Multidisciplinary management of hepatocellular carcinoma with portal vein tumor thrombus - Eastern Hepatobiliary Surgical Hospital consensus statement.

Hepatocellular carcinoma (HCC) complicated by portal vein tumor thrombus (PVTT) is associated with poor prognosis, early recurrence of HCC, and limited treatment options. Current guidelines do not have standardized diagnostic and treatment modalities, thus creating a need for a multidisciplinary treatment model for standardization of the treatment. Eastern Hepatobiliary Surgical Hospital (China) convened two working parties of experts from all the departments, to consolidate the current evidence, prevailing vision for the future, and experience of the practicing clinicians engaged in HCC management, so as to develop a consensus for PVTT diagnosis and treatment according to the GRADE system. Based on the quality of the existing evidence and the strength of recommendations, the consensus statements were categorized into 3 evidence levels (A/B/C) and 5 classes (I/II/IIa/IIb/III).The panel discussed and provided clarity on the management and research options in the field of HCC with PVTT. In addition, the panel also assessed the quality of the cited studies and assigned grades to the recommendation statements. Among the group of experts, there was excellent agreement with regard to effective diagnosis and treatment of HCC with PVTT. The recommendations of this consensus will provide guidance to physicians and clinical researchers on the effective management of HCC with PVTT.

Controlled and Targeted Drug Delivery by a UV-responsive Liposome for Overcoming Chemo-resistance in Non-Hodgkin Lymphoma.

Although chemotherapy plays a vital role in treating non-Hodgkin lymphomas, the clinical applications are limited because of intolerable side-effects and multidrug resistance at the beginning or during the course of therapy. In this study, we successfully fabricated a CD20-targeting immuno-liposome based on 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC-8,9PC), which can form intermolecular cross-linking through the diacetylenic group by ultraviolet irradiation. This immuno-liposome showed appropriate size distribution, well-defined regular spherical structure, favorable biocompatibility, high serum stability, and prolonged circulation time in blood vessels. The in and ex vivo experiments demonstrate enhanced tumor suppression abilities against both wild-type and resistant non-Hodgkin lymphomas for liposomal doxorubicin when compared with free drugs. The outstanding antitumor activities are attributed to the accumulation and retention of liposomal drugs in malignant tissues and cells, which are realized by the co-operation of active targeting via antibody-antigen reaction and passive targeting via enhanced permeability and retention effect.

[Relationship between metastasis or recurrence of hepatocellular carcinoma and hepatitis B virus DNA or double mutation at 1762/1764 in the basic core promoter].

OBJECTIVE: To study the relationship between metastasis or recurrence of hepatocellular carcinoma (HCC) and hepatitis B virus (HBV) DNA load or the presence of double mutation at 1762/1764 in the basic core promoter (BCP). METHODS: One-hundred-and-fifty-seven patients with HCC were included in the study. Events of tumor metastasis or recurrence were recorded during 120 weeks of clinical follow-up after treatment by surgery or transarterial chemoembolization (TACE). The 1-year follow-up included monthly alpha fetoprotein (AFP) measurement and abdominal ultrasonography (US), as well as helical computed tomographic (CT) scan performed every 3 months. Follow-up beyond 1-year (surveillance) included AFP measurement and abdominal US every 2 months and helical CT scan every 6 months. Suspected metastasis or recurrence was investigated by hepatic angiography and confirmed according to the combined imaging findings. Serum HBV DNA level was measured by real-time PCR. HBV genotypes were determined by PCR-restriction fragment length polymorphism analysis. RESULTS: Of the 157 HCC cases 110 experienced tumor metastasis or recurrence; the cumulative probability of post-treatment HCC metastasis or recurrence was 4 (2.55%) at week 12, 14 (8.92%) at week 24, 28 (17.83%) at week 48, 64 (40.76%) at week 72, 92 (58.60%) at week 96, and 110 (70.06%) at week 120. Multivariate analysis indicated that both the BCP 1762/1764 double mutations and HBV DNA levels were risk factors for HCC recurrence or metastasis. In particular, the incidence of HCC recurrence or metastasis increased with baseline serum HBV DNA levels in a dose-response manner, ranging from 8/19 (42.1%) for less than 3 log10 copies/ml HBV DNA to 35/61 (57.3%) for 3-5 log10 copies/ml and 67/77 (87.0%) for more than 5 log10 copies/ml. After adjusting for potential confounders, serum HBV DNA level remained independently associated with HCC metastasis or recurrence. HCC recurrence or metastasis occurred in 22/43 (51.2%) of patients without BCP 1762/1764 mutations and 88/114 (77.2%) of patients with BCP 1762/1764 mutations. The adjusted odds ratio for patients infected with BCP 1762/1764 double mutation HBV, compared with those infected with non-BCP 1762/1764 mutation HBV, was 5.264 (95% CI: 1.436-12.574, P less than 0.05). CONCLUSION: Infection with HBV carrying the BCP 1762/1764 double mutation and presence of high HBV DNA load are independent risk factors for developing HCC metastasis or recurrence after surgery or TACE.

Diagnosis and surgical treatment of mucin-producing bile duct tumors.

BACKGROUND/AIMS: To summarize the experience of diagnosis and surgical treatment of mucin-producing bile duct tumors (MPBTs). METHODOLOGY: A retrospective analysis was undertaken to determine the radiography characteristics and results of surgical treatment of MPBTs over the past 9 years. Only eight patients underwent such treatment. The detailed data of diagnosis, treatment and prognosis were carefully studied. RESULTS: Intermittent jaundice was the most frequently clinical manifestation of MPBTs, with unique characteristics on magnetic resonance cholangiopancreatography (MPCP) when compared with gallbladder carcino-ma, hilar cholangiocarcinoma and distal bile duct can-cer. All the 8 patients with MPBTs received appropriate surgical procedure and were cured. CONCLUSIONS: Appropriate diagnosis and curative hepatectomy for MPBTs made it possible to achieve long-term survival.

Analysis of the relationships between clinicopathologic factors and survival in gallbladder cancer following surgical resection with curative intent.

BACKGROUND: This study elucidated the relationships between various clinicopathologic factors and the outcome of patients with gallbladder cancer (GBC) treated by surgical resection with curative intent. METHODS: Between January 2003 and January 2011, 76 patients with GBC underwent surgical resection with curative intent at our department. We then conducted a retrospective analysis of clinicopathologic data. Fourteen clinicopathological variables were selected for univariate and multivariate analysis to evaluate their influence on the outcome. RESULTS: The actuarial 1-, 3-, and 5-year survival rates in the 76 resected cases were 56.6%, 32.7%, and 23.8%, respectively. The univariate analysis revealed that curative resection (P<0.001), lymph node metastasis (P<0.001), AJCC stage (P = 0.030), tumor location (P = 0.008), histologic differentiation (P = 0.028), intraoperative blood loss (P = 0.011), and preoperative jaundice (P = 0.012) were significant risk factors for survival. Multivariate analysis revealed that noncurative resection and tumor location on gallbladder neck were significant risk factors for poor outcome. Among jaundiced patients, we discovered that gallbladder carcinoma with tumor thrombus in common bile duct (CBD) was very rare but with relatively special clinical manifestation and characteristic radiography manifestation. The prognosis of gallbladder carcinoma with tumor thrombus in CBD after surgical procedure was apparently better than gallbladder carcinoma with invasion of hilar tissues. CONCLUSIONS: Curative surgical resection remains the only effective approach to the treatment of GBC. This series confirm that jaundice is a poor prognostic factor. However, the presence of jaundice does not preclude resection, especially in highly selected patients (when R0 resection is achievable). Gallbladder carcinoma with tumor thrombus in CBD has special clinical characteristics, which need to be awared by radiologists and clinicians.

Patt1, a novel protein acetyltransferase that is highly expressed in liver and downregulated in hepatocellular carcinoma, enhances apoptosis of hepatoma cells.

Protein acetylation is increasingly recognized as an important post-translational modification. Although a lot of protein acetyltransferases have been identified, a few putative acetyltransferases are yet to be studied. In this study, we identified a novel protein acetyltransferase, Patt1, which belongs to GNAT family. Patt1 exhibited histone acetyltransferase activity and auto-acetylation activity. Deletion and mutation analysis of the predicted acetyltransferase domain in Patt1 showed that the conserved Glu139 was an important residue for its protein acetyltransferase activity. Furthermore, we found that Patt1 was highly expressed in liver and significantly downregulated in hepatocellular carcinoma tissues. In addition, we showed that overexpression of Patt1 enhanced the apoptosis of hepatoma cells dependent on its acetyltransferase activity, whereas knockdown of Patt1 significantly protected Chang liver cells from apoptosis. These data suggest that Patt1 might be involved in the development of hepatocellular carcinoma, and could be served as a potential therapy target for hepatocellular carcinoma.

Aberrant splicing of Hugl-1 is associated with hepatocellular carcinoma progression.

PURPOSE: Lethal giant larvae functions as a cell polarity regulator and a tumor suppressor in Drosophila. Its evolutionary conservation implies a tumor suppressor role for its human homologue, Hugl-1. The aims of this study were to characterize Hugl-1 and to determine the clinical significance of Hugl-1 alterations in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Sequence alterations of Hugl-1 from 80 HCC specimens and 5 HCC cell lines were characterized by reverse transcription-PCR and sequence analysis. Western blot was used for determining Hugl-1 expression. The biological activities of Hugl-1 and its aberrant variants were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound healing assay, Boyden chamber assay, and tumorigenicity assay. RESULTS: In 32.5% (26 of 80) of the specimens and 20.0% (one of five) of HCC cell lines, 23 unique aberrant Hugl-1 transcripts were identified, most of which resulted from skipping part of and/or entire exon or insertion of intron sequences. The majority of these aberrant Hugl-1 transcripts encoded truncated proteins lacking one or more conserved WD-40 repeat motifs. Two truncated Hugl-1 proteins were found exclusively in HCC tissues. Aberrant Hugl-1 transcripts (78.3%, 20 of 23) had a short "direct repeat" sequence flanking their deleted regions. The abnormal Hugl-1 was significantly correlated with poor differentiation and large tumor size of HCC. Overexpression of two representative HCC-derived aberrant Hugl-1 variants promoted HCC cell migration, invasion, and tumorigenicity in nude mice. CONCLUSIONS: We provide the first evidence that Hugl-1 mRNA is frequently mutated by aberrant splicing exclusively in HCC, which may be involved in HCC progression.

Gene expression profile analysis of human hepatocellular carcinoma using SAGE and LongSAGE.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the second cancer killer in China. The initiation and malignant transformation of cancer result from accumulation of genetic changes in the sequences or expression level of cancer-related genes. It is of particular importance to determine gene expression profiles of cancers on a global scale. SAGE and LongSAGE have been developed for this purpose. METHODS: We performed SAGE in normal liver and HCC samples as well as the liver cancer cell line HepG2. Meanwhile, the same HCC sample was simultaneously analyzed using LongSAGE. Computational analysis was carried out to identify differentially expressed genes between normal liver and HCC which were further validated by real-time quantitative RT-PCR. RESULTS: Approximately 50,000 tags were sequenced for each of the four libraries. Analysis of the technical replicates of HCC indicated that excluding the low abundance tags, the reproducibility of SAGE data is high (R = 0.97). Compared with the gene expression profile of normal liver, 224 genes related to biosynthesis, cell proliferation, signal transduction, cellular metabolism and transport were identified to be differentially expressed in HCC. Overexpression of some transcripts selected from SAGE data was validated by real-time quantitative RT-PCR. Interestingly, sarcoglycan-epsilon (SGCE) and paternally expressed gene (PEG10) which is a pair of close neighboring genes on chromosome 7q21, showed similar enhanced expression patterns in HCC, implicating that a common mechanism of deregulation may be shared by these two genes. CONCLUSION: Our study depicted the expression profile of HCC on a genome-wide scale without the restriction of annotation databases, and provided novel candidate genes that might be related to HCC.

[Proteomic analysis on portal vein tumor thrombus-associated proteins for hepatocellular carcinoma].

OBJECTIVE: A comparative proteomic approach was used to analyze proteins relevant to portal vein tumor thrombus forming. METHODS: proteins extracted from five pairs of matched primary tumor/tumor thrombus samples in the same patient were separated by two-dimensional gel electrophoresis (2-DE). Selected proteins exhibiting statistically significant alternations were identified by mass spectrometry. Western blotting was further performed to examine the expression of the candidate proteins. RESULT: There were 20 significant proteins were identified in total, Among the 20 spots, 12 proteins were up-regulated proteins in primary tumor tissue, including Galectin-1, HMGBI, peroxiredoxin 1, Cyclophilin B, PCNA. whereas 8 were up-regulated proteins in tumor thrombus samples, including Annexin V, Triosephosphate Isomerase. Western blotting Confirmed the difference of Annexin V on protein level. CONCLUSION: There are many proteins associated with the formation of PVTT in HCC. The overexpression of Annexin V may serve as a biomarker for early detection and therapeutic targets to HCC with PVTT.

Gradual upregulation of OCI-5 expression during occurrence and progression of rat hepatocellular carcinoma.

BACKGROUND: OCI-5, the rat homologene of human glypican 3 (GPC3), is confirmed upregulated in hepatocellular carcinoma (HCC). The present study was undertaken to detect gene expression change of OCI-5 during occurrence and progression of rat HCC. METHODS: Male Sprague-Dawley rats were given diethylnitrosamine (DENA) to induce HCC. Three DENA-induced rats and one control rat were sacrificed every week for 18 weeks during the development of HCC. Tissues specimens were snap-frozen in liquid nitrogen and total RNA was isolated. Sk-Hep1 cells were treated with DENA at different concentrations. The gene expression levels of OCI-5 and GPC3 were detected with the RT-PCR method. RESULTS: OCI-5 was not expressed in normal rat liver tissues. When HCC occurred and aggravated, OCI-5 expression was gradually elevated to a very high level. GPC3 was not expressed in the DENA-treated Sk-Hep1 cells. CONCLUSIONS: OCI-5 was not expressed in normal rat liver tissues but in rat HCC tissues. High-expression of OCI-5 in DENA-induced rat HCC model was the gene expression change of HCC not the DENA-induced gene expression. The expression level of OCI-5 was not only elevated in rat HCC but also gradually along the occurrence and progression of HCC, indicating that GPC3 might serve as a sensitive marker of early stage HCC.

Protein-tyrosine phosphatase PCP-2 inhibits beta-catenin signaling and increases E-cadherin-dependent cell adhesion.

beta-Catenin is a key molecule involved in both cell adhesion and Wnt signaling pathway. However, the exact relationship between these two roles has not been clearly elucidated. Tyrosine phosphorylation of beta-catenin was shown to decrease its binding to E-cadherin, leading to decreased cell adhesion and increased beta-catenin signaling. We have previously shown that receptor-like protein-tyrosine phosphatase PCP-2 localizes to the adherens junctions and directly binds and dephosphorylates beta-catenin, suggesting that PCP-2 might regulate the balance between signaling and adhesive beta-catenin. Here we demonstrate that PCP-2 can inhibit both the wild-type and constitutively active forms of beta-catenin in activating target genes such as c-myc. The phosphatase activity of PCP-2 is required for this effect since loss of catalytic activity attenuates its inhibitory effect on beta-catenin activation. Expression of PCP-2 in SW480 colon cancer cells can lead to stabilization of cytosolic pools of beta-catenin perhaps, by virtue of their physical interaction. PCP-2 expression also leads to increased membrane-bound E-cadherin and greater stabilization of adherens junctions by dephosphorylation of beta-catenin, which could further sequester cytosolic beta-catenin and thus inhibit beta-catenin mediated nuclear signaling. Furthermore, SW480 cells stably expressing PCP-2 have a reduced ability to proliferate and migrate. Thus, PCP-2 may play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may be an alternative mechanism for activating beta-catenin signaling.

Upregulation of Glypican-3 expression in hepatocellular carcinoma but downregulation in cholangiocarcinoma indicates its differential diagnosis value in primary liver cancers.

BACKGROUND/AIM: Expression alteration of Glypican-3 (GPC3) is associated with several malignancies and has been identified as an overexpressed gene in hepatocellular carcinoma (HCC). In this study GPC3 expression in intrahepatic chanlangiocarcinoma (ICC), gallbladder cancer and HCC was quantitatively detected. METHODS: Real-time quantitative reverse transcription polymerase chain reaction was used to detect the expression level of GPC3. RESULTS: GPC3 expression was elevated more than two-fold in HCC compared with adjacent tissue in 90 of 100 HCC cases. The average expression level of GPC3 was significantly higher in HCC than that in adjacent liver tissues (P<0.0001). Only in four of 21 ICC cases GPC3 expression was upregulated more than two-fold in tumor tissues. GPC3 expression was downregulated in gallbladder cancer in 12 of 13 cases and the average expression level was significantly lower than that in normal gallbladder tissues (P<0.05). CONCLUSION: The different expression patterns of GPC3 in HCC and ICC suggested that it might play a different role in theses tumors and could serve as a biomarker for differential diagnosis of HCC and ICC.

Proteomic analysis of hepatitis B virus-associated hepatocellular carcinoma: Identification of potential tumor markers.

Hepatocellular carcinoma (HCC) is a malignancy of both underdeveloped and developing countries. Proteomes of ten pairs of clinical hepatitis B virus associated HCC tissue samples were obtained by high resolution two-dimensional gel electrophoresis. Comprehensive analyses of proteins associated with B-type HCC were focused on total differentially expressed proteins (> or = two-fold increase or decrease, Student's t-test, p < 0.05) from one pair of samples. Protein identification was done by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Comparative analyses of proteins associated with B-type HCC included repeat statistics in ten cases. A total of 100 protein spots, corresponding to 80 different gene products, were identified. Proteins whose expression levels were different by more than 2-fold in at least 50% of the cases (five of ten cases) were further analyzed and 45 proteins were selected out as candidates for HCC-associated proteins. Western blotting further validated up-regulated expressions of two candidate proteins in tumor tissues: proliferating cell antigen and stathmin 1. This comprehensive and comparative analyses of proteins associated with B-type HCC could provide useful molecular markers for diagnostics and prognostics and for therapeutic targets. The physiological significance of the differential expressions for several candidate proteins are discussed.

Expression of cytochrome P4502E1 gene in hepatocellular carcinoma.

AIM: To investigate cytochrome P4502E1 (CYP2E1) gene expression in occurrence and progression of hepatocellular carcinoma (HCC). METHODS: The human liver arrayed library was spotted onto the nylon membranes to make cDNA array. Hybridization of cDNA array was performed with labeled probes synthesized from RNA isolated from HCC and adjacent liver tissues. Sprague-Dawley rats were administrated diethylnitrosamine (DENA) to induce HCC. CYP2E1 expression was detected by the method of RT-PCR and Northern blot analysis. RESULTS: CYP2E1 was found by cDNA array hybridization to express differently between HCC and liver tissues. CYP2E1 only expressed in liver, but did not express in HCC tissues and expressed lowly in cirrhotic tissues. In the progression of cirrhosis and HCC, the expression level of CYP2E1 was gradually decreased and hardly detected until the late stage of HCC. CONCLUSION: Using arrayed library to make cDNA arrays is an effective method to find differential expression genes. CYP2E1 is a unique gene expressing in liver but did not express in HCC. CYP2E1 expression descended along with the initiation and progression of HCC, which is noteworthy further investigations in its significance in the development of HCC.

Effects of SIRPalpha1 on liver regeneration after partial hepatectomy in rat.

BACKGROUND: SIRPalpha1 is well known as a negative regulator for cell proliferation through the regulation of the activity of receptor tyrosine kinase with ITAM motif. No investigation to data was undertaken on SIRPalpha1 involving liver regeneration. MATERIALS AND METHODS: Adult male Sprague-Dawley rats underwent approximately 70% partial hepatectomy (PH) or sham operation (SO). Liver specimens were collected at 2, 6, 12, 24, 30, 48, 72, 120, 168, and 240 h after PH or SO. SIRPalpha1 expression was determined in mRNA level by Northern blotting as well as in protein levels via immunohistochemical staining. RESULTS: SO treatment did not induce remarkable changes in SIRPalpha1 expression; however, the level of a 3.9-kb transcript for SIRPalpha1 was significantly up-regulated after PH (versus SO, P < 0.05). SIRPalpha1 mRNA expression in the regenerating liver displayed a biphasic response with its first large peak at as early as 12h followed by a second phase of up-regulation from 48 to 120 h post-PH. SIRPalpha1 mRNA expression returned to its physiological level 168 h later. As seen from immunohistochemistry experiments, SIRPalpha1 protein mainly located in membrane was expressed uniquely in regenerating hepatocytes. Similarly, PH-induced overexpression for SIRPalpha1 protein occurred between 12 and 168 h with a peak level at 24h after surgery. CONCLUSION: SIRPalpha1, a principle negative regulator for cell proliferation, may also play a role in the termination of hepatic proliferation during liver regeneration induced by physiological stress or pathological states, such as PH, drugs, toxins, etc.

Different alterations of cytochrome P450 3A4 isoform and its gene expression in livers of patients with chronic liver diseases.

AIM: To determine whether parenchymal cells or hepatic cytochrome P450 protein was changed in chronic liver diseases, and to compare the difference of CYP3A4 enzyme and its gene expression between patients with hepatic cirrhosis and obstructive jaundice, and to investigate the pharmacologic significance behind this difference. METHODS: Liver samples were obtained from patients undergoing hepatic surgery with hepatic cirrhosis (n=6) and obstructive jaundice (n=6) and hepatic angeioma (controls, n=6). CYP3A4 activity and protein were determined by Nash and western blotting using specific polychonal antibody, respectively. Total hepatic RNA was extracted and CYP3A4cDNA probe was prepared according the method of random primer marking, and difference of cyp3a4 expression was compared among those patients by Northern blotting. RESULTS: Compared to control group, the CYP3A4 activity and protein in liver tissue among patients with cirrhosis were evidently reduced. (P<0.01) Northern blot showed the same change in its mRNA levels. In contrast, the isoenzyme and its gene expression were not changed among patients with obstructive jaundice. CONCLUSION: Hepatic levels of P450s and its CYP3A4 isoform activity were selectively changed in different chronic liver diseases. CYP3A4 isoenzyme and its activity declined among patients with hepatic cirrhosis as expression of cyp3a4 gene was significantly reduced. Liver's ability to eliminate many clinical therapeutic drug substrates would decline consequently. These findings may have practical implications for the use of drugs in patients with cirrhosis and emphasize the need to understand the metabolic fate of therapeutic compounds. Elucidation of the reasons for these different changes in hepatic CYP3A4 may provide insight into more fundamental aspects and mechanisms of imparied liver function.