Identification of whole-genome mutations and structural variations of bile cell-free DNA in cholangiocarcinoma.

Bile cell-free DNA (cfDNA) has been reported as a promising liquid biopsy tool for cholangiocarcinoma (CCA), however, the whole-genome mutation landscape and structural variants (SVs) of bile cfDNA remains unknown. Here we performed whole-genome sequencing on bile cfDNA and analyzed the correlation between mutation characteristics of bile cfDNA and clinical prognosis. TP53 and KRAS were the most frequently mutated genes, and the RTK/RAS, homologous recombination (HR), and HIPPO were top three pathways containing most gene mutations. Ten overlapping putative driver genes were found in bile cfDNA and tumor tissue. SVs such as chromothripsis and kataegis were identified. Moreover, the hazard ratio of HR pathway mutations were 15.77 (95% CI: 1.571-158.4), patients with HR pathway mutations in bile cfDNA exhibited poorer overall survival (P = 0.0049). Our study suggests that bile cfDNA contains genome mutations and SVs, and HR pathway mutations in bile cfDNA can predict poor outcomes of CCA patients.

HMGB2 upregulation promotes the progression of hepatocellular carcinoma cells through the activation of ZEB1/vimentin axis.

Background: High mobility group box 2 (HMGB2) is abnormally expressed in human cancers and participated in multiple biological behaviors, such as proliferation, invasion and prognosis. However, its role in hepatocellular carcinoma (HCC) is largely unknown. Methods: In clinical sample analysis, 62 HCC patients were enrolled in this study. The expression of HMGB2 was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical method, clinical prognosis data were analyzed by Kaplan-Meier analysis. In cellular and molecular biology experiments, HMGB2 expression was analyzed in HCC cells. HMGB2 knockdown model was constructed by small interfering RNA (siRNA). Cell counting kit-8 (CCK-8) and cell migration & invasion assay were used to evaluate cell proliferative potential and motility. Recombinant human vimentin protein was used to partially restore the expression and function of vimentin. Western blot and immunochemical staining were performed to detect HMGB2 protein, zinc finger E-box binding homeobox 1 (ZEB1) and vimentin. Flow cytometry analyses were performed to determine the alteration of cell cycle in different groups. Results: HMGB2 was abnormally overexpressed in HCC. HMGB2 knockdown reduced malignant behaviors especially the proliferative potential and motility of HCC cells. The inhibition of HCC cells proliferation and mobility could be partially restored via treatment with recombinant vimentin protein. Our findings confirmed abnormal activation of HMGB2-ZEB1 vimentin axis facilitates HCC malignant proliferation and motility. The elevated HMGB2 expression in clinical samples was related to postoperative survival time of HCC patients. It indicated HMGB2 promotes the proliferation and motility potential of HCC via HMGB2-ZEB1-vimentin axis activation. Conclusions: HMGB2 is up-regulated in HCC and affects the malignant transformation by modulating HMGB2-ZEB1-vimentin signaling pathway, which may provide a research basis for evaluating the disease progression and developing clinical treatment strategies of HCC.

Immune index: A gene and cell prognostic signature for immunotherapy response prediction in hepatocellular carcinoma.

The heterogeneity of tumor immune microenvironment (TIME) plays important roles in the development and immunotherapy response of hepatocellular carcinoma (HCC). Using machine learning algorithms, we introduced the immune index (IMI), a prognostic model based on the HCC immune landscape. We found that IMI low HCCs were enriched in stem cell and proliferating signatures, and yielded more TP53 mutation and 17p loss compared with IMI high HCCs. More importantly, patients with high IMI exhibited better immune-checkpoint blockade (ICB) response. To facilitate clinical application, we employed machine learning algorithms to develop a gene model of the IMI (IMIG), which contained 10 genes. According to our HCC cohort examination and single-cell level analysis, we found that IMIG high HCCs exhibited favorable survival outcomes and high levels of NK and CD8+ T cells infiltration. Finally, after coculture with autologous tumor infiltrating lymphocytes, IMIG high tumor cells exhibited a better response to nivolumab treatment. Collectively, the IMI and IMIG may serve as powerful tools for the prognosis, classification and ICB treatment response prediction of HCC.

Propofol suppresses hepatocellular carcinoma by inhibiting NET1 through downregulating ERK/VEGF signaling pathway.

Hepatocellular carcinoma (HCC) is the leading cause of tumor death in China with high mortality since its strong metastatic potential. Currently, treatment against advanced HCC is poorly efficient and thus screening new drugs to prevent the HCC invasion is of great significance to improve the survival rate of patients with HCC. From the results of this study, we concluded that propofol, a widely used anesthetics could prevent the proliferation by MTT assay. The scratch wound and invasion assays showed that migratory property and invasiveness in HCC cells SMMC-7721 was inhibited by propofol. This process was probably mediated by NET1 since NET1 overexpression offset the repressive effect of propofol on the invasiveness and migratory ability of SMMC-7721 cells. Furthermore, propofol treatment also reduced p-ERK1/2 and VEGF level by western blot analysis. Similar observation was found when NET1 was silenced. Thus, the results of this study provided valuable clinical therapy potential of propofol against liver cancer. We also disclosed molecular mechanism underlying the regulation of invasion and migration in HCC cells by NET1.

The SNP of rs6854845 suppresses transcription via the DNA looping structure alteration of super-enhancer in colon cells.

Single-nucleotide polymorphisms (SNPs) identified by Genome-Wide Association Studies (GWASs) have been determined to closely connect with multiple diseases. Previous studies revealed one underlying mechanism that SNPs located within the regulatory elements could affect the encoding gene expression through long-range regulation. SNP rs6854845 was suggested to be a risk of colon cancer in human population. Nevertheless, the underlying molecular mechanism for colon carcinogenesis remains largely unknown. In present study, rs6854845 with G > T mutation in situ in FHC, HCT-116 and SW-480 cells were generated by Crispr/Cas9. The nearby chromatin organization was investigated by chromatin conformation capture (3C). And the expression of coding gene regulated by super-enhancer (SE) was detected by real-time PCR. We observed a significantly different pattern of the genome organization upon rs6854845 generation in colon epithelial cells but not in colon cancer cells. Moreover, we observed the shifted enrichment of H3K4me1 and H3K27ac at the SE (chr4:75.7M-76.0 M) where rs6854845 located. Furthermore, we observed that the transcription of the gene clusters regulated by SE were affected by rs6854845 in colon cells. Overall, our results demonstrated that SNP rs6854845 located in SE could destroy the long-range chromosomal interaction between SE and target gene clusters thereby affecting the transcription of these genes.

miR-194 inhibits liver cancer stem cell expansion by regulating RAC1 pathway.

Liver cancer stem cells (CSCs) contribute to tumorigenesis, progression, drug resistance and recurrence of hepatocellular carcinoma (HCC). However, the underlying mechanism for the propagation of liver CSCs remains unclear. Herein, we observed low expression of miR-194 in chemoresistant HCC cells. A remarkable decrease of miR-194 was detected in EpCAM or CD133-positive liver CSCs and CSC-enriched hepatoma spheres. Interference miR-194 facilitated liver CSCs expansion by enhancing the self-renewal of liver CSCs. While up-regulating miR-194 inhibited liver CSCs expansion by suppressing the self-renewal of liver CSCs. Furthermore, hepatoma cells with miR-194 overexpression performed more sensitivity to sorafenib treatment. Mechanistically, functional studies found that Ras-related C3 botulinum toxin substrate 1 (RAC1) was a direct target of miR-194. Overexpression of miR-194 inhibited the expression of RAC1 in liver CSCs. Special RAC1 siRNA diminished the discrepancy in liver CSC proportion and the self-renewal capacity between miR-194 overexpression hepatoma cells and control cells, which further confirmed that RAC1 was required in miR-194-inhibited liver CSCs expansion. More importantly, downregulated expression of miR-194 was a predictor of poor prognosis of HCC patients.

Prognostic importance of bile duct invasion in surgical resection with curative intent for hepatocellular carcinoma using PSM analysis.

There is not yet a consensus regarding a difference in prognosis for patients with hepatocellular carcinoma (HCC) with and without bile duct invasion (BDI). The present study aimed to clarify the prognostic importance of BDI on the short and long-term outcome of patients with HCC who underwent surgical resection. The present study evaluated HCC with BDI, including peripheral microscopic biliary invasion and revealed that the prognosis of patients with BDI was poorer compared with those without BDI. It should be noted that peripheral BDI also had a negative impact on the prognosis of patients with HCC. The clinical prognosis assessment revealed that BDI should be considered when assigning a disease stage. BDI, either macroscopic or microscopic, indicated a poor prognosis in patients with HCC who underwent curative resection, however it was not a surgical contraindication. Macroscopic BDI and hyperbilirubinemia were significantly associated with a dismal prognosis, which should alert surgeons.

Small molecule metabolite biomarkers for hepatocellular carcinoma with bile duct tumor thrombus diagnosis.

Hepatocellular carcinoma with bile duct tumor thrombus (BDTT) is a malignant disease. The most commonly used diagnosis methods for BDTT are MRCP/ERCP, ultrasonic diagnosis or CT scan. However, BDTT is often misdiagnosed as other bile duct diseases, such as extrahepatic cholangiocarcinoma (EHCC), choledochal cyst (Cyst) and common bile duct stone (Stone). Diagnostic methods, which are more accurate and less destructive, are urgently needed. In this paper, we analyzed the small molecule metabolites in the serum of BDTT, Stone, Cyst and EHCC patients and normal people using untargeted GC-MS, and identified 21 metabolites that show different levels among different samples. Using targeted UHPLC-QQQ-MS analysis, we found that several metabolites are significantly changed. ROC curve analysis revealed two metabolites, L-citrulline and D-aspartic acid, as potential biomarkers that can distinguish BDTT from other bile duct diseases.

CCL20 is overexpressed in hepatocellular carcinoma with bile duct tumor thrombus and correlates negatively with surgical outcome.

There is not yet a consensus regarding prognosis of hepatocellular carcinoma (HCC) with bile duct tumor thrombus (BDTT) versus without bile duct tumor thrombus. Chemokine (C-C motif) ligand 20 (CCL20) plays critical roles in the progress of many types of tumor. But the clinicopathological and prognostic value of this marker in HCC with BDTT is unceratin. In this study, we reported that the overall survival (OS) and disease-free survival (DFS) in HCC with BDTT were significantly shorter than in those without BDTT (P<0.05). CCL20 was expressed at a significantly higher level in bile duct tumor thrombus by real-time PCR, western blot, and immunohistochemistry. Patients with high CCL20 expression levels had a poor prognosis. Multivariate survival analysis indicated that CCL20 was an independent prognostic factor for OS. The presence of bile duct tumor thrombus indicateda poor prognosis in HCC patients, but was not a surgical contraindication. CCL20 was associated with tumor progression and high CCL20 expression was correlated with worse surgical outcomes in HCC with BDTT. Inhibition of CCL20 expression might offer novel promising molecular targets for treatment.

Surgical treatment of double primary liver cancer: An observational study for a rare type of tumor.

Double primary liver cancer (DPLC) is a special type of clinical situation. As such, a detailed analysis of the surgical management and prognosis of patients with DPLC is lacking. The objective of the current study was to define the management and outcome of patients undergoing surgery for DPLC at a major hepatobiliary center.A total of 87 patients treated by surgical resection at the Eastern Hepatobiliary Surgery Hospital from January 1st, 2007 to October 31st, 2013 who had DPLC demonstrated by final pathological diagnosis were identified. Among these, 50 patients had complete clinical and prognostic data. Demographic and tumor characteristics as well as the prognosis were analyzed.The proportion of hepatitis B surface antigen (HBsAg) (+) and hepatitis B virus e antigen (HBeAg) (+), HBsAg (+), and HBeAg (-) hepatocirrhosis in all patients was 21.84%, 67.82%, and 63.22%, respectively. Incidental findings accounted for 58.62% of patients; among those who had symptoms, the main symptom was abdominal pain (31.03%). Nonanatomic wedge resection was the main operative approach (62.07%). Postoperatively, the main complications included seroperitoneum (11.49%), hypoproteinemia (10.34%), and pleural effusion (8.05%). Factors associated with disease-free survival (DFS) included intrahepatic cholangiocarcinoma (ICC) tumor size (P = 0.002) and use of postoperative prophylactic transcatheter arterial chemoembolization (TACE) treatment (P = 0.015). Meanwhile, hepatocellular carcinoma (HCC) size (P = 0.045), ICC size (P < 0.001), and liver function (including aspartate aminotransferase [P = 0.001] and r-glutamyl transferase [P < 0.001]) were associated with overall survival (OS).Hepatitis B virus (HBV)-related hepatitis or cirrhosis is also an important factor in the pathogenesis of DPLC and surgical treatment is safe for it with low complication rates. In addition, it is effective to prolong DFS that DPLC patients undergo postoperative prophylactic TACE treatment.

The significance of RDW in patients with hepatocellular carcinoma after radical resection.

BACKGROUND: Red blood cell distribution width (RDW) is a parameter reported in blood routine examination, and has been reported as an inflammatory biomarker. The objective of this study was to investigate the significance of RDW in patients with hepatocellular carcinoma after radical resection. METHOD: The relationship between the preoperative serum RDW value and clinic pathologic characteristics was analyzed in 106 HCC patients who underwent curative resection of hepatocellular carcinoma. Kaplan-Meier survival analysis and Cox proportional hazard models were used to examine the effect of RDW on survival. RESULTS: The RDW levels were divided into two groups: high RDW (> 14.5%) and low RDW (≤ 14.5%), (n= 28 vs n= 78). The patients with preoperative high levels of RDW had significantly worse survival than those with low RDW (p< 0.05). According to multivariate analysis, high RDW (HR = 1.89, p= 0.002), TNM stage (HR = 1.70, p= 0.019), tumor size (HR = 1.33, p= 0.045), tumor number (HR = 1.42, p= 0.027) and vascular invasion (HR = 1.64, p= 0.009) were independent prognostic factors of OS. CONCLUSIONS: Preoperative high levels of RDW are associated with poor survival. It might be an independent prognostic factor in patients with hepatocellular carcinoma.

Successful en bloc resection of recurrent hepatocellular carcinoma directly invading the abdominal wall: a case report.

INTRODUCTION: Repeat hepatic resection has previously been reported as the most effective treatment for recurrence of intrahepatic carcinoma. To the best of our knowledge, en bloc resection of recurrent hepatocellular carcinoma directly invading the abdominal wall has not been previously reported. CASE PRESENTATION: In September 2012, a 64-year-old Chinese male patient was referred to our hospital because of primary hepatocellular carcinoma located in Couinaud's segments III and V. Our patient first had a hepatectomy of the liver. Ten months later, he presented with an abdominal wall mass and upper abdominal pain. Computed tomography and magnetic resonance imaging scans demonstrated a 10cm tumor in his left liver with extrahepatic metastases in his abdominal wall. It was determined that he had recurrent hepatocellular carcinoma associated with direct invasion into his abdominal wall. He had an en bloc left hepatectomy with resection of the tumor in his abdominal wall. A pathological examination of the resected specimen confirmed the diagnosis of hepatocellular carcinoma involving the abdominal wall. Disease-free margins of resection were achieved. Our patient's postoperative course was uneventful. Eight months after the last surgery, our patient died owing to recurrence and distal metastasis. CONCLUSION: Direct invasion of hepatocellular carcinoma into the abdominal wall is rarely encountered. Complete surgical resection should be considered in patients with an appropriate hepatic functional reserve, with consideration of the technical difficulty relating to tumor involvement with surrounding tissues.

Surgery for pregnancy-associated primary hepatocellular carcinoma: Report of four cases.

INTRODUCTION: Hepatocellular carcinoma (HCC) occurring in pregnancy is quite rare. The prognosis is usually poor because of a delay in diagnosis. Reported cases of HCC in pregnancy are largely isolated with no large experience. Thus the effect of pregnancy on the prognosis of patients with HCC and the risk factors of developing HCC in pregnancy are not well documented. Our aim was to review our experience with management of four young pregnant women with HCC. PRESENTATION OF CASE: Laboratory tests were performed before surgery. We analyzed the effects of age, hepatitis B surface antigens status, cirrhosis at presentation, gestational age of fetus, and maternal outcome. DISCUSSION: Increase in alpha-fetoprotein (AFP) level was somewhat useful for diagnosis. Three patients died in 5 months, 6 months, and 24 months from HCC recurrence, and another patient is alive without disease 12 months postoperatively. CONCLUSION: Surgery for HCC during pregnancy should be similar to that for non-pregnant women. Complete excision of tumor without termination of pregnancy provides the greatest chance of survival for women with HCC during pregnancy but depends on gestational age of the fetus. Adjuvant treatments are required to improve the long-term results of this type of surgery. The 28-week gestational week is a critical point of fetal maturation which is very important in deciding whether pregnancy should be terminated or not. The pregnancy was terminated in two of our patients when spontaneous rupture of HCC was diagnosed to save the mother's life.

Inhibition of autophagy significantly enhances combination therapy with sorafenib and HDAC inhibitors for human hepatoma cells.

AIM: To clarify whether histone deacetylase inhibitors histone deacetylase inhibitors (HDACIs) can sensitize hepatocellular carcinoma (HCC) cells to sorafenib treatment. METHODS: Bax, Bcl-2, ATG5-ATG12, p21, and p27 protein levels in Hep3B, HepG2, and PLC/PRF/5 cells were examined by Western blot. CCK8 and a fluorometric caspase-3 assay were used to examine cellular viability and apoptosis levels. The effect of Beclin-1 on sensitization of HCC cells to sorafenib was examined by transfecting Beclin-1 siRNA into Hep3B, HepG2, and PLC/PRF/5 cells. RESULTS: Autophagy inhibition enhances the inhibitory effects of vorinostat and sorafenib alone or in combination on HCC cell growth. Vorinostat and sorafenib synergistically induced apoptosis and cell cycle alterations. Western blot data indicated that HDACIs and Beclin-1 knockdown increased the p53 acetylation level. The knockdown of Beclin-1 enhanced the synergistic effect of the combination of vorinostat with sorafenib. CONCLUSION: HDACIs can sensitize HCC cells to sorafenib treatment by regulating the acetylation level of Beclin-1.

Genetic polymorphisms of XRCC1 gene and susceptibility to hepatocellular carcinoma in Chinese population.

Hepatocellular carcinoma (HCC) is a common cancer in the worldwide. Accumulated evidences indicate that genetic polymorphisms of human X-ray repair complementing group 1 gene (XRCC1) are associated with the susceptibility to HCC. This study aims to investigate the potential association between XRCC1 c.482C>T and c.1178G>A genetic polymorphisms and the susceptibility to HCC. A total of 1,069 Chinese Han subjects consisting of 530 HCC patients and 539 cancer-free controls were recruited in this case-control study. The created restriction site-polymerase chain reaction and directly DNA sequencing methods were utilized to analyze the genotyping of XRCC1 genetic polymorphisms. Our data suggested that the XRCC1 c.482C>T and c.1178G>A genetic polymorphisms were statistically associated with the increased risks of HCC [for c.482C>T, TT vs. CC: OR 2.05, 95% CI 1.26-3.32, P = 0.003; T vs. C: OR 1.26, 95% CI 1.04-1.51, P = 0.017; for c.1178G>A, AA vs. GG: OR 2.15, 95% CI 1.26-3.67, P = 0.004; A vs. G: OR 1.33, 95% CI 1.10-1.61, P = 0.004]. The allele-T and genotype-TT of c.482C>T and allele-A and genotype-AA of c.1178G>A genetic polymorphisms may enhance the susceptibility to HCC. Our findings indicate that the studied XRCC1 genetic polymorphisms may influence the risk of HCC in Chinese populations and might be used as molecular markers for assessing the risk of HCC.

Superior approach for the exclusion of hepatic veins in major liver resection: a safe and easy technique.

We describe a technique for isolating and excluding the hepatic veins during liver resection. First, the bare area near the right and left wall of the suprahepatic inferior vena cava (IVC) is dissected, exposing the right, left, and superior walls of the right hepatic vein (RHV) and the left-middle hepatic vein (LMHV). Two Satinsky clamps are used to clamp the roots of the right and common trunk of the LMHV, parallel to the IVC. It is not necessary to dissect the posterior wall of the hepatic veins. We used this method during major liver resection in 65 patients. The mean dissecting time of each hepatic vein was 7.31 +/- 3.6 min. No hepatic vein was lacerated during dissection and exclusion. The postoperative complication rate was 31.2%. Thus, the superior approach is a safe and easy maneuver when the posterior wall of the hepatic vein is difficult to dissect due to tumor invasion.

Management and surgical treatment for focal nodular hyperplasia in children.

Focal nodular hyperplasia (FNH) is rare in pediatric liver tumor. To investigate the experience in the appropriate management of pediatric FNH, the records of consecutive children who were managed at our institute from 2000 to 2007were reviewed for FNH. There were four males and nine females whose ages ranged from 26 months to 18 years with a mean of 11.7 years. FNH in most children was detected by medical examination for abdominal pain (61.5%) or other symptoms. All the 13 pediatric patients underwent liver resection. There was no operative death or postoperative complications. The children were regularly followed up ranging from 2 to 85 months and they were healthy without recurrence. For pediatric FNH patients with clinical symptoms or indefinite diagnosis, it is suggested that active surgical treatment by hepatectomy should be performed.