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Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Its high mortality rate is mainly a result of intra-hepatic metastases. We analyzed the expression profiles of HCC samples without or with intra-hepatic metastases. Using a supervised machine-learning algorithm, we generated for the first time a molecular signature that can classify metastatic HCC patients and identified genes that were relevant to metastasis and patient survival. We found that the gene expression signature of primary HCCs with accompanying metastasis was very similar to that of their corresponding metastases, implying that genes favoring metastasis progression were initiated in the primary tumors. Osteopontin, which was identified as a lead gene in the signature, was over-expressed in metastatic HCC; an osteopontin-specific antibody effectively blocked HCC cell invasion in vitro and inhibited pulmonary metastasis of HCC cells in nude mice. Thus, osteopontin acts as both a diagnostic marker and a potential therapeutic target for metastatic HCC., Author(s): Qing-Hai Ye [1]; Lun-Xiu Qin [1]; Marshonna Forgues [2]; Ping He [2]; Jin Woo Kim [2]; Amy C. Peng [3, 4]; Richard Simon [3]; Yan Li [1]; Ana I. [...]

Abstract Background Recent studies indicate that Transforming Growth Factor beta (TGF β) correlated with pulmonary metastasis of cancers. However, the correlation between TGF β and pulmonary metastasis of hepatocellular carcinoma (HCC) is till unknown. Methods We detected the in vitro and in vivo expression levels of TGF β1/Smads by Real-time PCR and Western blot in MHCC97-H and MHCC97–L cell lines, which are HCC cell lines and have higher and lower pulmonary metastatic potential respectively. Results TGF β1 mRNA level in MHCC97-L tumors were higher than that in MHCC97-H tumors, (2.81±1.61 vs. 1.24±0.96, P=0.002), TGF β1 protein level in MHCC97-L tumors were also higher than that in MHCC97-H tumors (1.37±0.95 vs. 0.32±0.22, P2=0.12 and 0.40, respectively). Conclusions Our results suggest that TGF β/ Smads promote pulmonary metastasis of HCC.

Abstract Background The prognosis of patients with hepatocellular carcinoma (HCC) still remains very dismal, which is mainly due to metastasis. In our previous studies, we found that chromosome 8p deletions might contribute to metastasis of HCC. In this study, we aimed to identify the candidate metastatic suppressor gene on chromosome 8p. Methods Oligo-nucleotide microarrays which included 322 genes on human chromosome 8p were constructed to analyze the difference in gene expression profiles between HCC tissues with and without metastasis. The leading differentially expressed genes were identified and selected for further analysis by real-time PCR and Western blotting. Recombinant expression plasmid vectors for each target gene were constructed and transfected into HCC cells and its in vitro effects on proliferation and invasion of HCC cells were also investigated. Results Sixteen leading differentially expressed genes were identified from the HCC tissues with metastasis compared with those without metastasis (p < 0.01, q < 16 %). Among of the 10 significantly down-regulated genes in HCC with metastasis, methionine sulfoxide reductase A (MSRA) had the lowest p value and false discovery rate (FDR), and was considered as a potential candidate for metastasis suppressor gene. Real-time PCR and Western blotting confirmed that the mRNA and protein expression levels of MSRA were significantly decreased in HCC with metastasis compared with those without metastasis (p < 0.001), and MSRA mRNA level in HCCLM6 cells (with high metastatic potential) was also much lower than that of other HCC cell lines. Transfection of a recombinant expression plasmid vector and overexpression of MSRA gene could obviously inhibit cell colony formation (4.33 ± 2.92 vs. 9.17 ± 3.38, p = 0.008) and invasion (7.40 ± 1.67 vs. 17.20 ± 2.59, p= 0.0001) of HCCLM6 cell line. Conclusion MSRA gene on chromosome 8p might possess metastasis suppressor activity in HCC.

Hepatocellular carcinoma (HCC) is a genetically heterogeneous disease for which a dominant actionable molecular driver has not been identified. Patients with the stem cell-like EpCAM + AFP + HCC subtype have poor prognosis. Here, we performed a genome-wide RNAi screen to identify genes with a synthetic lethal interaction with EpCAM as a potential therapeutic target for the EpCAM + AFP + HCC subtype. We identified 26 candidate genes linked to EpCAM/Wnt/β-catenin signaling and HCC cell growth. We further characterized the top candidate PMPCB, which plays a role in mitochondrial protein processing, as a bona fide target for EpCAM + HCC. PMPCB blockage suppressed EpCAM expression and Wnt/β-catenin signaling via mitochondria-related reactive oxygen species production and FOXO activities, resulting in apoptosis and tumor suppression. These results indicate that a synthetic lethality screen is a viable strategy to identify actionable drivers of HCC and identify PMPCB as a therapeutically vulnerable gene in EpCAM + HCC subpopulations. SIGNIFICANCE: This study identifies PMPCB as critical to mitochondrial homeostasis and a synthetic lethal candidate that selectively kills highly resistant EpCAM + HCC tumors by inactivating the Wnt/β-catenin signaling pathway., (©2019 American Association for Cancer Research.)

Growing evidences support the concept that peritumoral microenvironment gene expression is an important element for physicians to make an accurate prognosis. Nonetheless, the correlation between peritumoral ubiquitin ligases and the hepatocellular carcinoma (HCC) survival remains unclear till this present. The expression of intratumoral and peritumoral Casitas B-lineage Lymphoma (Cbl) and epidermal growth factor receptor (EGFR) in hepatocellular carcinomas (HCCs) followed by curative resection was assessed by tissue microarray-based immune-histochemistry in two independent cohorts (n = 352). Their respective prognostic values and other clinicopathologic factors were then evaluated. The peritumoral Cbl density, much higher than that in intratumoral tissue, was an independent prognostic factor for overall survival (P < 0.001) and time to recurrence (P < 0.001) of HCCs after curative resection. The hazard ratio were 1.587 and 1.689, respectively. However, there was no correlation between intratumoral Cbl and prognosis. The peritumoral Cbl was also associated with prognosis even in HCC subgroups with small tumor size, negative AFP, without microvascular invasion and negative HBeAg. After a thorough analysis pertaining to the key role of Cbl on ubiquitination and degradation of activated receptor tyrosine kinases, we eventually discovered the negative correlation between peritumoral Cbl and EGFR (P = 0.015). Furthermore, the combination of peritumoral Cbl and EGFR serves as a much stronger indicator to make an accurate prognosis, especially during early recurrence (P < 0.001). These findings suggest that low expression of peritumoral Cbl and EGFR were positively associated with tumor size, microvascular invasion and patients survival after hepatectomy, highlighting the key role of peritumoral liver milieu in HCC progression.

Purpose: Our previous studies have shown that chromosome 8p deletion correlates with metastasis of hepatocellular carcinoma (HCC). This study was to determine whether 8p deletion could be used in predicting the prognosis of patients with HCC, particularly in those with early stage of HCC., Experimental Design: A total of 131 patients with tumor-node-metastasis (TNM) stage I of HCC who underwent curative liver resection were enrolled. Loss of heterozygosity (LOH) was examined using 10 microsatellite markers at chromosome 8p, as well as 14 microsatellites at chromosome 1p, 17p, 4q, 13q, and 16q, and their association with 5-year overall survival (OS) and disease-free survival (DFS) of patients was analyzed., Results: In the entire cohort of patients, the mean LOH frequency at these 24 loci was 43.2%; LOH frequencies at D8S298 and D1S199 were 31.5% and 33.7%, respectively. LOH at D8S298 was associated with a worse 5-year OS (P = 0.008) and DFS (P = 0.038) in patients with TNM stage I of HCC. Likewise, the patients with LOH at D1S199 had a worse 5-year OS (P < 0.001) and DFS (P = 0.014) compared with those without LOH at D1S199. In multivariate analyses, LOH at D8S298 was an independent predictor of decreased DFS (hazard ratio, 0.372; 95% 95% confidence interval, 0.146-0.948; P = 0.038), whereas LOH at D1S199 was an independent predictor of decreased OS (hazard ratio, 0.281; 95% confidence interval, 0.123-0.643; P = 0.003)., Conclusions: LOH at D8S298 and D1S199 is independently associated with a worse survival in patients with TNM stage I of HCC after curative resection and could serve as novel prognostic predictors for this subgroup of patients.

Aim: To quantify the circulating DNA in plasma from patients with hepatocellular carcinoma (HCC) and to evaluate its prognostic value., Methods: Blood samples were collected from 79 patients with HCC before operation, 20 patients with liver cirrhosis, and 20 healthy volunteers. Circulating DNA was extracted from plasma and quantified. The association between circulating DNA level and prognosis of HCC patients was evaluated., Results: Compared with the healthy volunteers (17.6 +/- 9.5 ng/mL), a significant higher circulating DNA level was found in the patients with HCC (47.1 +/- 43.7 ng/mL, P = 0.000) or with liver cirrhosis (30.0 +/- 13.3 ng/mL, P = 0.002). The circulating DNA level was closely associated with tumor size (P = 0.008) and TNM stage (P = 0.040), negatively associated with the 3-year disease-free survival (DFS) (P = 0.017) and overall survival (OS) (P = 0.001)., Conclusion: Large or invasive tumor may release more circulating DNA, and higher level of circulating DNA may be associated with poor prognosis of HCC patients.

Objective: To explore the features of microsatellite alterations of circulating DNA in the plasma of patients with hepatocellular carcinoma (HCC) and whether they are in concordance with those in the carcinoma tissues., Methods: Peripheral blood samples were collected from 62 HCC patients and the corresponding tumor tissues were obtained during operation. Three high-polymorphic microsatellite markers located at chromosome 8p, D8S277, D8S298, and D8S1771, were selected to be used to detect the loss heterozygosity (LOH) and microsatellite instability (MSI) by PCR and sequencing., Results: Joint detection showed that 39 out of the 62 tissue samples showed LOH at all the 3 loci, and the same alterations at the same loci were seen in 33 matched plasma samples with a concordance rate of 84.6%. Nineteen tissue samples showed MSI at all 3 loci, and the same alterations were shown in 14 matched plasma samples with a concordance rate of 73.3%. The rate of LOH for at least one locus in the plasma DNA was 58.1% (36/62), significantly higher than the rate of MSI at at least one locus in the plasma samples (29.0%, 18/62, P < 0.01). The MSI positive rate in the loci D8S277 of the plasma DNA was 22.6%, significantly higher than those of the other 2 loci (4.8% and 4.8% respectively, both P < 0.05). The MSI positive rate at the loci D8S277 of the cancer tissue was 46.8%, significantly higher than those of the other 2 loci (38.7% for D8S298 and 37.1% for D8S1771, both P < 0.05)., Conclusion: Microsatellite alterations show a high concordant pattern between the tissue and plasma DNA in HCC, which indicates that the microsatellite alterations of tumor tissue are reflected by plasma DNA, LOH may play an important role in hepatocarcinogenesis whereas MSI may also contribute to this progress in a less significant way, and D8S277 is a sensitive locus to MSI in HCC.

Aim: To evaluate the effect of postoperative adjuvant transcatheter arterial chemoembolization (TACE) on the prognosis of hepatocellular carcinoma (HCC) patients with or without risk factors for the residual tumor., Methods: From January 1995 to December 1998, 549 consecutive HCC patients undergoing surgical resection were included in this research. There were 185 patients who underwent surgical resection with adjuvant TACE and 364 patients who underwent surgical resection only. Tumors with a diameter more than 5 cm, multiple nodules, and vascular invasion were defined as risk factors for residual tumor and used for patient stratification. Kaplan-Meier method was used to analyze survival curve and Cox proportional hazard model was used to evaluate the prognostic significance of adjuvant TACE., Results: In the patients without any risk factors for the residual tumor, the 1-, 3-, 5-year survival rates were 93.48%, 75.85%, 62.39% in the control group and 97.39%, 70.37%, 50.85% in the adjuvant TACE group, respectively. There was no significant difference in the survival between two groups (P = 0.3956). However, in the patients with risk factors for residual tumor, postoperative adjuvant TACE significantly prolonged the patients' survival. There was a statistically significant difference in survival between two groups (P = 0.0216). The 1-, 3-, 5-year survival rates were 69.95%, 49.86%, 37.40% in the control group and 89.67%, 61.28%, 44.36% in the adjuvant TACE group, respectively. Cox proportional hazard model showed that tumor diameter and cirrhosis, but not the adjuvant TACE, were the significantly independent prognostic factors in the patients without risk factors for residual tumor. However, in the patients with risk factors for residual tumor adjuvant TACE, and also tumor diameter, AFP level, vascular invasion, were the significantly independent factors associated with the decreasing risk for patients' death from HCC., Conclusion: Postoperative adjuvant TACE can prolong the survival of patients with risk factors for residual tumor, but can not prolong the survival of patients without risk factors for residual tumor.

Aim: Hepatocellular carcinoma (HCC) with bile duct tumor thrombosis (BDT) is a rare event. The prognosis of this type of patients is very dismal. The aim of this study was to share the experience in the diagnosis and treatment of HCC with BDT, to further improve the prognosis of these patients., Methods: Thirty-four patients of HCC with BDT received surgical treatment in authors' institute from July 1987 to January 2003 were reviewed retrospectively. The experience in the diagnosis and treatment, and the outcome of this type of HCC patients were summarized., Results: Thirty of the 34 patients (88.2%) were positive for alpha-fetoprotein (AFP) (>20 microg/L), and 12 patients (35.3%) were found having obstructive jaundice before operation, 18 cases were suspected of "obstruction of bile duct" preoperatively. The primary tumors were frequently located at the left medial (13 cases) or right anterior lobe (14 cases). Thirty-one patients received liver resections and removal of BDT, while the other 3 patients received removal of BDT combined with hepatic artery ligation and cannulation (HAL+HAI), or only removal of BDT because their liver function reservation and general condition could not tolerate the primary tumor resection. The 1-year survival rate was 71.4%(20/28). The longest disease-free survival was over 15 years. The intrahepatic tumor recurrence within 1 year after operation was found in 14 patients (14/28, 50.0%)., Conclusion: Surgical removal of primary tumors and BDT is safe and beneficial to the HCC patients with BDT. Early detection, diagnosis, and surgical treatment are the key points to prolong the survival time of patients.

Aim: To confirm if p53 mutation could be a routine predictive marker for the prognosis of hepatocellular carcinoma (HCC) patients., Methods: Two hundreds and forty-four formalin-fixed paraffin-embedded tumor samples of the patients with HCC receiving liver resection were detected for nuclear accumulation of p53. The percent of P53 immunoreactive tumor cells was scored as 0 to 3+ in P53 positive region (<10% -, 10-30% +, 31-50% ++, >50% +++). Proliferating cell nuclear antigen (PCNA) and some clinicopathological characteristics, including patients' sex, preoperative serum AFP level, tumor size, capsule, vascular invasion (both visual and microscopic), and Edmondson grade were also evaluated., Results: In univariate COX harzard regression model analysis, tumor size, capsule status, vascular invasion, and p53 expression were independent factors that were closely related to the overall survival (OS) rates of HCC patients. The survival rates of patients with 3+ for P53 expression were much lower than those with 2+ or + for P53 expression. Only vascular invasion (P<0.05) and capsule (P<0.01) were closely related to the disease-free survival (DFS) of HCC patients. In multivariate analysis, p53 overexpression (RI 0.5456, P<0.01) was the most significant factor associated with the OS rates of patients after HCC resection, while tumor size (RI 0.5209, P<0.01), vascular invasion (RI 0.5271, P<0.01) and capsule (RI-0.8691, P<0.01) were also related to the OS. However, only tumor capsular status was an independent predictive factor (P<0.05) for the DFS. No significant prognostic value was found in PCNA-LI, Edmondson's grade, patients' sex and preoperative serum AFP level., Conclusion: Accumulation of p53 expression, as well as tumor size, capsule and vascular invasion, could be valuable markers for predicting the prognosis of HCC patients after resection. The quantitative immunohistochemical scoring for P53 nuclear accumulation might be more valuable for predicting prognosis of patients after HCC resection than the common qualitative analysis.