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Purpose Alpha-fetoprotein-producing gastric cancer (AFPGC) and hepatoid adenocarcinoma of stomach (HAS) are rare types of gastric cancer, with specific clinical manifestations and poor prognosis. The standardized treatment process of such cancers remains elusive. We aim to investigate the efficacy of immunotherapy combined with chemotherapy on patients with AFPGC or HAS. Patients and methods AFPGC and HAS patients who underwent immunotherapy and/or chemotherapy as the first-line treatment at our institute from June 2016 to December 2018 were enrolled in this observational study. Their clinicopathological characteristics, serum AFP level and treatment methods were collected. The progression-free survival (PFS) and overall survival (OS) were analyzed and compared between patients who received immunotherapy plus chemotherapy and those received chemotherapy. Results A total of 21 patients with advanced AFPGC or HAS were included in the study and the median follow-up time was 28.0 months. Of the 21 patients, 7 patients received immunotherapy of PD-1 antibody (nivolumab) plus chemotherapy and 14 patients as control received chemotherapy with or without Herceptin/Apatinib. The median progression-free survival (mPFS) time was 5.0 months (4.3 months in the control group and 22.0 months in the immunotherapy group). The median overall survival (mOS) time of the control group was 16.0 months (14.0 months in chemotherapy alone subgroup, 20.0 months in chemotherapy plus Apatinib or Herceptin subgroup), while the mOS of patients receiving immunotherapy was not reached. Conclusion This study suggested PD-1 checkpoint inhibitor plus chemotherapy could benefit AFPGC and HAS patients. Its mechanism of action warrants further investigation.

Background The indistinctive effects of antiangiogenesis agents in gastric cancer (GC) can be attributed to multifaceted gene dysregulation associated with angiogenesis. Angiopoietin‐like (ANGPTL) proteins are secreted proteins regulating angiogenesis. They are also involved in inflammation and metabolism. Emerging evidences have revealed their various roles in carcinogenesis and metastasis development. However, the mRNA expression profiles, prognostic values, and biological functions of ANGPTL proteins in GC are still elucidated. Methods We compared the transcriptional expression levels of ANGPTL proteins between GC and normal gastric tissues using ONCOMINE and TCGA‐STAD. The prognostic values were evaluated by LinkedOmics and Kaplan–Meier Plotter, while the association of expression levels with clinicopathological features was generated through cBioPortal. We conducted the functional enrichment analysis with Metascape. Results The expression of ANGPTL1/3/6 was lower in GC tissues than in normal gastric tissues. High expression of ANGPTL1/2/4 was correlated with short overall survival and post‐progression survival in GC patients. Upregulated ANGPTL1/2 was correlated with higher histological grade, non‐intestinal Lauren classification, and advanced T stage, while ANGPTL4 exhibited high expression in early T stage, M1 stage, and non‐intestinal Lauren classification. Conclusions Integrative bioinformatics analysis suggests that ANGPTL1/2/4 may be potential therapeutic targets in GC patients. Among them, ANGPTL2 acts as a GC promoter, while ANGPTL1/4’s role in GC is still uncertain., Integrative bioinformatics analysis suggests that ANGPTL1/2/4 may be potential therapeutic targets in GC patients. Among them, ANGPTL2 acts as a GC promotor, while ANGPTL1/4’s role in GC is still uncertain.

High-throughput messenger RNA (mRNA) analysis has become a powerful tool for exploring tumor recurrence or metastasis mechanisms. Here, we constructed a signature to predict the recurrence risk of Stages II and III gastric cancer (GC) patients. A least absolute shrinkage and selection operator method Cox regression model was utilized to construct the signature. Using this method, a 16-mRNA signature was identified to be associated with the relapse-free survival of Stages II and III GCs in training dataset GSE62254 (n = 194). Then this signature was validated in an independent Gene Expression Omnibus cohort GSE26253 (n = 297) and a dataset of The Cancer Genome Atlas (TCGA; n = 235). This classifier could successfully screen out the high-risk Stages II and III GCs in the training cohort (hazard ratio [HR] = 40.91; 95% confidence interval [CI] = 5.58-299.7; p < .0001). Analysis in two independent validation cohorts yielded consistent results (GSE26253: HR = 1.69, 95% CI = 1.17-2.43,; p = .0045; TCGA: HR = 2.01, 95% CI = 1.13-3.56, p = .0146). Cox regression analyses revealed that the risk score derived from this signature was an independent risk factor in Stages II and III GCs. Besides, a nomogram was constructed to serve clinical practice. Through gene set variation analysis, we found several gene sets associated with chemotherapeutic drug resistance and tumor metastasis significantly enriched in high-risk patients. In summary, this 16-mRNA signature can be used as a powerful tool for prognostic evaluation and help clinicians identify high-risk patients.

Gastric cancer (GC) is a common disease globally with high mortality rate. It is therefore necessary to develop novel therapies targeting specific events in the pathogenesis of GC. Some hnRNP family members are involved in multiple cancer biological behaviors. However, the potential function and mechanism of hnRNPR, a new molecule of hnRNP family in GC remains unknown. We found that the expression of hnRNPR was significantly overexpressed in multiple cancers compared to the normal tissues. Functionally, hnRNPR promoted cancer cell proliferation, migration, and invasion. Knockdown of hnRNPR in two type mice models, with two types of tumors models decreased the tumor aggressiveness and metastasis. Mechanistically, hnRNPR targeted oncogenic pathways by stabilizing the expression of CCNB1 and CENPF mRNA level. Knockdown of CCNB1 and CENPF abolished the hnRNPR-induced cell growth and invasion, respectively. Furthermore, the protein level of hnRNPR in the tumor was positively correlated with the expression of CCNB1 and CENPF in clinical samples. Together, these results indicate that overexpression of hnRNPR promoted the aggressiveness of GC by increasing the mRNA expression of CCNB1 and CENPF. HnRNPR-CCNB1/CENPF axis may be a potential therapeutic target for GC treatment.

Our previous study demonstrated that heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) is a key gene that facilitates metastasis of hepatocellular carcinoma (HCC). However, the molecular mechanisms behind this relationship are not fully understood. In our study, we utilized long-noncoding RNA (lncRNA) microarrays to identify a HNRNPAB-regulated lncRNA named lnc-ELF209. Our findings from chromatin immunoprecipitation assays indicate that HNRNPAB represses lnc-ELF209 transcription by directly binding to its promoter region. We also analyzed clinical samples from HCC patients and cell lines with quantitative real-time polymerase chain reactions, RNA in situ hybridization and immunohistochemistry, and found that there is a negative relationship between HNRNPAB and lnc-ELF209 expression. Up/downregulation assays and rescue assays indicate that lnc-ELF209 inhibits cell migration, invasion and epithelial-mesenchymal transition regulated by HNRNPAB. This suggests a new regulatory mechanism for HNRNPAB-promoted HCC progression. RNA pull-down and LC-MS/MS were used to determine triosephosphate isomerase, heat shock protein 90-beta and vimentin may be involved in the tumor-suppressed function of lnc-ELF209. Furthermore, we found lnc-ELF209 could stabilize TPI protein expression. We also found that lnc-ELF209 overexpression in HCCLM3 cell resulted in a lower rate of lung metastatic, which suggested a less aggressive HCC phenotype. Collectively, these findings offer new insights into the regulatory mechanisms that underlie HNRNPAB cancer-promoting activities and demonstrate that lnc-ELF209 is a HNRNPAB-regulated lncRNA that may play an important role in the inhibition of HCC progression.

Natural killer (NK) cell can inhibit tumor initiation and regulates metastatic dissemination, acting as key mediators of the innate immune response. Intrinsic factors modulating NK cells infiltration and its anticancer activity remain poorly characterized. We investigated the roles of dysregulation of micro(mi)RNAs and NK cells in progression of hepatocellular carcinoma (HCC). Methods: Small RNA sequencing were used to detect the miRNA profiles of tumor tissues from HCC patients with (n=14) or without (n=13) pulmonary metastasis and HCC cell lines with different pulmonary metastatic potentials. Chemokine expression profiling and bioinformatics were used to detect the downstream target of candidate target. In gain- and loss-of-function assays were used to investigate the role of miRNA in HCC progression. Different subsets of NK cells were isolated and used for chemotaxis and functional assays in vivo and in vitro. In situ hybridization and immunohistochemical analyses were performed to detect the expression of miRNA in tumor tissues from 242 HCC patients undergoing curative resection from 2010. Results: Three miRNAs (miR-137, miR-149-5p, and miR-561-5p) were identified to be associated with pulmonary metastasis in patients with HCC. miR-561-5p was most highly overexpressed in metastatic HCC tissues and high-metastatic-potential HCC cell lines. In gain- and loss-of-function assays in a murine model, miR-561-5p promoted tumor growth and spread to the lungs. Yet, miR-561-5p did not appear to affect cellular proliferation and migration in vitro. Bioinformatics and chemokine expression profiling identified chemokine (C-X3-C motif) ligand 1 (CX3CL1) as a potential target of miR-561-5p. Furthermore, miR-561-5p promoted tumorigenesis and metastasis via CX3CL1-dependent regulation of CX3CR1+ NK cell infiltration and function. CX3CR1+ NK cells demonstrated stronger in vivo anti-metastatic activity relative to CX3CR1- NK cells. CX3CL1 stimulated chemotactic migration and cytotoxicity in CX3CR1+ NK cells via STAT3 signaling. Blockade of CX3CL1, CX3CR1, or of pSTAT3 signaling pathways attenuated the antitumor responses. Clinical samples exhibited a negative correlation between miR-561-5p expression and levels of CX3CL1 and CX3CR1+ NK cells. High miR-561-5p abundance, low CX3CL1 levels, and low numbers of CX3CR1+ NK cells were associated with adverse prognosis. Conclusion: We delineated a miR-561-5p/CX3CL1/NK cell axis that drives HCC metastasis and demonstrated that CX3CR1+ NK cells serve as potent antitumor therapeutic effectors.

Alpha-fetoprotein (AFP)-producing gastric cancer (AFPGC) is regarded as a rare but highly malignant gastric adenocarcinoma subtype and its clinic pathological presentation mimics hepatocellular carcinoma. However, the underlying mechanism of this disease remains elusive. The level of ANGPTL6 in AFPGC cell lines is much higher than that of common types of gastric cancer cells. A high level of ANGPTL6 confers a poor prognosis and is correlated with the expression of CD34 (an endothelial cell marker). ANGPTL6 promotes endothelial cell migration and tube formation, Moreover, ANGPTL6 knockdown inhibits cancer cell apoptosis and invasiveness. Mechanistically, ANGPTL6 activates the ERK1/2 and AKT pathways. Treatment of ERK1/2 or AKT inhibitor can attenuated cell migration and tube formation. ANGPTL6 loss results in tumor growth in vivo. Our study revealed that ANGPTL6 is an important driver gene of angiogenesis in AFPGC development. These findings provide not only an effective biomarker for diagnosis but also an attractive therapeutic target for use in AFPGC patients.

Background Due to the phenotypic and molecular diversity of hepatocellular carcinomas (HCC), it is still a challenge to determine patients’ prognosis. We aim to identify new prognostic markers for resected HCC patients. Methods 274 patients were retrospectively identified and samples collected from Zhongshan hospital, Fudan University. We analyzed the gene expression patterns of tumors and compared expression patterns with patient survival times. We identified a “9-gene signature” associated with survival by using the coefficient and regression formula of multivariate Cox model. This molecular signature was then validated in three patients cohorts from internal cohort (n = 69), TCGA (n = 369) and GEO dataset (n = 80). Results We identified 9-gene signature consisting of ZC2HC1A, MARCKSL1, PTGS1, CDKN2B, CLEC10A, PRDX3, PRKCH, MPEG1 and LMO2. The 9-gene signature was used, combined with clinical parameters, to fit a multivariable Cox model to the training cohort (concordance index, ci = 0.85), which was successfully validated (ci = 0.86 for internal cohort; ci = 0.78 for in silico cohort). The signature showed improved performance compared with clinical parameters alone (ci = 0.70). Furthermore, the signature predicted patient prognosis than previous gene signatures more accurately. It was also used to stratify early-stage, HBV or HCV-infected patients into low and high-risk groups, leading to significant differences in survival in training and validation (P

The semaphorins were originally identified as having roles as guidance cues during neural development. Class 3 semaphorins are involved in cancer progression. However, the roles of class 3 semaphorins in hepatocellular carcinoma (HCC) are unknown. We examined the expression levels of class 3 semaphorins in HCC cell lines with different metastatic potential and in carcinoma tissue samples. The results indicated that Semaphorin 3A expression was up-regulated in metastatic cell lines and in samples from patients with tumor recurrence. Cell functional studies revealed that Semaphorin 3A promoted HCC cell proliferation, migration, and invasion. Animal studies indicated that Semaphorin 3A overexpression enhanced tumor growth and lung metastasis. Semaphorin 3A also acted as a chemoattractant involved in direct recruitment of macrophages in vitro, and facilitated tumor-associated macrophage (TAM) infiltration in vivo. Multivariate analysis revealed that Semaphorin 3A expression alone, or combined with the number of TAMs, can be an independent predictor for overall survival time and time to recurrence. Overall, the results suggested that Semaphorin 3A increased TAM infiltration and promoted HCC progression. Semaphorin 3A expression alone, or combined with the number of TAMs, is a new prognostic factor and potential target for the treatment of HCC.

Background: In recent years, hepatoid adenocarcinoma (HAC), a rare tumor subtype, has attracted more and more attention because of its malignant nature, especially its poor prognosis due to frequent liver metastasis. HAC, recognized as an extrahepatic cancer that resembles hepatocellular carcinoma (HCC), is characterized by the solid sheet-like growth structures of hepatoid differentiation and excessive production of alpha-fetoprotein (AFP). Hepatoid adenocarcinoma of the stomach (HAS) is the most common site of HAC, however, the differences between HAS and HAC of other organs are unknown. Methods: Our Chinese HAC cohort consisted of 31 samples (25 HAS patients and 6 HAC of other organs patients) that were collected in the form of formalin-fixed, paraffin-embedded (FFPE) tumor tissues at Zhongshan Hospital from July 2013 to August 2017 for whole exome sequencing (WES) (OrigiMed, Shanghai). Results: A total of 31 patients with HAC had surgical treatment, 25 patients with HAS (median age 66.5 years, range 48-82) and 6 patients with HAC of other organs (median age 62.5 years, range 52-67). Most HAS patients were male (18/25, 72%), and most HAC of other organs patients were female (4/6, 66.7%). Survival analysis revealed that compared with patients with HAS, patients with HAC of other organs had a significantly poorer overall survival (OS) (p=0.0081) and disease-free survival (DFS) (p=0.0011). The most frequently mutated genes in the HAS tumor samples were TP53 (42%), MUC19 (35%), and TTN (35%). The substitution mutations of CDK4, KRT34 and GOLGA6L3 were only found in HAC from other organs samples but not from HAS samples. Copy number variations (CNVs) of CCNE1, VSTM2B, PLEKHF1 and POP4 were only identified in HAS samples. Interestingly, these 4 genes were located at chr19 and were co-mutated in 5 patients who had a significantly poorer OS than those without these CNVs (11.1 months vs. 19.3 months, respectively, p=0.045). The tumor mutational burden (TMB) of HAS was significantly higher compared with HAC of other organs was also significantly different (4 muts/Mb vs. 1.2 muts/Mb, respectively, p=0.012). Conclusions: Our study revealed the genomic profiling of Chinese HAC patients. Compared to patients with HAS, patients with HAC of other organs had a significantly poorer OS and DFS. The origin for HAC was associated with specific gene mutations and the TMB value. Patients with CNVs in chr19 were significantly associated with a poorer OS. Citation Format: Tianshu Liu, Erbao Chen, Chen Xu, Wei Li, Mengling Liu, Yichou Wei, Yiyi Yu, Yuehong Cui, Qian Li, Shuirong Zhang, Lujia Huang, Yueting Qu. Genomic alterations and clinical correlations of Chinese hepatoid adenocarcinoma patients [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 4746.

e13514 Background: Hepatoid adenocarcinoma of stomach (HAS) is a rare subtype of gastric cancer (GC) with poor prognosis due to frequent liver metastasis. HAS, recognized as an extrahepatic cancer that resembles hepatocellular carcinoma (HCC), is characterized by solid sheet-like growth structures with hepatoid differentiation and excessive production of alpha-fetoprotein (AFP). This HAS subtype of GC (AFPGC) is genetically distinct compared to other common GC but requires more evidences for better characterization. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor tissues were collected from 25 HAS and 9 AFPGC patients at Zhongshan Hospital from July 2013 to August 2017 for whole exome sequencing (WES) test. The testing was carried out by a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Results: Our cohort included 25 males and 9 females who had undergone surgical treatment. The 25 HAS patients had a median age of 66.5 years (range 48-82) and the 9 AFPGC patients had a median age of 64 years (range 52-76). Patients with AFP>20 had a significantly better overall survival (OS) compared with patients with AFP≤20. The most frequently mutated genes were TP53 (44%), TTN (44%), and MUC19 (30%) in HAS tumor samples and CDC27 (44%), NKX2-3 (44%), and TTN (44%) in AFPGC samples. Patients with URI1 or POP4 alterations had a significantly poorer OS than patients without those genes mutations (7.6 months vs. 19.8 months, p = 0.016, and 10.9 months vs. 19.7 months, p = 0.038, respectively). However, patients with SPTA1 alterations had a significantly better OS compared to patients without SPTA1 alterations (27.6 months vs. 15.8 months, p = 0.042). Patients with higher tumor mutational burden (TMB) (≥3.9 muts/Mb) had a significantly better OS compared with patients with lower TMB (

Additional file 2: Table S2. The coefficient and regulation of 9-gene signature.

Backgroud: Gastric cancer (GC) has a high mortality rate worldwide, this calls for urgent novel therapies targeting specific underlying oncogenic events. However, the potential function and mechanism of hnRNPR in GC remains unknown. Methods: Data from The Cancer Genome Atlas (TCGA) and GEO database were used to investigate the expression of hnRNPR in multiple cancers. The functional effects of up/down-regulated hnRNPR levels were assessed via in vitro and in vivo assays (CCK-8, colony formation, wound scratch, transwell, subcutaneous tumor model and metastasis model). Gene Set Enrichment Analysis (GSEA) were performed to identify hnRNPR-downstream signaling pathways in TCGA GC cohort. RNA immunoprecipitation-PCR was used to validate the direct binding between hnRNPR and the downstream genes. Findings: The expression of hnRNPR is significantly overexpressed in multiple cancers. Functionally, hnRNPR increased cancer cell proliferation, migration, and invasion. Concordantly, in in vivo orthotopic mice models, knockdown of hnRNPR was associated with decreased features of tumor aggressiveness and metastasis. Mechanistically, hnRNPR leads to typical oncogenic pathways activated via stabilizing CCNB1 and CENPF mRNA. Knockdown pf CCNB1 and CENPF abolished the hnRNPR-induced cell growth and invasion. Furthermore, the protein level of hnRNPR in the tumor was positively correlated with the expression of CCNB1 and CENPF in clinical samples. Interpretation: The results indicated that overexpression of hnRNPR facilitated GC aggressiveness by stabilizing CCNB1 and CENPF mRNA. HnRNPR/CCNB1/CENPF axis may represent a potential therapeutic target for GC treatment. This finding casts new light on developing more rational therapies to treat this highly aggressive tumor. Funding: This work was supported by the National Natural Science Foundation of China (81502003) and the Shanghai Science and Technology Commission (17411951400, 15411961900). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The study was approved by the Research Ethics Committee of Zhongshan Hospital.

Background Liver is the most frequent metastatic site of gastric cancer (GC), especially in patients with HER2 positive GC. Exosomal integrin αvβ5 has been shown to promote liver metastasis (LM), and the cross talk between integrins and HER2 during breast cancer metastasis has been reported. However, whether there's an association between HER2 and integrin αvβ5 (ITGAvB5), and whether their association has predictive value in GC liver metastasis (GCLM) remains unknown. Methods The association between ITGβ5 and HER2 were accessed by RT-PCR, western blot and ELISA. We tested the function of ITGβ5 on HER2 positive GC cells using Transwell assays and scratch assays. Besides, we detect ITGβ5 expression in tumor tissue of GC patients and exosomes derived from advanced GC to analyze the association between HER2 and LM. Results In our study, we found that ITGβ5, rather than ITGAV, was highly upregulated by HER2 through PI3K-AKT pathways in HER2 positive GC. Overexpression of ITGβ5 promoted the migration and invasion of HER2 positive GC cells in vitro. ITGβ5 was found to be an independent prognostic factor for GC. Besides, ITGβ5 level was only associated with LM. Detection of exosomal ITGβ5 and HER2 in the serum of GC patients revealed that exosomal ITGβ5 and HER2 levels are in accordance with that in tissue, and exosomal ITGβ5 level was higher in GCLM than other metastasis. Conclusions Our study demonstrated ITGβ5 is regulated and functions in accordance with HER2 in promoting GCLM. Exosomal ITGβ5 levels might be a potential liquid biopsy biomarker for GCLM.

Gui-Qi Zhu,1,2,* Kang Wang,3,* Biao Wang,1,2,* Yu-Jie Zhou,4,* Yi Yang,1,2 Er-Bao Chen,5 Zheng-Jun Zhou,1,2 Shao-Lai Zhou,1,2 Ying-Hong Shi,1,2 Jian Zhou,1,2 Zhi Dai1,2 1Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China; 2State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, 200032, China; 3Department of Breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing Medical University, Chongqing, 400016, China; 4Department of Gastroenterology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, Shanghai, China; 5Department of Medical Oncology, Zhongshan Hospital, Fudan University, Shanghai, China *These authors contributed equally to this work Background and aim: To investigate the value of the aspartate aminotransferase-to-platelet ratio index (APRI) and build a new nomogram for hepatocellular carcinoma (HCC) patients undergoing postoperative adjuvant transarterial chemoembolization (PATACE).Methods: We retrospectively reviewed 351 patients with HCC undergoing PATACE. We collected baseline HCC patient characteristics to obtain the risk factors for determining poor disease-free survival (DFS) and early time to recurrence (TTR) after PATACE. The multivariate Cox proportional hazards model was used to build new nomogram based on significant prognostic factors of outcomes.Results: We generated the cutoff value of the APRI as 0.50 using the X-tile to distinguish patients with different outcomes in the whole cohort. Two hundred seventeen patients with high APRI had poorer survival (P

A high preoperative peripheral blood neutrophil-to-lymphocyte ratio (NLR) has been reported to be a predictor of poor survival in patients with various cancers. The aim of this study was to evaluate the predictive significance of the NLR in patients undergoing hepatectomy for intrahepatic cholangiocarcinoma (ICC). From 2005 to 2011, 322 patients who underwent hepatectomy for ICC were enrolled in this retrospective study. Clinicopathological parameters, including NLR, were evaluated to identify predictors of overall and recurrence-free survival after hepatectomy. The best cutoff for NLR was 2.49, and 177 of 322 patients (54.9 %) had an NLR ≥ 2.49. The 5-year survival rate after hepatectomy was 51.1 % in patients with NLR < 2.49 and 24.8 % in those with NLR ≥ 2.49 (P = 0.0001). Univariate analyses revealed that NLR was significantly associated with recurrence-free survival (RFS) and overall survival (OS; both P < 0.05). Multivariable analyses revealed that elevated NLR independently predicted poorer OS (P = 0.003, hazard ratio [HR] = 1.600). In summary, our results indicate that elevated NLR is a promising independent predictor of poor survival after hepatectomy in patients with ICC.

Background: Due to the phenotypic and molecular diversity of hepatocellular carcinomas (HCC), it is a challenge to determine patients' prognosis. We aimed to identify new biomarkers of resected patients. Methods: We collected 274 HCC samples from patients at Zhongshan hospital, Fudan university. Gene expression analyses were performed using NanoString technology and compared with patient survival times. Using the coefficient and regression formula of the multivariate Cox model, we identified a "9-gene signature" associated with survival times. This molecular signature was then validated in three groups of patients from internal cohort (n=69), TCGA dataset (n=369) and HCC genomic consortium (n=80). Results: We identified 9-gene signature consisting of ZC2HC1A, MARCKSL1, PTGS1, CDKN2B, CLEC10A, PRDX3, PRKCH, MPEG1 and LMO2. The 9-gene signature was used, combined with clinical parameters, to fit a multivariable Cox model to the training data (concordance index, ci=0.85), which was successfully validated (ci=0.86 for internal cohort; ci=0.78 for external cohort). The signature showed improved performance compared with clinical parameters alone (ci=0.70). Furthermore, the signature more accurately predicted patient outcomes than gene signatures reported previously. It was also used to stratify early-stage, HBV or HCV-infected patients into groups with low and high risk of survival, leading to significant differences in survival in training and validation (P

Semaphorin 3A (SEMA3A), a secretory protein, is a founding member of the semaphorin family and functions in both the biological behavior of tumor cells and the modulation of tumor-associated macrophages. However, the role of SEMA3A in hepatocellular carcinoma (HCC) is still not well established. In the present study, we investigated the expression levels of SEMA3A in 80 HCC tissues and cell lines, using RT-qPCR, western blotting and immunohistochemistry. Expression profile analysis revealed that SEMA3A was significantly overexpressed in human HCC patients and positively correlated with the metastatic potential of HCC cells. Lentiviral transfection into PLC/PRF/5 and HCCLM3 cells was performed to stably upregulate and downregulate the expression of SEMA3A in HCC cells. Cell Counting Kit-8 (CCK-8), wound-healing and invasion assays revealed that SEMA3A promoted the proliferation and migration of HCC cells in vitro. Proteome profiler antibody microarray analysis revealed that overexpression of SEMA3A in HCC cells induced a significant increase in the expression levels of gelsolin-like capping protein (CapG), galectin-3, enolase 2 and epithelial cell adhesion molecule (EpCAM). Furthermore, the upregulation of SEMA3A in HCC cells promoted tumor growth and progression in an HCC mouse model. These results indicate that SEMA3A enhances CapG, galectin-3, enolase 2 and EpCAM expression to promote HCC progression and is a potential therapeutic target for HCC.

// Er-Bao Chen 1, 2, * , Shao-Lai Zhou 1, * , Xu-Guang Pang 3, * , Dan Yin 1 , Pei-Zhen Miao 1 , Yi Yang 1 , Qing Chen 1 , Kai Zhu 1 , Dong-Mei Gao 1 , Tian-Shu Liu 4 , Xiao-Yi Wang 1 , Ying-Hong Shi 1 , Wei-Zhong Wu 1 , Jian Zhou 1, 2 , Zheng-Jun Zhou 1 and Zhi Dai 1, 2 1 Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Labolatory of Carcinogenesis and Cancer Invasion, Fudan University, Ministry of Education, Shanghai, China 2 State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China 3 Department of Thoracic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China 4 Department of Medical Oncology, Zhongshan Hospital, Fudan University, Shanghai, China * These authors have contributed equally to this work Correspondence to: Zhi Dai, email: dai.zhi@zs-hospital.sh.cn Zheng-Jun Zhou, email: zhou.zhengjun@zs-hospital.sh.cn Keywords: PDEF, HCC, EMT, invasion, proliferation Received: August 25, 2016 Accepted: December 27, 2016 Published: January 31, 2017 ABSTRACT Prostate-derived E-twenty-six (ETS) factor (PDEF), an epithelium-specific ETS transcription factor, regulates carcinogenesis and tumor progression. The prognostic importance and biologic functions in hepatocellular carcinoma (HCC) have not been established. We investigated PDEF expression in 400 HCC patients using quantitative real-time polymerase chain reaction, western blot and immunohistochemistry analysis. PDEF expression was significantly lower in tumors than in peritumoral tissues. Lower PDEF levels were associated with poorer prognosis in patients. PDEF was an independent predictor of overall survival in multivariate analysis. PDEF expression was suppressed in highly metastatic HCC cell lines, and shRNA-mediated down-regulation of PDEF in low-metastatic HCC cell lines (with high PDEF) significantly increased cellular proliferative and invasive capacity in vitro and in vivo . RNA sequencing analysis indicated that PDEF may mediate transcription of several genes involved in apoptosis and the cell cycle. PDEF modulated epithelial-mesenchymal transition by up-regulating E-cadherin expression and down-regulating Slug and Vimentin expression, thereby lowering migration and invasion abilities of HCC cells. In conclusion, PDEF is associated with proliferation and invasiveness of HCC cells. It may serve as an independent predictor of prognosis in patients with HCC.

The solubility of nitrogen in Fe-C-B-N system was measured at 1 758 K, and the computational model on activity (action concentration) of nitrogen and boron was established based on phase diagram and the coexistence theory about metal melt structure model. Comparing the computed results with the experimental results, satisfactory conclusion can be obtained. The result shows that BN and B 4 C can exist in Fe-C-B-N molten metal at high temperature, which consequently restrains the nitrogen removal from the melt. However, B 4 C content is extremely low. Before graphite is precipitated, the influence of carbon on activity of nitrogen in melt is higher in ternary system than in binary system; however, this effect is contrary to that after graphite is precipitated.

Carbon solubility in Fe-Mn melts (xFe = 0.102 3 − 0.789 9, xMn = 0.055 1 − 0.638 0) was measured experimentally at various temperatures. Using Gibbs-Duhem equation, in combination with the experimental results in this work, quoting experimental data reported in references, and by strict thermodynamic derivation and calculation, the relation equations between the activity interaction parameters in Fe-C system and temperature were obtained. The calculation equation of lnγFe in Fe-C system was also obtained. The calculated results show that these relation equations can be used to calculate the activity coefficients of carbon and iron in Fe-C system and can satisfy the necessary condition to satisfy Gibbs-Duhem equation and the necessary condition to satisfy the stability condition of system at high carbon content. The calculation formula for ln/c in Fe-Mn-C system was also obtained.

The solubility of nitrogen in the Fe-C-V-N system was measured at 1 708 K and the model of calculating activity (action concentration) of nitrogen (N) and vanadium (V) was derived according to the phase diagram and the coexistence theory of the metal melt structure. The solubility expression of nitrogen in the Fe-C-V-N system at 1 708 K was wN=0.058 194 − 0.010 367wC + 0.005 543 4wV. Comparing the computing results with the experimental results, a satisfactory conclusion could be obtained. The analysis of the Fe-C-V-N system using this model showed that VN was present in a high temperature metal melt, which would reduce the action concentration of nitrogen obviously. It was consequently disadvantageous to the removal of nitrogen from hot metal.

Carbon solubility in Mn-Fe melts ( X Mn = 0. 161 — 0. 706, X Fe = 0. 034 — 0. 633) was measured experimentally at various temperatures. By thermodynamic derivation and calculation, the relationship between activity coefficient of carbon in infinite dilute solution of manganese in Mn-C system and temperature was obtained. Using Gibbs-Duhem relationship, the experimental results of this study, and experimental data reported in references, the relationship between other thermodynamic properties in Mn-C system and temperature were obtained by thermodynamic derivation and calculation.

The saturated solubility of carbon and nitrogen in Fe-C-N and Fe-C-B-N melts was measured experimentally at 1 485 °C to obtain the activity interaction coefficients between components in these melts. A new method was used to treat experimental results. Using thermodynamic derivation and calculation, some important interaction coefficients between components in these melts were obtained.

Background & Aims Neutrophils can either promote or inhibit tumor progression, depending on the tumor microenvironment, via release of cytokines. Neither the factors produced by tumor-associated neutrophils (TANs) nor their effects on tumor progression have been characterized. We investigated the roles of TANs in progression of hepatocellular carcinoma (HCC) using cell lines and immune cells isolated from patients. Methods We performed studies with HepG2, PLC/PRF/5, MHCC97H, and HCCLM3 human and Hepa1-6 and H22 mouse HCC cell lines; expression of chemokines and cytokines were knocked down with small hairpin RNAs. Cells were analyzed in chemotaxis assays and as growth as tumors in mice. HCC tissues and peripheral blood were collected from 20 patients undergoing curative resection or 20 healthy individuals (controls) in 2012 at Zhongshan Hospital in China. TANs and peripheral blood neutrophils (PBNs) were isolated and exposed to conditioned media from HCC cell lines; reverse-transcription polymerase chain reaction was used to quantify the expression of cytokines and chemokines. We collected neutrophils from another 60 patients undergoing curative resection for HCC in 2012 to measure the production of C-C motif chemokine ligand 2(CCL2) and CCL17. Patients were followed up until March 15, 2014. For immunohistochemical analyses, we collected HCC tissues and paired, adjacent, nontumor cirrhotic liver tissues from 832 HCC patients undergoing curative resection from 2006 through 2008. All patients were followed up until March 15, 2013. To study the effects of sorafenib, we collected clinical and pathology data from 46 patients who underwent curative resection in 2010. Results CCL2 and CCL17 were the cytokines most highly expressed by TANs and HCC cell-activated PBNs. Levels of CCL2 and CCL17 messenger RNAs and proteins were significantly higher in TANs than in PBNs, and increased in patients with HCC recurrence. CCL2 and CCL17 messenger RNA and proteins also increased when PBNs were exposed to conditioned media from HCC cell lines. Immunohistochemical analysis of a tissue microarray showed that CCL2+ and CCL17+ cells, which also expressed the neutrophil marker CD66b, were distributed throughout the HCC stroma, but not in tumor cells or the adjacent nontumor liver cells. The number of CCL2+ or CCL17+ TANs correlated with tumor size, microvascular invasion, tumor encapsulation, tumor differentiation, and stage. Patients whose tumors had lower levels of CCL2+ or CCL17+ cells had longer survival times than those with higher numbers of these cells. TAN-conditioned media, as well as recombinant CCL2 and CCL17, increased the migratory activity of the macrophages and T-regulatory (Treg) cells from patients or mice with HCC to a greater extent that PBN-conditioned media. Neutralizing antibodies against CCL2 and CCL17, or their receptors C-C chemokine receptor 2 and C-C chemokine receptor 4, reduced the migratory activities of macrophage and Treg cells. HCC cell lines injected into mice formed larger tumors when they were co-injected with TANs and formed more pulmonary metastases; these tumors were infiltrated by Ly6G+ cells, F4/80+ macrophages, and Foxp3+ Treg cells. In a phosphokinase array of human PBNs, levels of phosphorylated AKT and P38 increased after exposure to conditioned media from all 4 HCC cell types. Pharmacologic inhibitors of AKT and P38 inhibited secretion of CCL2 and CCL17 by these PBNs. In tumor-bearing mice, sorafenib increased the numbers of TANs and levels of CCL2 and CCL17 in tumors. HCC tissues from patients who received sorafenib before surgery contained more TANs than tissues from patients who did not receive sorafenib. In knockdown cells, HCC cell–derived CXCL5 was the strongest effector of neutrophil migration under hypoxic conditions. In mice, the combination of sorafenib and TAN depletion inhibited tumor growth and neovascularization to a greater extent than sorafenib alone. Conclusions TANs recruit macrophages and Treg cells to HCCs to promote their growth, progression, and resistance to sorafenib.