Abstract 533: S100A10 as a novel biomarker in hepatocellular carcinoma

Background Studies show that patients with hepatocellular carcinoma (HCC) have poor prognosis, particularly when patients are diagnosed at late stages of the disease development. The flap endonuclease 1 (FEN1) gene is overexpressed in multiple malignant tumors and may promote tumor aggressiveness. However, its expression profile and functional roles in HCC are still unclear. Here, we evaluated the molecular mechanisms of FEN1 in HCC. Methods The expression of FEN1 in HCC was evaluated using HCC mRNA expression data from TCGA and GEO databases. The expression of FEN1 was also confirmed by immunohistochemistry (IHC) using a tissue microarray (TMA) cohort with a total of 396 HCC patients. Kaplan-Meier analysis and univariate and multivariate Cox regression analyses were used to determine the correlation between FEN1 expression and survival rate of HCC patients. The molecular mechanism and biological functions of FEN1 in HCC were predicted using functional and pathway enrichment analysis in vitro experiments. Results FEN1 was overexpressed in multiple HCC cohorts at both mRNA and protein levels. The receiver operating characteristic (ROC) curve showed that FEN1 can serve as a diagnostic predictor of HCC. Meanwhile, patients with high FEN1 expression levels showed lower overall survival (OS) and relapse-free survival (RFS) rates than those with low FEN1 expression. More importantly, we found that FEN1 elevation was an independent prognostic factor for OS and RFS in HCC patients based on univariate and multivariate analyses, indicating that FEN1 might be a potential prognostic marker in HCC. Furthermore, knocking down FEN1 resulted in suppressed cell proliferation and migration in vitro. This could have been due to regulation expressions of c-Myc, survivin, and cyclin D1 genes, indicating that FEN1 may function as an oncogene through its role in the cell cycle and DNA replication pathway. Conclusion Our study indicated that high FEN1 expression might function as a biomarker for diagnosis and prognosis. In addition, the study confirms that FEN1 is an oncogene in HCC progression.

Objective: Transcription elongation factor 1 (TCERG1) is a nuclear protein consisted of multiple protein structural domains that plays an important role in regulating the transcription, extension, and splicing regulation of RNA polymerase II. However, the prognostic and immunological role of TCERG1 in human cancer remains unknown. In this study, we analyzed the expression of TCERG1 gene in hepatocellular carcinoma (HCC) patients, its clinical significance, and its possible prognostic value by bioinformatics.Methods: RNA sequencing data and clinicopathological characteristics of patients with HCC were collected from TCGA and CCLE databases. The Wilcoxon rank-sum test was used to analyze the expression of TCERG1 in HCC tissues and normal tissues. The protein levels of TCERG1 between normal and liver cancer tissues were analyzed by the Human Protein Atlas Database (HPA) (www.proteinatlas.org). Validation was performed using the Gene Expression Omnibus (GEO) dataset of 167 samples. The expression of TCERG1 in HCC cells were verified by qRT-PCR, and CCK-8, scratch assay and Transwell assay were performed to detect cell proliferation, migration and invasion ability. According to the median value of TCERG1 expression, patients were divided into high and low subgroups. Logistic regression, GSEA enrichment, TME, and single-sample set gene enrichment analysis (ssGSEA) were performed to explore the effects of TCERG1 on liver cancer biological function and immune infiltrates. TCERG1 co-expression networks were studied through the CCLE database and the LinkedOmics database to analyze genes that interact with TCERG1.Results: The expression levels of TCERG1 in HCC patient tissues were significantly higher than in normal tissues. Survival analysis showed that high levels of TCERG1 expression were significantly associated with low survival rates in HCC patients. Multifactorial analysis showed that high TCERG1 expression was an independent risk factor affecting tumor prognosis. This result was also verified in the GEO database. Cellular experiments demonstrated that cell proliferation, migration and invasion were inhibited after silencing of TCERG1 gene expression. Co-expression analysis revealed that CPSF6 and MAML1 expression were positively correlated with TCERG1. GSEA showed that in samples with high TCERG1 expression, relevant signaling pathways associated with cell cycle, apoptosis, pathways in cancer and enriched in known tumors included Wnt signaling pathway, Vegf signaling pathway, Notch signaling pathway, MAPK signaling pathway and MTOR pathways. The expression of TCERG1 was positively correlated with tumor immune infiltrating cells (T helper two cells, T helper cells).Conclusion:TCERG1 gene is highly expressed in hepatocellular carcinoma tissues, which is associated with the poor prognosis of liver cancer, and may be one of the markers for the diagnosis and screening of liver cancer and the prediction of prognosis effect. At the same time, TCERG1 may also become a new target for tumor immunotherapy.

Background: In HCC, hypoxia and MET can promote tumor progression and induce resistance to radiation, chemo or targeted therapies. The aim of this study was to correlate MET and hypoxia marker carbonic anhydrase 9 (CA9) expression levels by immunohistochemistry with clinicopathological characteristics and disease-free survival (DFS) in patients with HCC. Material and methods: One-hundred HCC resection specimens were evaluated by immunohistochemistry for MET (clone sp44, Ventana) and CA9 (rabbit polyclonal) expression. For automated evaluation, we elaborated our own macro using ImageJ software, and compared it with H- and MetMab validated scores. METhigh and CA9high expression were defined as moderate to strong staining. Staining results were correlated with clinicopathological characteristics. Univariate analyses were performed using Fisher's exact or chi square tests, and multivariate analyses using Cox regression model. Median DFS (mDFS) were calculated by Kaplan-Meier method. MET amplification was assessed by FISH (zytovision probe) and centromere 7 (CEN7) used as an internal control. Amplification was defined as a mean ratio of MET/CEN7> 4, counting 100 nuclei. Results: Tumors were BCLC A1 (92%), uninodular (53%), moderately differentiated (63%), with pathological vascular invasion (71%), and had low AFP expression (82%). METhigh expression was observed in 53% of tumors being higher in patients with viral hepatitis-associated HCC (p = 0.02) and in patients with AFP>400UI/L (p = 0.03). CA9high expression was observed in 41% of tumors and was correlated with viral hepatitis (p = 0.002), pathological vascular invasion (p = 0.007), and poor differentiation (p = 0.007). MET and CA9 expression levels significantly correlated with each other (p = 0.008). mDFS was shorter in METhigh (12.9 vs >80 months in METlow patients; p = 0.018) and CA9high (10.2 vs 34.4 months in CA9low patients; p = 0.02) populations. Combination of MET and CA9 status discriminated 3 prognostic groups. In the METhigh/CA9high group mDFS was 10.7 months, whereas mDFS was not reached in the METlow/CA9low group (p = 0.003). Using H- and MetMab scores, mDFS of the METhigh group were 12.9 months and 12.4 months respectively, and not reached in the METlow group. The METhigh/CA9low and METlow/CA9high groups were defined as intermediate risk populations (mDFS = 19 months). In multivariate analysis, tumor < 5cm (p = 0.025), uninodular morphology (p = 0.004), and CA9-low levels (p = 0.007) were independently associated with prolonged DFS. METhigh expression level was associated with shorter DFS when CA9 was excluded from the model (p = 0.024). No amplification was detected in METhigh HCC samples. Conclusion: MET expression is a useful prognostic marker in HCC. Patients with HCC overexpressing MET and CA9 represent a subgroup with poor prognosis that might benefit from MET inhibition therapy. Citation Format: Annemilai Tijeras-Raballand, Cindy Neuzillet, Miguel Albuquerque, Nathalie Colnot, Friedhelm Bladt, Manfred Klevesath, Christian Ihling, Hongxia Zheng, Maryse Baia, Christiane Copie-Bergman, Eric Raymond, Armand de Gramont, Valérie Paradis, Sandrine Faivre. High MET and CA9 expressions define a subgroup of hepatocellular carcinoma (HCC) patients with poor prognosis candidates for MET inhibition strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1911. doi:10.1158/1538-7445.AM2015-1911

Background: In HCC, acquired resistance to receptor tyrosine kinase receptors has been associated with an increased expression and activation of the c-MET pathway. In addition, initial data suggest that carbonic anhydrase 9 (CA9), a hypoxia marker, is associated with HCC development. The aim of this study was to correlate c-MET and CA9 expression levels by immunochemistry with clinicopathologic characteristics and disease-free survival (DFS) in patients with HCC. Materials and Methods: One-hundred HCC resection specimens were evaluated by immunohistochemistry. Staining results were qualitatively and quantitatively assessed and correlated with clinicopathologic parameters. We elaborated our own dedicated macro, using ImageJ software, for automated evaluation. c-MET high and CA9 high expression were defined as moderate to strong staining. Univariate analyses were performed using Fisher's exact or chi square test, and multivariate analyses using Cox regression model. Median DFS were calculated by Kaplan-Meier method. Results: Patients were mainly males (84%). Tumors were classified as BCLC A1 (92%), uninodular (53%), low-AFP-expressing (82%), moderately differentiated (63%), and with pathologic vascular invasion (71%). c-MET high expression was observed in 51% of tumors and was higher in patients with viral hepatitis-associated HCC (p=0.02) and in patients with AFP>400UI/L (p=0.03). CA9 high expression was observed in 41% of tumors and was correlated with viral hepatitis (p=0.002), pathological vascular invasion (p=0.007), and poor differentiation (p=0.007). c-MET and CA9 expression levels significantly correlated with each other (p=0.008). Median DFS was shorter in the c-MET high (12.9 months, vs >80 months in c-MET low patients; p=0.018) and CA9 high (10.2 months, vs 34.4 months in CA9 low patients; p=0.02) populations. Comparison of c-MET and CA9 status discriminated 3 groups with distinct prognosis. The shortest median DFS was 10.7 months in the c-MET high / CA9 high group, whereas median DFS was not reached in the c-MET low / CA9 low group (p=0.003). The group comprising c-MET high / CA9 low and c-MET low/ CA9 high expressing patients was defined as an intermediate risk populations (DFS=19 months). In a multivariate analysis, smaller tumor size (p=0.025), uninodular morphology (p=0.004), and CA9-low levels (p=0.007) were independently associated with a prolonged DFS. In addition, c-MET high expression level was significantly associated with shorter DFS when CA9 was excluded from the model (p=0.024). Data on c-MET amplification will be added. Conclusion: c-MET expression is a useful prognostic marker in HCC that could be used for patient stratification. Patients with HCC overexpressing c-MET and CA9 represent a subgroup with a particularly poor prognosis who might benefit from therapy with c-MET inhibitors. Citation Format: Annemilai Tijeras-Raballand, Miguel Albuquerque, Cindy Neuzillet, Nathalie Colnot, Friedhelm Bladt, Christian Ihling, Manfred Klevesath, Hongxia Zheng, Eric Raymond, Armand de Gramont, Sandrine Faivre, Valérie Paradis. Biomarker selection defines a subgroup of hepatocellular carcinoma (HCC) patients with poor prognosis who are candidates for MET inhibition strategy. [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 4706. doi:10.1158/1538-7445.AM2014-4706

Simple SummaryLiver cancer is the sixth most common cancer world-wide and hepatocellular carcinoma (HCC), the most common form of primary liver cancer, accounts for 90% of the cases. The diagnosis of HCC is usually based on non-invasive criteria using detection of a liver nodule in abdominal ultrasonography or high serum alpha-fetoprotein (AFP) levels. However, as it is only elevated in 60% of patients with HCC, AFP has limited accuracy, especially in early stages, as both a diagnostic and prognostic test. We investigated hPG80 (circulating progastrin), which is associated with liver cancer biology, and found that hPG80 levels is both an independent prognostic marker in HCC and used in combination with AFP, it improves the stratification of the patients in good and poor prognosis, especially for those patients at early-stage. This will help stratify HCC patients more accurately in the future and improve the management of these patients.Alpha-fetoprotein (AFP) is the most widely used biomarker for hepatocellular carcinoma (HCC) prognosis. However, AFP is not useful in establishing a prognosis for patients with a tumor in the early stages. hPG80 (circulating progastrin) is a tumor promoting peptide present in the blood of patients with various cancers, including HCC. In this study, we evaluated the prognostic value of plasma hPG80 in patients with HCC, alone or in combination with AFP. A total of 168 HCC patients were tested prospectively for hPG80 and analyzed retrospectively. The prognostic impact of hPG80 and AFP levels on patient survival was assessed using Kaplan-Meier curves and log-rank tests. hPG80 was detected in 84% of HCC patients. There was no correlation between hPG80 and AFP levels in the training and validation cohorts. Both cohorts showed higher sensitivity of hPG80 compared to AFP, especially at early stages. Patients with high hPG80 (hPG80+) levels (optimal cutoff value 4.5 pM) had significantly lower median overall survival (OS) compared to patients with low hPG80 (hPG80−) levels (12.4 months versus not reached respectively, p < 0.0001). Further stratification by combining hPG80 and AFP levels (cutoff 100 ng/mL) improved prognosis in particular for those patients with low AFP level (hPG80−/AFP+ and hPG80−/AFP−, 13.4 months versus not reached respectively, p < 0.0001 and hPG80+/AFP+ and hPG80+/AFP−, 5.7 versus 26 months respectively, p < 0.0001). This was corroborated when analyses were performed using the BCLC staging especially at early stages. Our findings show that hPG80 could serve as a new prognostic biomarker in HCC. Used in combination with AFP, it improves the stratification of the patients in good and poor prognosis, especially for those patients with negative AFP and early-stage HCC.

Aim: Some pluripotent genes and epithelial cell adhesion molecule (EpCAM) have been reported as cancer stem cell markers in hepatocellular carcinoma (HCC). Nanog was recently reported to regulate self-renewal of cancer stem cells though the insulin-like growth factor pathway in HCC. However, the expression status of the pluripotent stem cell genes and their clinicopathological significance in HCC from different etiologies remain unexplored. Here, we compared the expression status of stem cell markers in HCCs derived from hepatitis C virus (HCV)-related liver disease and from nonalcoholic steatohepatitis (NASH), and also determined the clinical characteristics of Nanog-positive HCCs in NASH patients. Methods: Resected tissues from 21 NASH-related HCC (NASH-HCC) patients with histologically proven steatohepatitis and 24 HCV-related HCC (HCV-HCC)CV-HCC)HCV patients were analyzed with all patients' written informed consent. The NASH group was defined as patients who consumed less than 20 g/day of alcohol. The expression status of Nanog, Oct4, Sox9, and EpCAM were determined by immunohistochemistry. The clinicopathological features of Nanog-positive NASH-HCC patients were analyzed. Results: 1) Nanog was strongly expressed in 14.3/8.3% of NASH-HCC/HCV-HCC patients, and in 66.7/25% of noncancerous tissues of NASH/HCV (p&amp;lt;0.05). Strong positive expression (defined as a positive area of over 50% of the examined tissues) was observed in most in NASH-HCC cases. Nanog-positive HCC patients showed no expression of EpCAM, and were Oct4-positive in 50% of cases and Sox9-positive in 75%. 2) EpCAM was strongly expressed in 5.0/8.3% of NASH-HCC/HCV-HCC, and in 10/41.7% of noncancerous tissues of NASH/HCV (p&amp;lt;0.05). EpCAM-positive HCC patients showed no expression of Nanog, Oct4, or Sox9. 3) The expression of Sox9 was more dominant in noncancerous tissues in both NASH and HCV patients. 4) In NASH-HCC, Nanog-positive patients showed a significantly higher indocyanine green retention rate at 15 min (ICG-R15), were more obese, and showed advanced fibrosis of noncancerous tissues. Conclusions: The expression status of stem cell markers differed between NASH- and HCV-related liver diseases. The expression of Nanog and EpCAM in HCC were mutually exclusive and their expression in noncancerous liver tissues may be an independent risk factor in NASH- and HCV-related HCCs, respectively. Citation Format: Tomomi Kogiso, Etsuko Hashimoto, Kazuhisa Kodama, Maki Tobari, Noriko Matsushita, Nobuyuki Torii, Makiko Taniai, Katsutoshi Tokushige, Keiko Shiratori. Differential expression of cancer stem cell markers and their clinicopathological features in hepatocellular carcinoma patients with different etiologies. [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 3878. doi:10.1158/1538-7445.AM2014-3878

HCC1 was originally cloned as a cell nuclear autoantigen in a patient with liver cirrhosis that progressed to HCC. The full-length sequence of HCC1encodes a protein of 530 amino acids and migrating at 64 kDa in SDS-PAGE. HCC1 has two isoforms of HCC1.4 and HCC1.3. The difference is that HCC1.3 has 6-amino acid deletion located in the RNP-1-like region of the putative third RNP-CS domain. The nucleotides and protein sequences of HCC1 were not found at that time in searching available databases and, therefore, it was considered as a novel gene with unknown function. Several years later, a group showed that HCC1 was a co-activator of activating protein-1 and estrogen receptors and proposed naming it CAPER. Further study by other group has demonstrated that HCC1/CAPER is a novel transcriptional co-activator for v-Rel that strongly suppresses its transforming activity, uncovering a tumor suppressor role for HCC1/CAPER in regulating Rel's oncogenic activity. Our previous studies have demonstrated that autoantibodies against tumor-associated antigens (TAAs) can be regarded as serological biomarkers in cancer immunodiagnosis. Whether autoantibodies to HCC1/CAPER can be also used as one of the markers in HCC remains to be investigated. In the present study, autoantibody responses to HCC1.3 and HCC1.4 were firstly evaluated by ELISA, western blotting and indirect immunofluorescence assay in sera from patients with HCC, liver cirrhosis and chronic hepatitis, as well as from normal human individuals. Immunohistochemistry (IHC) with HCC tissue array slides was also performed to analyze protein expression profiles of HCC1. The prevalence of anti-HCC1.4 antibodies was 13.5% (23/171) in HCC, which was significantly higher than that in normal human sera (1.1%). Whereas the significant difference regarding frequency of anti-HCC1.3 antibodies between HCC patients and normal individuals was not found. The frequency of HCC1 expression in HCC tissues was also significantly higher than that in normal liver tissues (57.5% vs. 0%; P&amp;lt;0.001). The data suggests that anti-HCC1.4 autoantibody may be a potential biomarker for HCC, but not HCC1.3. Further functional study of HCC1 may provide more evidence to address the question why these two isoforms of HCC1 can induce different humoral immune response in HCC patients. Citation Format: Liping Dai, Pengfei Ren, Xinxin Liu, Rosalia Ortega, Wu Yao, Kaijuan Wang, Mingan Wang, Eng M. Tan, Jianying Zhang. Humoral autoimmune response to a RNA-binding protein HCC1/CAPER in hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1256. doi:10.1158/1538-7445.AM2013-1256

Objective: Hepatocellular carcinoma (HCC) is a highly heterogeneous disease with a dismal prognosis. However, driver genes and prognostic markers in HCC remain to be identified. It is hoped that in-depth analysis of HCC genomes in relation to available clinicopathological information will give rise to novel molecular prognostic markers.Methods: We collected genomic data of 1,061 HCC patients from previous studies, and performed integrative analysis to identify significantly mutated genes and molecular prognosticators. We employed three MutSig algorithms (MutSigCV, MutSigCL and MutSigFN) to identify significantly mutated genes. The GISTIC2 algorithm was used to delineate focally amplified and deleted genomic regions. Nonnegative matrix factorization (NMF) was utilized to decipher mutational signatures. Kaplan-Meier survival and Cox regression analyses were used to associate gene mutation and copy number alteration with survival outcome. Logistic regression model was applied to test association between gene mutation and mutational signatures.Results: We discovered 11 novel driver genes, including RNF213, VAV3 and TNRC6B, with mutational prevalence ranging from 1% to 3%. Seven mutational signatures were also identified in HCC, some of which were associated with mutations of classical driver genes (e.g., TP53, TERT) as well as alcohol consumption. Focal amplifications of TERT and other druggable targets, including AURKA, were also revealed. Targeting AURKA by a small-molecule inhibitor potently induced apoptosis in HCC cells. We further demonstrated that HCC patients with TERT amplification displayed shortened overall survival independent of other clinicopathological parameters. In conclusion, our study identified novel cancer driver genes and prognostic markers in HCC, reiterating the translational importance of omics data in the precision medicine era.

Hepatocellular carcinoma (HCC) is a common malignancy in the world with high morbidity and mortality rate. Identification of novel biomarkers in HCC remains impeded primarily because of the heterogeneity of the disease in clinical presentations as well as the pathophysiological variations derived from underlying conditions such as cirrhosis and steatohepatitis. The aim of this study is to search for potential metabolite biomarkers of human HCC using serum and urine metabolomics approach. Sera and urine samples were collected from patients with HCC (n = 82), benign liver tumor patients (n = 24), and healthy controls (n = 71). Metabolite profiling was performed by gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography-quadrupole time of flight mass spectrometry in conjunction with univariate and multivariate statistical analyses. Forty three serum metabolites and 31 urinary metabolites were identified in HCC patients involving several key metabolic pathways such as bile acids, free fatty acids, glycolysis, urea cycle, and methionine metabolism. Differentially expressed metabolites in HCC subjects, such as bile acids, histidine, and inosine are of great statistical significance and high fold changes, which warrant further validation as potential biomarkers for HCC. However, alterations of several bile acids seem to be affected by the condition of liver cirrhosis and hepatitis. Quantitative measurement and comparison of seven bile acids among benign liver tumor patients with liver cirrhosis and hepatitis, HCC patients with liver cirrhosis and hepatitis, HCC patients without liver cirrhosis and hepatitis, and healthy controls revealed that the abnormal levels of glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, and chenodeoxycholic acid are associated with liver cirrhosis and hepatitis. HCC patients with alpha fetoprotein values lower than 20 ng/ml was successfully differentiated from healthy controls with an accuracy of 100% using a panel of metabolite markers. Our work shows that metabolomic profiling approach is a promising screening tool for the diagnosis and stratification of HCC patients.

Chapter 2: Diagnosis and surveillance

Background: Hepatocellular carcinoma (HCC) is the most prevalent cancer in the liver, the 5th more common cause of deaths by cancer in the US, and the 3th worldwide. Tumor heterogeneity, few targeted therapies, and late detection make the overall prognosis of patients unsatisfactory and demonstrate the need for better early-detection biomarkers. Liver, by its nature, is the major site of amino acid processing and highly regulated by metabolic signaling. In particular, the majority of ingested tryptophan is processed in the liver through the kynurenine pathway, the endpoint of which is de novo NAD+ biosynthesis. Cumulative evidence shows that dysregulation tryptophan-kynurenine metabolism may promote tumor reprogramming and carcinogenesis. In this work, we provide new insight on how kynurenine pathway can be used as biomarker for patient prognosis. Methods: Using a publicly available gene expression dataset from liver hepatocellular carcinoma (LIHC) samples available through The Cancer Genome Atlas (TCGA), we employ Principal Component Analysis (PCA) to show that expression of kynurenine pathway gene in cancer cells can be used to cluster patients with particular clinical profiles and predict prognosis. Hierarchical clustering and dimensionality reduction demonstrate the segregation of cluster patients based on gene-pattern expression profiles. Kaplan-Meier survival curves were used to determine patient overall survival and Wilcoxon Signed Rank test to prove statistical significance. Results: Principal Component 1 (PC1) explains 14.5% gene variance among the patients while PC2 explain 9.2% of variance. Clustering genes by expression across patients, IDO, IDO2, AADAT, KMO, CCBL1, CCBL2, KYNU and ACMSD form one cluster, HAAO and AFMID form a second, and QPRT and TDO2 fall outside of clear cluster boundaries. Hierarchical k-means clustering of the patients based on the kynurenine gene-expression profiled segregated patients into four clusters. Heatmap representation of z-score values reveal that each cluster represents tumors with elevated expression of a specific gene or pair of genes: TDO, HAAO/AFMID, QPRT, and KYNU. Other kynurenine pathway genes appear to have stable expression among the population of HCC patients. Dimensionality reduction using t-Distributed Stochastic Neighbor Embedding (t-SNE) identified the same distribution as hierarchical clustering. One of the outcomes that was clearly determined by this segregation among HCC patients was overall survival. Using the total number of patients from the dataset (n = 371), patients with high QPRT expression had poor prognosis with increased median mortality, with no effect in the maximum survival. There is a significant decrease in the survival between patients in cluster 3 (with high QPRT expression) and those in cluster 2 (with high HAAO/AFMID expression) (HR = 1.2, [95% CI 0.5-1.8] P = 0.0181, Gehan-Breslow-Wilcoxon Test). Furthermore, patients with high vs low QPRT expression have significantly different survival rates (HR = 1.4, [95% CI 0.9-2.2] P = 0.0344, Gehan-Breslow-Wilcoxon Test). Conclusions: Kynurenine metabolism can be used to determine patient prognosis among HCC patients. In ongoing work, we are testing the hypothesis that inhibition of QPRT with phthalic acid will sensitize HepG2 to cisplatin-induced apoptosis, to validate our prediction model. Citation Format: Raul Castro-Portuguez, Samuel Freitas, George L. Sutphin. Kynurenine metabolism as a biomarker and therapeutic target in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-241.

Poly(C)‑binding protein2 (PCBP2) is a member of the PCBP family, and plays an important role in post‑transcriptional and translational regulation of various signaling molecules through direct binding to single‑stranded poly(C) motifs. PCBP2 has been reported to play a critical role in the development of multiple human tumors. However, whether PCBP2 participates in hepatocellular carcinoma(HCC) development remains largely elusive. Herein, we showed that PCBP2 was upregulated in human HCC tissues and cell lines. Overexpression of PCBP2 predicted significantly worsened prognosis in HCC patients, suggesting that PCBP2 may serve as a prognostic marker of HCC. In addition, we found that depletion of PCBP2 inhibited HCC cell proliferation, accompanying the increase in the cyclin‑dependent kinase inhibitor p27 level. Moreover, we found that high expression of PCBP2 may contribute to sorafenib resistance in HCC cells, involving dysregulated expression of Bax and Bcl‑2 proteins. In conclusion, our results suggest that PCBP2 may serve as a prognostic marker and potential therapeutic target of HCC.

BackgroundNumerous hepatocellular carcinoma (HCC) biomarkers have been assessed in the diagnosis and prognosis of HCC. The aim of this study was to assess the value of α-fetoprotein (AFP)-L3% and transforming growth factor B1 (TGFB1) as prognostic markers in hepatocellular carcinoma after radiofrequency ablation (RFA). This observational cohort study included 40 patients with HCC diagnosed by triphasic computed tomography criteria indicated for radiofrequency ablation. Serum AFP, AFP-L3%, and TGFB1 were measured in all patients before and 3 months after radiofrequency ablation.ResultsStatistically significant lower levels of TGFB1, AFP, and AFP-L3% were noted in the HCC patients after radiofrequency ablation. Significant lower levels of TGFB1, AFP, and AFP-L3% were found in the no recurrence group in comparison to the recurrence group. The cutoff value of TGFB1 > 56.87 ng/mL, AFP > 74.9 ng/mL, and AFP-L3% > 8.5% was the best in the discrimination of tumor recurrence with sensitivity of 85.7%, 57.1%, and 100%; specificity of 54.6%, 84.9%, and 100%; and diagnostic accuracy of 64.5%, 69%, and 100%, respectively.ConclusionTGFB1 and AFP-L3% are good prognostic markers for HCC. They could be used to monitor the response of HCC to treatment.

Liver cancer has the third highest mortality rate among all cancers in China and hepatocellular carcinoma (HCC) is the most common type of liver cancer. The mitogen-activated protein kinase (MAPK) signaling pathway is often constitutively active in HCC. The growth of HCC requires the formation of new blood vessels. and VEGF is critical in this process. Sorafenib, a multikinase inhibitor that targets both RAF and VEGF receptor, is to date the only approved drug to treat advanced stages of HCC. Despite sorafenib extended the survival in patients with HCC, its clinical benefits remain modest and drug resistance is common. BGB283 is a second generation RAF inhibitor with unique RAF dimer and VEGFR inhibition activity. Thus, there is good rationale to test BGB-283 in HCC. In this study, we compared the in vitro and in vivo activities of BGB-283 and sorafinib in human HCC cell lines and patient derived HCC xenograft models. In the biochemical assays, BGB-283 demonstrated great potency for BRAF-V600E, BRAF-WT, CRAF(IC50 = 32, 69 and 6.5 nM, respectively) and for VEGFR family enzymes, VEGFR1, VEGFR2 and VEGFR3 (IC50 = 25, 14 and 58 nM, respectively). In the cellular assays, the anti-proliferative effect of BGB-283 and sorafinib was evaluated in several HCC cell lines. A head-to-head comparison of in vivo anti-tumor activities of BGB-283 and sorafinib were also evaluated in human primary HCC xenograft mouse models. The patient derived xenograft (PDX) HCC models were established in house using HCC patient surgical samples. Sorafinib was not active in 2 out of 7 models. Oral administration of BGB-283 resulted in significant tumor growth inhibition in all 7 models and was significantly more efficacious than sorafinib in 4 models and similar in the other 3 models. In conclusion, BGB-283 is a unique RAF dimer inhibitor with VEGFR inhibition activity. BGB-283 has also demonstrated better anti-tumor activity than sorafinib in HCC PDX models, suggesting BGB-283 could be a promising drug candidate for treating HCC patients. Citation Format: Zhiyu Tang, Yong Liu, Beibei Jiang, Yajuan Gao, Wenfeng Gong, Xing Wang, Dan Su, Fenglong Yu, Ye Liu, Min Wei, Lai Wang. BGB-283: a novel RAF Dimer inhibitor, displays potent antitumor activity in HCC patient derived xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1230.

Hepatitis B virus (HBV) infection still remains to be one of the dominant risk factor for hepatocellular carcinoma (HCC). Both genetic as well as environmental factors influence the progression of chronic hepatitis B (CHB) to HCC. MHC class I polypeptide-related chain A and B (MICA and MICB) molecules are induced in response to viral infection as well as various stresses. We have recently revealed that SNP in MICA promoter was significantly associated with HCV-induced HCC risk as well as lower sMICA level in serum. To investigate the possible involvement of MICA in hepatocellular carcinogenesis caused by HBV-infection, we analyzed MICA gene polymorphisms and the levels of soluble MICA (sMICA) in HBV-induced HCC patients. The genetic association analysis revealed a nominal association at rs2596542 in which G allele was associated with susceptibility to HBV-induced HCC (P=0.029 with odds ratio of 1.19). We also found a significant elevation of sMICA in HBV-induced HCC cases. Moreover, genotypes at SNP rs2596542 located in the upstream of MICA promoter, was significantly associated with sMICA levels (P = 0.009). Interestingly. HCC patients with high sMICA (&amp;gt; 5 pg/ml) exhibited poor prognosis compared with patients with low sMICA (P = 0.008). Thus, our results highlight the importance of MICA genetic variations and sMICA as predictive biomarkers for HBV-induced HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1648. doi:1538-7445.AM2012-1648