.786. Combination of serum fucosylated haptoglobin and serum AFP at (200 ng/ ml) increased sensitivity that reached 95%. Conclusion: Serum fucosylated haptoglobin may serve as a novel diagnostic biomarker for the detection of HCC with higher sensitivity than AFP.

Identification of diagnostic long non‑coding RNA biomarkers in patients with hepatocellular carcinoma.

Liver cancer is a leading cause of cancer-associated mortality worldwide. Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer. The aim of the present study was to identify long non-coding RNA (lncRNAs) as diagnostic biomarkers for HCC. The lncRNA and mRNA expression profiles of a large group of patients with HCC were obtained from The Cancer Genome Atlas. The differentially expressed lncRNAs (DElncRNAs) and the differentially expressed mRNAs (DEmRNAs) were identified by bioinformatics analysis. Using feature selection procedure and a classification model, the optimal diagnostic lncRNA biomarkers for HCC were identified. Classification models, including random forests, decision tree and support vector machine (SVM), were established to distinguish between HCC and normal tissues. DEmRNAs co-expressed with the lncRNAs were considered as targets of DElncRNAs. Functional annotation of DEmRNAs co-expressed with these lncRNAs biomarkers was performed. Receiver operating characteristic curve analysis of lncRNAs biomarkers was conducted. A total of 3,177 lncRNAs and 15,183 mRNAs between HCC and normal tissues were obtained. RP11-486O12.2, RP11-863K10.7, LINC01093 and RP11-273G15.2 were identified as optimal diagnostic lncRNA biomarkers for HCC that were co-expressed with 273, 69, 76 and 1 DEmRNAs, respectively. The area under the curve values of the random forest model, decision tree model and SVM model were 0.992, 0.927 and 0.992, and the specificity and sensitivity of the three models were 100.0 and 95.6, 92.0 and 98.3 and 98.0 and 97.2%, respectively. ‘PPAR signaling pathway’ and ‘retinol metabolism’ were two significantly enriched target pathways of DElncRNAs. The present study identified four DElncRNAs, including RP11-486O12.2, RP11-863K10.7, LINC01093 and RP11-273G15.2, as potential diagnostic biomarkers of HCC. Functional annotation of target DEmRNAs provided novel evidence for examining the precise roles of lncRNA in HCC.