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Rationale: The utility of the dorsal approach has been reported for laparoscopic left hemi-hepatectomy., Patient Concerns: The aim of the present study is to show the usefulness of the dorsal approach for laparoscopic extended left-hemi-hepatectomy while ensuring safe identification of hepatic veins and dissection of the dorsal tumor margin., Diagnoses: Tumors requiring extended left hemi-hepatectomy., Interventions: After mobilization of the lateral sector and division of the Arantius plate, parenchyma above the Arantius plate is removed to expose the root of the middle hepatic vein and left hepatic vein. Each of these veins can be isolated separately either intra- or extra-hepatically. After removing the parenchyma on the cranial side of the left Glissonean pedicle continuous with the exposed hepatic veins, the left Glissonean pedicle is isolated using the Glissonean pedicle transection method. After division of the left hepatic vein and Glissonean pedicle, segment 4 (in which the main part of the tumor is commonly located) is dissected from the anterior plane of the paracaval portion of the caudate lobe by the dorsal approach, along with the hepatic hilum. Following dissection of the dorsal side of the tumor, and division of parenchyma from the anterior edge of the liver, the anterior Glissonean branches and middle hepatic vein are divided safely and the specimen is resected., Outcomes: Three patients underwent laparoscopic extended left hemi-hepatectomy, with no open conversions. Operative time and blood loss were 331 (concomitant with another partial hepatectomy), 277, and 315 minutes; and 200, 100, and 100 g, respectively. The postoperative courses were uneventful., Lessons: The dorsal approach maximizes the advantages of laparoscopic extended left hemi-hepatectomy and can be performed safely., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

A 57-year-old man was treated with lenvatinib for unresectable hepatocellular carcinoma(HCC). Thereafter, the tumor marker levels decreased, and the tumor became resectable. The patient underwent portal vein embolization followed by laparoscopic extended left lobectomy. The patient's postoperative course was uneventful, and the tumor marker levels remained within the normal range. No recurrence was observed 3 months after surgery. In recent years, the use of systemic chemotherapy with drugs, such as lenvatinib, followed by conversion surgery has been reported in some cases of unresectable HCC. The present case reports successful conversion surgery following lenvatinib treatment.

Purpose: To verify the hypothesis that extracellular volume fraction (ECV) and precontrast CT density are the main determinants of washout of hepatocellular carcinoma (HCC) at the equilibrium phase CT., Materials and Methods: Between 2018 and 2020, patients with surgically resected HCC were recruited who had undergone preoperative 4-phase CT. Those larger than 6 cm were excluded to minimize the possibility of intratumoral hemorrhage or degeneration. Two radiologists reviewed the whole images in consensus and divided cases into washout positive and negative groups. Washout positive group at the equilibrium phase was defined as "HCC showing relatively low density as compared to the surrounding background liver (BGL), irrespective of the presence of early enhancement or fibrous capsule". Several clinico-pathological and radiological features, including ECV and precontrast CT density, were correlated to the presence of washout, using uni- and multi-variable analyses., Results: 27 HCC in 24 patients met the inclusion criteria. 22 (82%) and five HCC belonged to washout positive and negative groups, respectively. Univariable analysis revealed ECV of HCC and BGL, ECV difference between HCC and BGL, and presence of fibrous capsule on the equilibrium phase CT were the significant factors. Multivariable analysis showed ECV of HCC and BGL, and precontrast CT density of BGL, were the independently significant factors related to washout, suggesting washout is more likely observed with lower HCC ECV, higher BGL ECV, and higher BGL precontrast CT density., Conclusion: Major determinants of washout of HCC may be ECV of HCC and BGL, and precontrast CT density of BGL., (© 2022. The Author(s).)

Background/aim: Cancer is the most fatal disease worldwide whose most lethal characteristics are invasion and metastasis. Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. HCC often shows encapsulation, which is related to better prognosis. In this study, proteomic analysis of HCC tissues with and without encapsulation was performed, in order to elucidate the factors which play important roles in encapsulation., Materials and Methods: Five HCC tissues surrounded by a capsule and five HCC tissues which broke the capsule were obtained from patients diagnosed with HCC who underwent surgical liver resection. Protein samples from these tissues were separated by two-dimensional gel electrophoresis (2-DE), and the protein spots whose expression was different between encapsulated and non-encapsulated HCC tissues were identified through gel imaging analysis software. The selected protein spots were analyzed and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: Two-DE analysis showed 14 spots whose expression was different between encapsulated and non-encapsulated HCC tissues. Of these, 9 were up-regulated and 5 were down-regulated in HCC tissues without encapsulation. The validation by Western blot confirmed that leucine aminopeptidase 3 (LAP3) and phosphoenolpyruvate carboxykinase mitochondrial (PCK2) were up-regulated significantly in HCC tissues with a capsule, compared to HCC tissues that broke the capsule., Conclusion: These findings suggest that LAP3 and PCK2 could be factors responsible for the maintenance of encapsulation in HCC tissues., (Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The most common sites of recurrence after liver transplantation for hepatocellular carcinoma (HCC) have been reported to be the liver, lung, bone, and adrenal glands, but there have also been many reports of cases of multiple recurrence. The prognosis after recurrence is poor, with reported median survival after recurrence of HCC ranging from 9 to 19 months. Here, we report a case of long-term survival after recurrence of pharyngeal metastasis following living-donor liver transplantation (LDLT) for HCC within the Milan criteria, by resection of the metastatic region and cervical lymph node dissection., Case Presentation: A 47-year-old man with a Model End-stage Liver Disease (MELD) score of 11 underwent LDLT for HCC within the Milan criteria for liver cirrhosis associated with hepatitis B virus infection, with his 48-year-old elder brother as the living donor. One year and 10 months after liver transplantation, he visited a nearby hospital with a chief complaint of discomfort on swallowing. A pedunculated polyp was found in the hypopharynx, and biopsy revealed HCC metastasis. We performed pharyngeal polypectomy. Two years later, cervical lymph node metastasis appeared, and neck lymph node dissection was performed. Although recurrence subsequently occurred three times in the grafted liver, the patient is still alive 12 years and 10 months after recurrence of pharyngeal metastasis. He is now a tumor-free outpatient taking sorafenib., Conclusion: It is necessary to recognize that the nasopharyngeal region is a potential site of HCC metastasis. Prognostic improvement can be expected with close follow-up, early detection, and multidisciplinary treatment, including radical resection.

Background/aim: The cytolytic activity (CYT) score is a new index of cancer immunity calculated from the mRNA expression levels of GZMA and PRF1. We assessed the clinical significance of the CYT score in HCC., Materials and Methods: The calculated CYT scores of peripheral blood cells (GSE24759), cell lines (CCLE) and HCC tissues (TCGA, GSE14520 and Kyushu cohorts) were assessed. Then, immunohistochemical analysis (IHC) of GZMA and PRF1 was performed., Results: The CYT scores of HCC tissues were lower than those of non-cancerous tissues. The 5-year recurrence-free survival of patients with low CYT scores was significantly shorter than that of patients with high CYT scores. Multivariate analysis indicated that the CYT score was an independent prognostic factor for RFS in TCGA and GSE14520 cohorts., Conclusion: CYT score could be a useful prognostic biomarker in HCC, possibly through reflecting the host immune status., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The prognosis of hepatocellular carcinoma (HCC) patients with tumor thrombus (TT) in the inferior vena cava (IVC) or right atrium (RA) is extremely poor. We reviewed the recent surgical treatments and outcomes of this form of advanced HCC. TT is classified into three types according to its anatomic location relative to the heart: the inferior hepatic type (type I), where the TT is in the IVC below the diaphragm; the superior hepatic type (type II), where the TT is in the IVC above the diaphragm, but still outside the RA; and the intracardiac type (type III), where the TT is above the diaphragm and has entered the RA. Type I can be treated relatively easily by standard radical hepatectomy. For type II, the intrathoracic IVC is approached via the abdominal cavity and an incision in the diaphragm with total hepatic vascular exclusion (THVE). For type III, hepatectomy plus thrombectomy is generally performed under cardiopulmonary bypass. If the TT is only just inside the RA, THVE can be performed by mobilizing the liver caudally. The median overall survival of HCC patients with TT in the IVC or RA, who undergo curative resection, is 19.0-30.8 months. As postoperative recurrence is likely to develop, even after curative surgery, effective postoperative adjuvant chemotherapy is required.

Cancer stem cells (CSCs) are thought to play important roles in cancer malignancy. Previously, we successfully induced sphere cancer stem-like cells (CSLCs) from several cell lines and observed the property of chemoresistance. In the present study, we examined the metastatic potential of these induced CSLCs. Sphere cancer stem-like cells were induced from a human hepatoma cell line (SK-HEP-1) in a unique medium containing neural survival factor-1. Splenic injection of cells into immune-deficient mice was used to assess hematogenous liver metastasis. Transcriptomic strand-specific RNA-sequencing analysis, quantitative real-time PCR, and flow cytometry were carried out to examine the expression of epithelial-mesenchymal transition (EMT)-related genes. Splenic injection of CSLCs resulted in a significantly increased frequency of liver metastasis compared to parental cancer cells (P < .05). In CSLCs, a mesenchymal marker, Vimentin, and EMT-promoting transcription factors, Snail and Twist1, were upregulated compared to parental cells. Correspondingly, significant enrichment of the molecular signature of the EMT in CSLCs relative to parental cancer cells was shown (q < 0.01) by RNA-sequencing analysis. This analysis also revealed differential expression of CD44 isoforms between CSLCs and parental cancer cells. Increasing CD44 isoforms containing an extra exon were observed, and the standard CD44 isoform decreased in CSLCs compared to parental cells. Interestingly, another CD44 variant isoform encoding a short cytoplasmic tail was also upregulated in CSLCs (11.7-fold). Our induced CSLCs possess an increased liver metastatic potential in which promotion of the EMT and upregulation of CD44 variant isoforms, especially short-tail, were observed., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Anatomical hepatectomy is an ideal curative treatment for hepatocellular carcinoma (HCC). We have standardized our laparoscopic anatomical hepatectomy (LAH) procedure, gradually extending its indications. In the present study, we describe our experience and the perioperative and oncological outcomes of LAH for HCC compared to those of open anatomical hepatectomy (OAH) during the gradual introduction of LAH., Methods: Seventy patients with primary HCC underwent anatomical hepatectomy in our institution from November 2008 to April 2014. As we gained experience with LAH, our indications for choosing LAH over OAH gradually expanded. Ultimately, 40 and 30 patients underwent LAH and OAH, respectively. Perioperative and oncological outcomes were compared between the two groups., Results: There were no significant differences in age, sex, background of liver disease, liver function, tumor size, tumor number, or type of liver resection between the two groups. Major complications and mortality rates were similar between the LAH and OAH groups (12.5% vs. 20%; p = 0.582, and 0% vs. 3.3%; p = 0.429, respectively). The median follow-up time after surgery was 40.5 months in the LAH group and 32.9 months in the OAH group (p = 0.835). The 1-, 3-, and 5-year overall survival rates were 89.9, 84.7, and 70.9%, in the LAH group, and 89.8, 68.0, and 63.1% in the OAH group, respectively (p = 0.255). The 1-, 3-, and 5-year disease-free survival rates were 79.5, 58.0, and 42.5%, in the LAH group, and 72.4, 56.1, and 50.4% in the OAH group, respectively (p = 0.980)., Conclusions: Through gradual introduction of LAH, we obtained comparable results to those achieved with OAH. LAH can be a feasible surgical treatment for primary HCC, with good oncological outcomes.

Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzyme in gluconeogenesis, is reduced in expression in certain cancers where it has been hypothesized to act as a tumor suppressor, including in hepatocellular carcinoma (HCC). Here, we report functional evidence supporting this hypothesis, providing a preclinical rationale to develop FBP1 as a therapeutic target for HCC treatment. Three independent cohorts totaling 594 cases of HCC were analyzed to address clinical significance. Lower FBP1 expression associated with advanced tumor stage, poor overall survival, and higher tumor recurrence rates. In HCC cell lines, where endogenous FBP1 expression is low, engineering its ectopic overexpression inhibited tumor growth and intracellular glucose uptake by reducing aerobic glycolysis. In patient specimens, promoter methylation and copy-number loss of FBP1 were independently associated with decreased FBP1 expression. Similarly, FBP1 downregulation in HCC cell lines was also associated with copy-number loss. HCC specimens exhibiting low expression of FBP1 had a highly malignant phenotype, including large tumor size, poor differentiation, impaired gluconeogenesis, and enhanced aerobic glycolysis. The effects of FBP1 expression on prognosis and glucose metabolism were confirmed by gene set enrichment analysis. Overall, our findings established that FBP1 downregulation in HCC contributed to tumor progression and poor prognosis by altering glucose metabolism, and they rationalize further study of FBP1 as a prognostic biomarker and therapeutic target in HCC patients. Cancer Res; 76(11); 3265-76. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: Downregulation of paired related homeobox 1 (PRRX1) is associated with the acquisition of cancer stem cell (CSC)-like properties and poor prognosis in cancers. The purpose of this study is to clarify the role of PRRX1 expression in predicting prognosis and mediating CSC-like properties in hepatocellular carcinoma (HCC)., Methods: The association between PRRX1 expression and overall survival (OS) of patients with HCC was analyzed in three independent datasets: 62 resected primary cases, 242 cases from GSE14520, and 162 cases from The Cancer Genome Atlas (TCGA). A cell line expressing PRRX1 (HuH7) was established for the functional analyses. The ability to form spheres, the expression levels of the hepatic CSC surface markers (CD13, CD133, and EpCAM), in vitro chemosensitivity to 5-fluorouracil (FU), and radiosensitivity were evaluated., Results: Univariate and multivariate analyses showed that the 5-year OS of the low PRRX1 expression group was significantly poorer than that of the high PRRX1 expression group (P = 0.024 and P = 0.045, respectively). Consistent with this, the low PRRX1 expression group in GSE14520 and TCGA datasets showed significantly shorter OS (P = 0.027 and P = 0.010, respectively). Gene set enrichment analysis on GSE14520 and TCGA datasets indicated that downregulation of PRRX1 was correlated with the stemness signature. The number of spheres and the expression levels of CSC markers were significantly decreased when PRRX1 was expressed. Moreover, PRRX1 impaired resistance to 5-FU and radiation., Conclusions: Downregulation of PRRX1 expression contributes to the poor prognosis of patients with HCC through acquisition of CSC-like properties.

Transcriptional coactivator with PDZ-binding motif (TAZ) and yes-associated protein (YAP) are equivalently placed downstream effectors of the Hippo pathway with oncogenic roles in human cancers. However, the expression profiles of TAZ/YAP differ depending on the cancer cell type, suggesting that these proteins have different roles during cancer progression, yet no studies have examined the biologic significance of the balance between TAZ and YAP expression levels. Here we examined the functional roles of TAZ/YAP in hepatocellular carcinoma progression. We found that TAZ, but not YAP, was predominantly expressed in HCC. TAZ knockdown under normal conditions attenuated cell growth in HCC cells; however, TAZ knockdown combined with 5-fluorouracil treatment significantly increased chemoresistance compared with control cells. Notably, TAZ knockdown induced compensatory YAP expression and was accompanied by upregulation of CD90, a HCC-specific cancer stem cell marker. Continuous treatment with 5-fluorouracil also induced YAP expression and promoted tumor formation in vivo. Conversely, double knockdown of TAZ/YAP reduced chemoresistance and tumorigenicity. Moreover, YAP knockdown aggravated HCC cell growth to a greater degree than TAZ knockdown, and YAP overexpression was strongly associated with poor prognoses in patients with HCC. Collectively, these studies demonstrate that TAZ and YAP exhibit different functional roles in cancer progression, and a shift to predominant YAP expression upon TAZ depletion conferred cancer stem cell-like properties including chemoresistance and tumorigenicity in HCC. Therefore, targeting of both TAZ/YAP will be required for a complete antitumor response in HCC., (©2015 American Association for Cancer Research.)

We report a case of hepatocellular carcinoma (HCC) with a Vp4 tumor thrombus treated successfully with multidisciplinary therapy. A 59-year-old woman who complained of abdominal distension was diagnosed as having HCC associated with hepatitis B virus (HBV) infection. The tumor was located in the left lobe, caudate lobe, and S6, and the tumor thrombus extended into the main portal and right portal veins. Extended left lobectomy with thrombectomy and S6 partial hepatectomy were performed after intrahepatic arterial infusion chemotherapy with radiation. During the surgery, the collateral circulation was preserved for bleeding control and residual liver function preservation. The patient was alive 12 months after surgery. Cases of advanced HCC with Vp4 can be successfully treated by multidisciplinary therapy.

Background: We previously reported overexpression of heat-shock protein (HSP) 70 in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) using proteomic profiling and immunohistochemical staining (IHS). This suggested that HSP70 could be a molecular target for treatment of HCC., Methods: Twelve patients with HCV-related HCC were enrolled in a phase 1 clinical trial. Dendritic cells (DCs) transfected with HSP70 mRNA (HSP70-DCs) induced by electroporation were injected intradermally. Patients were treated three times every 3 weeks. The number of HSP70-DCs injected was 1 × 10(7) as the lowest dose, then 2 × 10(7) as the medium dose, and then 3 × 10(7) as the highest dose. Immunological analyses were performed., Findings: No adverse effects of grade III/IV, except one grade III liver abscess at the 3 × 10(7) dose, were observed. Thus, we added three more patients to confirm whether 3 × 10(7) is an appropriate dose. Eventually, we chose 3 × 10(7) as the recommended dose of DCs. Complete response (CR) without any recurrence occurred in two patients, stable disease in five, and progression of disease in five. The two patients with CR have had no recurrence for 44 and 33 months, respectively. IHS in one patient who underwent partial hepatectomy showed infiltration of CD8+ T cells and granzyme B in tumors, indicating that the dominant immune effector cells were cytotoxic T lymphocytes with tumor-killing activity., Interpretation: This study demonstrated that HSP70-DCs therapy is both safe and feasible in patients with HCV-related HCC. Further clinical trials should be considered.

Background: The inactivation of the Hippo pathway lead to TAZ (PDZ-binding motif)/YAP (yes-associated protein) overexpression, and is associated with worse prognostic outcomes in various cancers including hepatocellular carcinoma (HCC). Although there are several reports of microRNA (miR) targeting for YAP, miR targeting for TAZ remains unclear. The aim of this study is to identify the miR targeting TAZ expression in HCC., Methods: MicroRNA expression was analysed using the Human miFinder 384HC miScript miR PCR array, and was compared between low and high TAZ expression cell lines. Then, we extracted miR-9-3p as a tumour-suppressor miR targeting TAZ. We examined the functional role of miR-9-3p using miR-9-3p mimic and inhibitor in HCC cell lines)., Results: In HCC cell lines and HCC clinical samples, there was the inverse correlation between miR-9-3p and TAZ expressions. TAZ expression was induced by treatment of miR-9-3p inhibitor and was downregulated by treatment of miR-9-3p mimic. Treatment of miR-9-3p mimic inhibited cell proliferative ability with downregulated phosphorylations of Erk1/2, AKT, and β-catenin in HLF. Inversely, treatment of miR-9-3p inhibitor accelerated cell growth compared with control in HuH1., Conclusions: MicroRNA-9-3p was identified as the tumour-suppressor miR targetting TAZ expression in HCC cells.

Background: The long-term prognosis of hepatocellular carcinoma (HCC) patients after hepatic resection (HR) remains poor because of limited liver function and frequent recurrences. We created a prognostic system of HCC based on tumor markers and Child-Pugh classification., Patients and Methods: This study investigated 427 HCC patients and three tumor markers (alpha-fetoprotein (AFP), Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3), des-γ -carboxyprothrombin (DCP)) in relation to Child-Pugh classification by the stepwise Cox regression model for establishing a tumor marker staging (TMS)., Results: The TMS shows four levels (0/1/2/3) with 5-year recurrence rate of each stage of 76.7, 72.3, 80.9 and 100%, respectively, and 5-year overall survival of 77.0, 68.7, 52.1 and 28.9%, respectively. This TMS appears to be a better model to predict the recurrence and survival of HCC patients after hepatectomy than only the number of positive tumor markers., Conclusion: TMS is a useful staging system to evaluate biological status and background liver function., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Aim: The aim of this study was to determine whether antiviral therapy with nucleotide/nucleoside analog (NA) is beneficial for Japanese patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) who underwent initial curative liver resection., Patients and Methods: In 162 patients with positive hepatitis B surface antigen and negative anti-hepatitis C virus antibody, sixty-two patients received antiviral therapy with NA (NA group) and the remaining 100 patients did not (non-NA group). Prognostic factors for disease-free survival (DFS) and overall survival (OS) were evaluated. Moreover, to equalize the background covariates, a one-to-one propensity case-matched analysis was used., Results: NA administered were lamivudine (LAM) solely for 21 patients, LAM plus adefovir dipivoxil (ADV) for 6, LAM switched to entecavir (ETV) for 5 and ETV solely for 31. DFS did not significantly differ between the NA group and non-NA group (p=0.19). However, OS was significantly different (p=0.0063); 1-,3- and 5-year OS were 100% and 85.9%, 88.3% and 61.9% and 65.1% and 58.0%, respectively. In multivariate analysis, no antiviral therapy with NA was an independent poor prognostic factor (hazard ratio (HR)=2.72; p=0.0229). However, after propensity case-matched analysis, disease-free and overall survival were not significantly different between the two groups., Conclusion: In a Japanese cohort, antiviral therapy with NAs might provide longer survival for postoperative HBV-related HCC patients compared to patients without antiviral therapy. However, deterministic evaluation was impossible by this study alone., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The aim of this study was to develop an accurate predictive system for prognosis of hepatocellular carcinoma (HCC) patients after hepatectomy. We pooled data of clinicopathological features of 234 HCC patients who underwent curative hepatectomy. On the basis of the pooled data, we established a simple predictive staging system (PS score) scored by the mathematical product of tumor number and size, and degree of liver function. We compared the prognostic abilities of the PS score (score 0-3) with those of six well-known clinical staging systems. Then, we found that there were significant differences (P<0.05) in both disease-free survival (DFS) and overall survival (OS) between patients with different PS scores (PS score 0 vs. 1; PS score 1 vs. 2), and there was a significant difference in DFS, but not OS, between patients with PS score 2 and those with PS score 3. Moreover, the PS score had smaller values of the Akaike information criterion for both DFS and OS than any of the six well-known clinical staging systems. These results suggest that the PS score serves as a simple, accurate predictor for the prognosis of HCC patients after hepatectomy.

Aim: This study aimed to investigate the relationship between prognosis after curative hepatectomy and serum methylation signature (SMS), defined by methylation levels of six specific genes (cyclin D2, Ras association (RalGDS/AF-6) domain family member 1, serine peptidase inhibitor Kunitz type 2, cystic fibrosis transmembrane conductance regulator, brain abundant membrane attached signal protein 1, and steroid-5-alpha-reductase alpha polypeptide 2)., Patients and Methods: Serum samples were collected preoperatively from 125 patients with hepatocellular carcinoma associated with hepatitis C virus infection who underwent curative hepatectomy. We measured the methylation levels of the preceding six genes. We defined the methylation of three genes or more in the serum as SMS-positive in this study. We investigated the prognosis of SMS-positive patients., Results: SMS-positive patients exhibited significantly shorter disease-free survival (DFS) and overall survival (OS) than SMS-negative patients (p=0.0002 and p<0.0001, respectively). Multivariate analysis showed that SMS positivity was an independent risk factor for shorter DFS (hazard ratio (HR)=2.182; p<0.001) and OS (HR=4.198; p<0.001)., Conclusion: SMS is useful as a prognostic predictor in patients with hepatocellular carcinoma after curative hepatectomy., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Prognosis of patients with hepatocellular carcinoma (HCC) with macrovascular invasion is extremely poor even after hepatic resection. Herein, the efficacy of short-term adjuvant hepatic arterial infusion chemotherapy (HAI) for HCC with macrovascular invasion has been studied., Patients and Methods: From 1997 to 2013, 39 patients with HCC (BVp3, Vv3, or B3) undergoing hepatic resection were enrolled(group A: adjuvant HAI, n=21, group B: hepatectomy alone, n=18)., Results: Clinical and demographic characteristics were similar in the 2 groups. Five-year disease-free survival (DFS) rates were 31.0% and 6.2% in group A and group B, respectively (p=0.008). In multivariate analysis for DFS, adjuvant HAI was the only independent prognostic factor (hazard ratio: 0.44, p=0.04). In group A, the intrahepatic recurrence rate was significantly lower (33% vs 72%, p=0.01) compared to group B, but rates of extrahepatic recurrence were similar in the 2 groups (19% vs 22%). The rate of overall survival was marginally higher in group A compared to group B (51% vs 19%, respectively, p= 0.06)., Conclusion: Short-term adjuvant HAI after hepatic resection may significantly improve DFS for HCC patients with macrovascular invasion.

Background: The purpose of this study was to determine the relationship between serum zinc (Zn) level and clinicopathological profiles in patients with hepatocellular carcinoma (HCC). Furthermore, we investigate the influence of serum Zn level on the long-term prognosis after hepatectomy., Methods: A retrospective analysis was conducted of 310 patients who underwent initial hepatectomy for HCC. The correlation between serum Zn level and preoperative liver functional indicator, pathological features, including the degree of hepatic fibrosis, and perioperative outcome were evaluated. The patients were divided into two groups (serum Zn ≤ 65 mg/dL; n = 71 and Zn > 65 mg/dL; n = 239), and their long-term prognosis were compared., Results: There were strong correlations between preoperative serum Zn level and serum albumin (r = 0.47, P < 0.0001), branched-chain amino acids (r = 0.28, P < 0.0001), and hyaluronic acid level (r = -0.33, P < 0.0001). The patients with low Zn group were characterized as hepatitis C virus (HCV) infection, impaired liver function, background hepatic fibrosis, and pathological vascular invasion. The disease-free and overall survivals in the low Zn group were significantly lower than those in the high Zn group, especially in subgroup with HCV-related liver disease (P = 0.041 and 0.0004, respectively). Multivariate analyses showed that serum Zn level was an independent prognostic factor for overall survival (hazard ratio 1.79, P = 0.038)., Conclusions: Serum Zn level reflects liver function and is useful for predicting the hepatic fibrosis before surgery. The prognosis of patients with HCC was found to be associated with preoperative serum Zn level, especially in patients with HCV-related chronic liver disease.

Anatomical hepatectomy (AH) is basically not required for metastatic tumors in terms of oncology, but is required for hepatocellular carcinoma [1-5]; however, the surgeon cannot secure the surgical margin by palpation via a laparoscopic approach. Therefore, AH or partial hepatectomy exposing the vessels around the tumor (PHev) is often better for deep-seated or invisible lesions [6, 7] because unexpected exposure of the tumor on the cutting plane can be avoided by creating a cutting plane on the side of exposed vessels. From August 2008 to December 2012, we performed totally laparoscopic AH or PHev for 29 patients (AH in 21 patients and PHev in 8 patients) to secure the surgical margin of metastatic tumors [8, 9]. The median operative time was 329 (range 147-519) min, with median blood loss of 141 (range 5-430) g. Conversion was performed for one patient whose stump of the Glissonean branch was positive in a frozen section. Additional hepatectomy was performed via an open approach. Postoperative morbidity rate was 20.7 % (peroneal palsy in two patients, ileus in one patient, biloma in one patient, and pulmonary embolism in one patient). Mortality was zero. The median length of hospital stay after surgery was 9 (range 4-21) days. Only one patient, who underwent extended posterior sectorectomy for a 4.2-cm tumor developing close to the right main Glissonean pedicle, had a microscopically positive margin, because the tumors were exposed on the cutting plane. The embedded video demonstrates hepatectomy of the dorsal half-segment of the right anterior sector, during which the liver was divided at the anterior fissure [10] and the border between the anterior and posterior sector. Totally laparoscopic hepatectomy exposing the vessels around the tumor can be performed safely and is useful to secure the surgical margin in patients with a metastatic tumor.

A 61-year-old man who complained of right hypochondralgia was diagnosed as hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The tumor was located in the right lobe and S3, and the tumor thrombus extended into the main portal and left portal veins. Preoperatively, real-time tumor-tracking radiation therapy was performed on the tumor thrombus (20 Gy/4 Fr),after vessel coils were placed at the anterior hepatic artery as a marker for the radiation. Ten days after radiation therapy, extended right hepatectomy with thrombectomy and S3 partial hepatectomy were performed. There were no postsurgical complications, and intrahepatic artery infusion chemotherapy was performed. The patient was alive with no recurrences 20 months after surgery. Radiation therapy before hepatectomy is an effective treatment for portal venous tumor thrombus in HCC.

The poor prognosis of hepatocellular carcinoma (HCC) can be explained largely by the high rate of intrahepatic recurrence (IHR). Identification of genes related to IHR is needed to improve the poor prognosis and important for personalized medicine. Eighty-one HCC specimens were used in this study. We screened for IHR-related genes by DNA microarray analysis. The validation of screening was performed by using real-time PCR. The methylation levels in genomic DNAs were measured by quantitative methylation-specific PCR. Six hepatoma cell lines were used for examination of ABCB6 expressional regulation. Time-to-event analyses for recurrence after surgery were analyzed by Kaplan-Meier analysis and Cox regression analysis with cutoff values obtained from receiver operating characteristic (ROC) analysis. We confirmed that ABCB6 mRNA levels were significantly higher in hepatitis C virus (HCV)-related HCCs with early IHR compared to HCV-related HCCs without early IHR (2.5-fold, P=0.01) and the corresponding non-cancerous livers (3.1-fold, P=0.05). Experiments with cell lines showed correlation between DNA methylation and mRNA levels of ABCB6. ROC analysis revealed that mRNA levels (0.81 area under the curve, 88% sensitivity and 72% specificity) and DNA methylation levels (0.81 area under the curve, 80% sensitivity and 80% specificity) of ABCB6 in HCV-related HCCs allowed for the accurate discrimination of the development of early IHR. Cox regression analysis revealed that ABCB6 mRNA levels was an independent risk factor for IHR of HCV-related HCC. Aberrant mRNA and DNA methylation levels of ABCB6 may serve as useful predictive biomarkers for early IHR of HCV-related HCC.

Objective: The purpose of this study was to elucidate the clinicoradiologic characteristics of pseudolesions of the liver in patients with hepatocellular carcinoma (HCC) treated with transarterial chemoembolization (TACE) as observed on gadoxetate disodium-enhanced MR images. A particular interest was correlation between the pseudolesion characteristics and TACE-MRI interval, during which sequential changes in pseudolesions may be revealed after TACE., Materials and Methods: Forty-eight patients with HCC who underwent gadoxetate disodium-enhanced MRI after TACE were retrospectively recruited. Pseudolesions were defined as areas of decreased signal intensity in treated areas on hepatocellular phase images that were confirmed to be nontumorous areas at follow-up. The prevalence and MRI features of pseudolesions were correlated with various clinical parameters, including TACE-MRI interval., Results: Pseudolesions were found in 14 patients (29%). Within 1 month of TACE, the prevalence of pseudolesions was 83%. All of the pseudolesions had arterial enhancement, mimicking residual HCC. After 1 month, the prevalence of pseudolesions decreased, and these pseudolesions tended to exhibit no abnormality in any sequence other than the hepatocellular phase. Results of multivariate analysis suggested that size of HCC (p < 0.0001), duration of postembolization syndrome (p = 0.012), and TACE-MRI interval (p = 0.038) are independent indicators of the presence of pseudolesions., Conclusion: The prevalence and appearance of pseudolesions differ at different intervals from TACE. Radiologists need to recognize the clinicoradiologic characteristics to differentiate pseudolesions from true residual or recurrent HCC.

Hepatocellular carcinoma (HCC) often exhibits a poor prognosis due to metastatic spread caused by portal vein invasion (PVI). In the present study, we attempted to identify a novel therapeutic target related to PVI of HCC. Based on pooled genomic data, we identified RD RNA binding protein (RDBP), a member of the negative elongation factor (NELF) transcription elongation regulatory complex, to be preferentially overexpressed in HCC with PVI. We used quantitative reverse transcription polymerase chain reaction (RT-PCR) and immuno-histochemical analyses to investigate the relationship between RDBP mRNA and protein with metastatic potential in sample sets of hepatitis C virus (HCV)-related HCC and corresponding non-HCC liver tissues. We also used the small interfering RNA technique to examine the role of RDBP in invasion and proliferation of HCC cells in vitro. Our data showed that both mRNA and protein levels of RDBP were significantly higher in HCC compared to non-HCC liver tissue, and that these levels were also significantly higher in HCC with PVI compared to HCC without PVI. Multivariate analysis revealed that RDBP protein levels were an independent risk factor for early intrahepatic recurrence of HCC within 2 years of surgery. Knockdown of RDBP protein significantly inhibited the proliferation and invasion of cells in vitro. These data demonstrate that RDBP is related to the metastatic potential of HCC, suggesting a possible candidate for prevention of HCC cell metastasis.

Unlabelled: The stress-activated mitogen-activated protein kinases (MAPKs), c-Jun NH2-terminal kinase (JNK), and p38 have been implicated in hepatocarcinogenesis. Although the many interrelated functions of JNK and p38 are precisely regulated by upstream signaling molecules, little is known about upstream regulators. We investigated the role of apoptosis signal-regulating kinase 1 (ASK1), a major player in the regulation of JNK and p38 activities, in hepatocarcinogenesis using a mouse hepatocellular carcinoma (HCC) model. ASK1-deficient (ASK1(-/-) ) and wildtype (WT) mice were treated with diethylnitrosamine on postnatal day 14. Strikingly, after 7 months, approximately three times as many tumors developed in ASK1(-/-) mice as in WT mice. Although JNK and p38 activation were attenuated in ASK1(-/-) HCCs relative to WT HCCs, cell proliferation was comparable in HCCs from both types of mice. On the other hand, both cancer cell apoptosis and hyperphosphorylation of BimEL, a proapoptotic Bcl-2 family member, were suppressed in the ASK1(-/-) HCCs. ASK1(-/-) mice showed remarkable resistance to Fas-induced hepatocyte apoptosis in vivo, probably because of attenuated JNK-mediated BimEL phosphorylation and mitochondrial apoptotic pathway activation. The reintroduction of ASK1 to ASK1(-/-) mouse liver using an adenoviral vector restored Fas-induced hepatocyte death and phosphorylation of JNK and BimEL. Similar findings were obtained in tumor necrosis factor alpha-induced hepatocyte apoptosis. Furthermore, ASK1 was involved in DNA damage-induced p21 up-regulation through a p38 pathway., Conclusion: ASK1 is involved in death receptor-mediated apoptosis and DNA-damage response by way of stress-activated MAPK in the liver, and thus acts as a tumor suppressor in hepatocarcinogenesis. This study provides new insight into the regulation of stress- activated MAPK signaling in hepatocarcinogenesis., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Background: There are few blood tests for an efficient detection of hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) infection., Methods: The abilities of quantitative analyses of 7 genes hypermethylation in serum DNA, α-fetoprotein (AFP) and prothrombin-induced vitamin K absence II (PIVKA-II), and various combinations to detect HCC were evaluated in a training cohort of 164 HCV-infected patients (108 HCCs; 56 non-HCCs). An optimal hybrid detector, built using data for 2 methylated genes (SPINT2 and SRD5A2), AFP, and PIVKA-II, achieved the most satisfactory ability to detect HCC in the training cohort. We evaluated the ability of the optimal hybrid detector to detect HCC in an independent validation cohort of 258 consecutive HCV-infected patients (112 HCCs; 146 non-HCCs) who were newly enrolled in 4 distinct institutes., Results: In the validation cohort of 258 patients, accuracy, sensitivity, and specificity of the hybrid detector for detection of HCC were 81.4%, 73.2%, and 87.7%, respectively. Notably, even when detecting HCC ≤ 2 cm in diameter, the hybrid detector maintained markedly high abilities (84.6% accuracy, 72.2% sensitivity, 87.7% specificity). Youden's index (sensitivity+specificity - 1) for HCC ≤ 2cm was 0.60, vastly much superior to the 0.39 for AFP at a cut-off value of 20 ng/ml and the 0.28 for PIVKA-II at a cut-off value of 40 mAU/ml., Conclusions: These results show that the optimal hybrid blood detector can detect HCV-related HCC more accurately., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The purpose of this report was to describe pseudolesions of the liver that mimicked residual hypervascular hepatocellular carcinoma (HCC), as observed on gadoxetate disodium-enhanced magnetic resonance imaging (EOB-MRI) obtained shortly after transarterial chemoembolization (TACE). Between June 2008 and December 2008, three patients underwent MRI within 12 days after TACE to rule out remaining viable cancerous tissue or to assess the treatment effect. In all three patients, nontumorous liver tissue adjacent to the treated HCC exhibited focal arterial enhancement on dynamic phase and subsequent diminished uptake of gadoxetate disodium on hepatocellular phase images, which mimicked residual HCC. All three patients had mild postembolization syndrome at the time of EOB-MRI and showed no evidence of residual or recurrent tumors on follow-up. The findings of these areas may represent transient focal hyperemia and damage to the liver cell function caused by TACE. Radiologists should be aware that EOB-MRI obtained shortly after TACE may show pseudolesions around the treated tumors and should not mistake them for residual or recurrent tumors.

We reported a case of hepatocellular carcinoma (HCC) with needle tract implantation of the right thoracic wall, which were strongly suspected to have been caused by percutaneous needle biopsy performed at the former hospital to diagnose HCC in September 2001. He visited our hospital at his age of 68 and underwent transcatheter arterial embolization in February 2002. In November 2007, a right 9th intercostal tumor measuring 20 x 15 mm was found on his computed tomography (CT) scan. He underwent resection of the right thoracic wall tumor in January 2008. Pathological diagnosis of the tumor was well differentiated HCC. Twenty one months after surgery, he remains alive without recurrence.

Background: Prognosis of hepatocellular carcinoma (HCC) remains poor because of high recurrence rate. We examined preoperatively the methylated CCND2 gene levels present in the serum following release from HCC cells as a prognosis predictor in patients undergoing curative hepatectomy., Methods: Quantitative real-time RT-PCR and quantitative methylation-specific PCR were used to measure methylated CCND2 gene and its mRNA levels., Results: The CCND2 mRNA levels were down-regulated in HCC with early intrahepatic recurrence (IHR) within 1year of curative hepatectomy. We also identified that this down-regulation was due to promoter hypermethylation. In 70 HCC patients who underwent curative hepatectomy, 39 patients sero-positive for the methylated CCND2 gene (>70pg/ml serum) exhibited a significantly shorter disease-free survival (DFS) period (P=0.02) than the 31 patients who were sero-negative for the methylated CCND2 gene. None of the sero-negative patients demonstrated early IHR, and this method of serum testing did not produce any false-negative predictions for early IHR. Multivariate analysis showed that the serum level of methylated CCND2 was an independent risk factor for DFS (hazard ratio of 1.866, 95% CI: 1.106-3.149)., Conclusion: Methylated CCND2 gene in the serum serves as a prognosis predictor of HCC after curative hepatectomy., (2010. Published by Elsevier B.V.)

We previously identified BASP1 and SRD5A2 as novel hepatocellular carcinoma (HCC) methylation markers from among more than 10,000 screened genes. The present study aimed to improve the diagnostic potential of these genes. We compared the methylation status at distinct regions of the BASP1 and SRD5A2 genes using quantitative methylation-specific PCR, in 46 sets of HCC and corresponding non-tumor liver tissues. We also examined how their epigenetic status affected transcript levels in tissues and several hepatoma cell lines. We found that BASP1 and SRD5A2 loci were methylated in greater than 50% of the HCC tissues. Inverse correlations were identified between the methylation status and transcript levels in the tissues. Assessment of CpG island methylation rate of BASP1 and SRD5A2 resulted in different diagnostic powers for discriminating HCC even in the same CpG island. A combination analysis of BASP1 and SRD5A2 resulted in the optimum diagnostic performance (84.8% sensitivity and 91.3% specificity) with a maximal area under the receiver operating characteristic curve of 0.878. Even in patients with early HCC (well-differentiated, TNM stage I and small in diameter) and those negative for serum alpha-fetoprotein, combination analysis enabled an accurate diagnosis of HCC. In vitro analysis also showed that BASP1 and SRD5A2 transcripts were epigenetically regulated by methylation and acetylation. These results suggest that combined analysis of methylated BASP1 and SRD5A2 may prove useful in the accurate diagnosis of HCC, especially early HCC.

The current study was conducted to identify robust methylation markers and their combinations that may prove useful for the diagnosis of early hepatocellular carcinoma (HCC). To achieve this, we performed in silico CpG mapping, direct sequencing and pyrosequencing after bisulfite treatment, and quantitative methylation-specific PCR (MSP) in HCC and non-HCC liver tissues. In the filtering group (25 HCCs), our direct sequencing analysis showed that, among the 12 methylation genes listed by in silico CpG mapping, 7 genes (RASSF1A, CCND2, SPINT2, RUNX3, GSTP1, APC and CFTR) were aberrantly methylated in stages I and II HCCs. In the validation group (20 pairs of HCCs and the corresponding non-tumor liver tissues), pyrosequencing analysis confirmed that the 7 genes were aberrantly and strongly methylated in early HCCs, but not in any of the corresponding non- tumor liver tissues (p < 0.00001). The results obtained using our novel quantitative MSP assay correlated well with those observed using the pyrosequencing analysis. Notably, in MSP assay, RASSF1A showed the most robust performance for the discrimination of HCC and non-HCC liver tissues. Furthermore, a combination of RASSF1A, CCND2 and SPINT2 showed 89-95% sensitivity, 91-100% specificity and 89-97% accuracy in discriminating between HCC and non-HCC tissues, and correctly diagnosed all early HCCs. These results indicate that the combination of these 3 genes may aid in the accurate diagnosis of early HCC., (Copyright 2009 UICC.)

Background: Human leukocyte antigen (HLA) class I expression is reportedly frequently reduced in cancer. We examined heat-shock protein (HSP) expression in hepatocellular carcinoma (HCC)., Patients and Methods: HCV-related HCC was examined in 73 patients who had undergone hepatectomy, and the relationship between HSP70 and HLA class I expressions, clinicopathological factors and survival was evaluated., Results: Immunohistochemically positive results for HSP70 and HLA class I were seen in 67 (92%) and 43 cases (59%), respectively, while 38 patients (52%) were positive for both. Increased HSP70 immunoreactivity was significantly associated with high histological grade of tumor differentiation (p = 0.0179), whereas reduced HLA class I immunoreactivity was significantly associated with large tumor size (p = 0.0082). No differences were apparent between disease-free and overall survival in regard to expression levels., Conclusion: These results suggest that HSP70 expression may be related to tumor differentiation and HLA class I loss may occur with tumor growth in HCV-related HCC.

A genome-wide study using expression profiles of 12,600 genes was conducted to identify methylated genes that could be used for early diagnosis of hepatocellular carcinoma (HCC). Of the 12,600 genes examined, we identified 23 genes with significantly lower expression levels in HCC tissues than in non-HCC liver tissues by our statistical and CpG mapping tests. Of these 23 genes, methylation analysis by direct sequencing with bisulfite treatment determined 4 genes that were aberrantly methylated in 20 HCC samples of TNM stages I and II. Further methylation analysis of the 4 genes by quantitative sequencing with 20 HCCs and the corresponding non-tumor liver tissues from an independent cohort of HCC patients revealed that 2 genes, BASP1 and SRD5A2, were aberrantly methylated in only HCC tissues, though not in any corresponding non-tumor liver tissues. Notably, in the cohort we found that BASP1 or SRD5A2 were aberrantly methylated when a cut-off value of 30% in the methylation rate was used, in all cases of 11 HCCs of TNM stages I and II, of 10 well-differentiated HCCs and of 4 small HCCs <2 cm in maximum diameter, but in none of the 20 corresponding non-HCC livers. Methylation-specific PCR for BASP1 and SRD5A2 reproduced the same results observed by direct sequencing. These results indicate that BASP1 and SRD5A2 might serve as useful biomarkers for early diagnosis of HCC.

Our study revealed that the level of circulating cell-free DNA (cfDNA) is increased in the serum of patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). To gain insight into the mechanism underlying this phenomenon, we examined the association between cfDNA levels and various clinicopathological factors in 96 patients with HCV-related HCC and 99 non-HCC patients with HCV. Using pooled DNA microarray data, we profiled the expression patterns of inflammatory cytokine genes in 14 primary tumors from the group of HCC patients. We found that there were positive associations between the cfDNA level, aspartate aminotransferase levels and the number of leukocytes and neutrophils in patients with HCV-related HCC but not in non-HCC patients with HCV. The serum cfDNA level was not associated with other clinicopathological factors in HCC or non-HCC patients. A cluster analysis based on the inflammatory cytokine gene data revealed that HCCs with a high serum cfDNA level had increased levels of several inflammatory cytokine genes, suggesting that the serum cfDNA level is associated with the inflammatory status in primary tumors in HCV-related HCC.

An increased level of glycolysis, an intracellular hallmark of neoplasms, enables cancer cells to survive under various conditions. To elucidate the role of increased glycolysis in the progression of hepatocellular carcinoma (HCC), we investigated the associations between the expression patterns of 14 glycolysis-related genes and clinicopathologic factors in 60 HCCs by using pooled transcriptome data. We then evaluated the therapeutic efficacy of the knockdown of ENO1, which is encoded by a glycolysis-related gene, in HCC cells. Among the 14 genes, levels of 8 genes (GPI, ALDOA, TPI1, GAPD, PGK, PGAM, ENO1 and PKM), all of which can be transcriptionally activated by hypoxia-inducible factor 1alpha (HIF-1alpha), were significantly higher in HCC with venous invasion (VI) than in HCC without VI. Our cluster analysis showed that HCC patients with activation of the 8 HIF-1alpha-regulated genes had significantly shorter overall survival (P=0.023) than did HCC patients without increased expression levels of these genes. The association between the levels of ENO1 and VI was confirmed in an independent sample set of 49 HCCs by real-time reverse-transcription PCR. The knockdown of ENO1 by small-interfering RNA significantly inhibited the proliferation of an HCC cell line (HLE cells) in both the glucose-rich and glucose-free conditions, accompanied by a decreased S phase and increased G2/M phase of the cell cycle. Collectively, these data suggest that activation of an HIF-1alpha-regulated glycolysis module is closely related to the aggressive phenotype of HCC, and that ENO1, a glycolysis module gene, might serve as a new target to circumvent HCC metastasis.

Purpose: We aimed to explore the molecular and biological functions of Inhibitor of DNA binding/differentiation 2 (ID2), which was found to be responsible for portal vein invasion of hepatocellular carcinoma (HCC)., Experimental Design: We measured ID2 mRNA levels in 92 HCC patients by real-time reverse transcription-PCR and examined the relation to clinicopathologic features. To clarify the precise roles of ID2, we did in vitro analysis with expression vectors and small interfering RNAs. Effects of ID2 on cell invasive potential and expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha were analyzed by Matrigel-coated invasion chamber, ELISA, and Western blot analysis, respectively., Results: ID2 mRNA level correlated inversely with portal vein invasion (P < 0.001), tumor-node-metastasis stage (P < 0.001), tumor size (P < 0.001), and early intrahepatic recurrence (P < 0.05). When limited to a cohort of hepatitis C virus-related HCCs, patients with low levels of ID2 had significantly shorter disease-free survival time than those with high levels of ID2. Invasive potential of cells transfected with ID2 expression vector was lower than that of empty vector-transfected cells. Cells overexpressing ID2 also showed decreased VEGF secretion and hypoxia-inducible factor-1alpha protein levels. The results of ID2-knockdown experiments were opposite to those of ID2 overexpression experiments., Conclusions: On the basis of our clinical and in vitro data, we suggest that ID2 plays a significant role in the metastatic process during progression of HCC. This action might be explained, at least in part, by altered cell mobility due to decreased secretion of VEGF.

We previously developed a DNA microarray-based system that out-performs traditionally used clinical parameters for prediction of early intrahepatic recurrence (IHR) of hepatocellular carcinoma (HCC). Because DNA microarray is too expensive for daily clinical use, we used a quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) to develop a lower-cost predictor for early IHR. From the 12 early IHR-related genes integrated in the previous predictor, we selected 6 genes whose levels showed the strongest association between data from the 2 distinct DNA microarray platforms with the same sample set. Expression of these 6 genes relative to that of GAPDH was measured by QRT-PCR in 82 HCCs. Of the 82 HCCs, 39 and 43 were assigned to training and independent test sets, respectively. By searching all combinations (n=2-6) of the 6 genes, we found an optimal combination of 3 genes (HLADRA, DDX17 and LAPTM5) that minimized the leave-one-out error for prediction of early IHR in the training set. The 3-gene predictor constructed with the Fisher linear classifier correctly predicted early IHR or non-recurrence in 35 (81.4%) of 43 HCCs in the independent test set and had a high positive predictive value of 72.7% and a high negative predictive value of 84.4%. Multivariate analysis with the stepwise logistic regression showed that the 3-gene predictor [F(x)<0] was an independent risk factor for early IHR (risk ratio, 13.6; p=0.006), indicating its potential as an easy-to-use predictor for accurate prediction of early IHR of HCC.

In a previous study, we showed that levels of cell-free DNA (cfDNA) were significantly higher in sera of patients with hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) than in sera of non-HCC patients with HCV. To confirm this finding, we analysed serum cfDNA levels in a cohort of 96 patients with HCV-related HCC and in 100 HCV carriers without known HCC. Again we found that serum cfDNA levels were significantly higher in HCC patients than in HCV carriers (115.9+/-98.3 vs 34.4+/-40.4 ng ml(-1) (mean+/-s.d.), P<0.0001). Of 87 eligible patients who underwent curative hepatectomy, those with a high cfDNA level had a significantly shorter overall survival (OS) time than those in whom the cfDNA level was not high. Cox proportional hazards model showed the cfDNA level to be an independent prognostic factor for OS and cancer recurrence in distant organs. Our results suggest that the serum cfDNA level reflects the metastatic potential of HCV-related HCC and that it can be a useful predictive biomarker for distant metastasis after curative surgery.

Portal vein invasion (PVI) is a hallmark of metastatic potential of hepatocellular carcinoma (HCC) and is frequently found at a stage of moderately differentiated HCC. To identify genes involved in PVI of HCC associated with hepatitis C virus (HCV), we performed a comprehensive analysis of 12,600 genes in 35 moderately differentiated HCV-related HCCs by DNA microarray. Our supervised learning method identified 35 genes involved in PVI. Among the 35 identified genes, we focused on the inhibitor of DNA binding 2 (ID2), because it encodes a liver-rich dominant-negative helix-loop-helix protein. The microarray results for ID2 were reproduced by quantitative real-time reverse transcription (QRT)-PCR and Western blot analyses. In an independent set of HCV-related HCCs (n=28) and HCV-unrelated HCCs (n=14), our QRT-PCR showed that decrease in ID2 mRNA levels were associated with PVI in HCV-related HCC but not HCV-unrelated HCC. In conclusion, our results strongly suggest that ID2 plays an important role in PVI process of HCV-related HCC.

Recent genome-wide screens have identified genes associated with the metastatic potential of hepatocellular carcinoma (HCC); however, there is little overlap between the identified genes, and interpretations of the results remain controversial. These inconsistencies may be related to differences in the sample populations, use of distinct microarray platforms and algorithms, and the complicated modes of HCC recurrence. We investigated the gene expression profiles of extrahepatic recurrence (EHR) and early intrahepatic recurrence (IHR), which are two representative modes of recurrence of HCC attributable to metastasis. We used DNA microarray analysis and identified 46 signature genes for EHR in 35 HCCs in a supervised learning manner. The obtained gene expression profile was compared with that for early IHR that was determined previously in the same manner. The 46 signature genes for EHR included many cell adhesion-related genes (ITGA6, SPP1, DNMBP, CD44 and POSTN), which all showed higher expression in HCC with EHR than in HCC without EHR. The 46 signature genes for early IHR included 10 immune response-related genes, which all showed lower expression in HCC with early IHR than in HCC without early IHR. The signature genes for EHR included only two immune response-related genes (P=0.013). These results suggest that alteration of the cell adhesion system plays a central role in EHR and that reduction of the immune response is a specific step in early IHR. These results indicate that the metastatic processes in EHR and early IHR involve different molecular pathways.

Background: Circulating cell-free DNA is present in increased amounts in the blood of patients with one of several forms of cancer., Materials and Methods: A real-time PCR assay with glutathione S-transferase pi (GSTP1) gene was used to measure cell-free DNA levels in the sera of 52 patients with hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV), which included 30 HCV carriers without known HCC and 16 HCV-negative non-cancer patients (controls)., Results: Cell-free DNA levels were significantly higher in the sera from HCC patients than in the sera from HCV carriers or the control subjects. Cell-free DNA levels were associated with the degree of tumor differentiation and size but not patient age, gender, TNM stage or levels of alpha-fetoprotein (AFP) or protein induced by vitamin K absence (PIVKA-II). The cell-free DNA assay had a sensitivity of 69.2% and a specificity of 93.3% in discriminating HCC and HCV carriers at the optimal cut-off value of 73.0 ng/ml, with an area of 0.90 (95% CI, 0.83-0.96) under the receiver operating characteristic curve. The discriminative power of cell-free DNA was superior to that of AFP or PIVKA-II., Conclusion: Our results showed that levels of circulating cell-free DNA are significantly increased in sera of patients with HCV-associated HCC, suggesting that circulating cell-free DNA may be a good biomarker specific for HCV-associated HCC.

Purpose: The purpose of this study was to elucidate the molecular basis of hepatocellular carcinoma (HCC) caused by genotype-C hepatitis B virus (HBV)., Methods: We compared molecular profiles of 15 HCCs and five non-tumorous livers, all of which were associated with genotype-C HBV infection, using DNA microarray technology., Results: Our supervised learning identified 237 genes whose expression differed between HCCs and non-tumorous livers. This result was validated by a false discovery rate of 0%. Levels of expression of 35 and 202 genes were higher and lower, respectively, in HCCs than in non-tumorous livers. Among the 237 genes, we highlighted the top 35 upregulated and top 35 down regulated genes in tumor. Interestingly, when overlapping genes were excluded, 12 (e.g., NM23-H2, MCM7, PARP1, YWHAH, HSPB1, and MSH2) of the top 34 upregulated genes and five (e.g., MT1A and MT3) of the top 33 downregulated genes were c-myc-regulated genes. The microarray data for five randomly selected genes (MCM7, UBE2L3, PPIA, CXCL12, and ASS) were confirmed by quantitative real-time reverse transcription-polymerase chain reaction., Conclusions: Our results indicate that many c-myc-regulated genes are involved in genotype-C-HBV-related HCC, suggesting that c-myc is related to the hepatocarcinogenic activity of genotype-C HBV.

Cytochrome P450 (CYP) genes are involved in the pathogenesis of hepatocellular carcinoma (HCC). To examine changes in expression of CYPs in HCC arising from hepatitis C virus (HCV)-infected liver, we used oligonucleotide array data of 27 CYPs from samples of 50 HCV-associated HCCs, five HCV-infected non-tumorous livers, and six HCV-negative normal livers. Progression of primary HCC can be characterized by decrease in the grade of tumor differentiation, increased frequency of venous invasion and increased tumor size. On the basis of tumor differentiation, the self-organizing map (SOM) classified the 27 CYPs into four groups. The first group contained 11 CYPs, including the CYP2C and CYP4F families, that showed decreased expression in parallel with progression of HCV-infected liver to HCC with less differentiation. The second group contained CYP-IID, CYP3A7 and CYP27A1, genes that showed high levels of expression specific to well differentiated HCC. The third group contained 5 sterol-metabolizing CYPs with levels lower in HCV-infected livers than in HCV-uninfected livers. The last group included the CYP2E1 and CYP3A families. Among the 27 CYPs, levels of 7 (CYP2B6, CYP-IIC, CYP2C9, CYP2C19, CYP3A5, CYP4F3 and CYP27A1) were significantly lower and levels of 2 (CYP2E1 and CYP4F2) were slightly lower in HCC with venous invasion than in HCC without venous invasion. Levels of CYP-IIC and CYP2C9 were inversely associated with tumor size. In contrast, levels of CYP51A1 were positively associated with tumor size. Our present study revealed that expression of specific CYPs was altered in conjunction with progression of HCV-associated HCC. These CYPs may serve as markers of progression and molecular targets for treatment of HCV-associated HCC.

The outcome of patients with hepatocellular carcinoma (HCC) remains poor because of the high frequency of intrahepatic recurrence (IHR), particularly early IHR within 1 year of hepatectomy. To search for genes involved in early IHR, we performed DNA microarray analysis in a training set of 33 HCCs and selected 46 genes linked to early IHR from approximately 6,000 genes by means of a supervised learning method. Gene selection was validated by a false discovery rate of 0.37%. The 46 genes included many immune response-related genes, which were all downregulated in HCCs with early IHR. Four of these genes (HLA-DRA, HLA-DRB1, HLA-DG and HLA-DQA), encoding MHC class II antigens, were coordinately downregulated in HCCs with early IHR compared to levels in HCCs with nonrecurrence. A cluster analysis reproduced expression patterns of the 4 MHC class II genes in 27 blinded HCC samples. To localize the major site of production of HLA-DR protein in the tumor, we used 50 frozen specimens from 50 HCCs. Immunofluorescence staining showed that HLA-DR protein levels in tumor cells, but not in stromal cells, were associated with the transcription levels of HLA-DRA determined by both DNA microarray analysis and real-time quantitative reverse transcription-PCR. Univariate analysis showed that tumor HLA-DR protein expression, pTNM stage and venous invasion were associated with early IHR. Multivariate analysis showed that tumor HLA-DR protein expression was one of the independent risk factors for early IHR, suggesting HLA-DR protein potential as a biomarker and a molecular target for therapeutic intervention., (Copyright 2005 Wiley-Liss, Inc)

It has been suggested that sex affects not only the incidence of hepatocellular carcinoma (HCC) but also the outcome after treatment. However, no sex-specific therapeutic targets for HCC have been identified. Identification of sex-specific genes will allow for the development of more personalized therapies. To this end, we investigated the expression of approximately 6000 genes in 50 samples of hepatitis C virus (HCV)-related HCC by oligonucleotide microarray. Our supervised learning method and subsequent random permutation test identified 27 genes that were differentially expressed in samples from male (n=34) and female (n=16) patients. Our gene selection was validated by a false discovery rate of only 0.5%. For the 27 genes, expression levels of 12 were higher and expression levels of 15 were lower in HCC samples from men than in HCC samples from women. For the cell proliferation-related genes identified, expression levels of PRDX1 were relatively high in HCC samples from men, and expression levels of PRDX3 were relatively high in HCC samples from women. The DNA microarray data for PRDX1 and PRDX3 were reproduced by reverse transcription-PCR analysis. Our results suggest that these 27 genes may serve as molecular targets or markers for sex-specific treatment of HCV-related HCC. Further studies are needed to elucidate their possible roles in male and female patients with HCV-related HCC.

Using high-density oligonucleotide array, we comprehensively analyzed expression levels of 12600 genes in 50 hepatocellular carcinoma (HCC) samples with positive hepatitis C virus (HCV) serology (well (G1), moderately (G2), and poorly (G3) differentiated tumors) and 11 non-tumorous livers (L1 and L0) with and without HCV infection. We searched for discriminatory genes of transition (L0 vs. L1, L1 vs. G1, G1 vs. G2, G2 vs. G3) with a supervised learning method, and then arranged the samples by self-organizing map (SOM) with the discriminatory gene sets. The SOM arranged the five clusters on a unique sigmoidal curve in the order L0, L1, G1, G2, and G3. The sample arrangement reproduced development-related features of HCC such as p53 abnormality. Strikingly, G2 tumors without venous invasion were located closer to the G1 cluster, and most G2 tumors with venous invasion were located closer to the G3 cluster (P=0.001 by Fisher's exact test). Our present profiling data will serve as a framework to understand the relation between the development and dedifferentiation of HCC.

Chronic infection with hepatitis B or C virus (HBV or HCV) is the most clearly established risk factor for hepato-cellular carcinoma (HCC). One type of HCC (non-B, non-C HCC) also appears to develop in patients negative for both HBV and HCV. Using a supervised learning method, we investigated gene expression in 11 non-B, non-C HCCs with high-density oligonucleotide microarrays, and compared the patterns of gene expression with those of HBV-infected HCCs (B-type HCCs) and HCV-infected HCCs (C-type HCCs) in the previous dataset. Our gene selection identified 112 and 64 genes that were differentially expressed in non-B, non-C HCC in comparison with B- and C-type HCCs, respectively. In both gene selections, we found that the false discovery rate, the percentage of genes identified by chance, was less than 5%. Additionally, in combination with the previous data, our present data revealed a set of genes specific to each type of B- and C-type HCCs and non-B, non-C HCC. Among these, an interferon-induced gene, IFI27, was differentially expressed among all three types of HCCs, and this result was confirmed by RT-PCR. Thus, our present study provides a framework to characterize the molecular features in the three subtypes of HCC with different viral origin.