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Elucidation of the basic genetic changes of human hepatocellular carcinoma is important for the understanding and treatment of this cancer. We used microsatellite polymorphism markers to study 30 cases of hepatocellular carcinoma (34 tumours) on all human chromosomes. DNA from 34 pairs of hepatocellular carcinomas and corresponding non-tumour parts was prepared. Loss of heterozygosity (LOH) and microsatellite instability on 23 chromosomes were investigated by 231 sets of microsatellite markers. More than 20% LOH was shown for loci on 16q (47.1%), 13q (32.4%), 17p (32.4%), 5q (26.5%), 11p (23.5%) and 9p (20.6%). The commonly affected regions were mapped to 16q12.1, 16q12.2, 16q24, 13q12.1–32, 17p13, 5q32, 5q34, 5q3, 11p15, 11q23–24 and 9p21. Hepatitis B virus carriers had a significantly higher frequency of LOH on chromosomes 5q, 11p and 16q. Furthermore, larger tumour size tended to have higher frequency of LOH at D16S409 locus (16q12.1). Microsatellte instability was only found in 12 of 231 markers and the frequency is very low. These data suggest that the chromosomes 16q, 13q, 17p, 5q, 11p and 9p might participate in hepatocarcinogenesis. However, microsatellite instability might play little role in the development of this cancer in Taiwan. © 1999 Cancer Research Campaign

Aims/background Activation of human MAGE genes leads to the expression of a set of tumor rejection antigens, which are recognized by cytotoxic T lymphocytes. The antigens may become the targets of immunotherapy. The expression of MAGE genes was originally found in, but is not restricted, to melanomas. The aim of this study was to investigate the expression of MAGE genes in human hepatocellular carcinomas. Methods The expression of MAGE-1, -2, -3, -4 genes in tumorous and corresponding non-tumorous liver tissue was studied using a reverse-transcription polymerase chain reaction. Results In the 50 hepatocellular carcinomas studied, MAGE-1, -2, -3, -4 mRNA expression was detected in 23 (46%), 17 (34%), 21 (42%) and 8 (16%), respectively. Seventy-four percent of the hepatocellular carcinomas expressed at least one of the MAGE genes. MAGE mRNAs were not detected in the corresponding non-tumor liver tissues. MAGE gene expression was not significantly correlated with clinicopathological factors. Conclusions The MAGE genes are expressed in a high percentage of hepatocellular carcinomas; the MAGE gene products are potential targets for tumor-specific immunotherapy.

Telomerase activity is activated and telomere length altered in various types of cancers, including hepatocellular carcinoma (HCC). A total of 39 HCC tissues and the corresponding non-tumour livers were analysed and correlated with clinical parameters. Telomere length was determined by terminal restriction fragment assay, and telomerase activity was assayed by telomeric repeat amplification protocol. Telomerase activity was positive in 24 of the 39 tumour tissues (1.15–285.13 total product generated (TPG) units) and in six of the 39 non-tumour liver tissues (1.05–1.73 TPG units). In the 28 cases analysed for telomere length, telomere length was shortened in 11 cases, lengthened in six cases, and unaltered in 11 cases compared with non-tumour tissues. Neither telomore length nor telomerase activity was correlated to any clinical parameters.

CDKN2A (p16INK4A/MTS1) and CDKN2B (p15INK4B/MTS2) have recently been shown to be potent inhibitors of the cyclin D/cyclin-dependent kinase-4 complex. Both genes are candidates for the putative tumour suppressor genes located at chromosome 9p21 and are frequently inactivated in many human cancers through homozygous deletion. More recently, another reported pathway of inactivation involves loss of transcription associated with de novo methylation of the 5' CpG island of p16/MTS1 and p15/MTS2 in human cancers. We examined a total of 34 tumours from 30 hepatocellular carcinoma (HCC) patients for deletion, mutation and DNA methylation of these two genes by polymerase chain reaction (PCR) amplification, sequence analysis and Southern blot. Homozygous deletions of P16/MTS1 exon 1 were only identified in 1 of 30 cases (3%). Homozygous deletions of p15 exon 1 or exon 2 were found in 7 of 30 cases (13%). Automated sequencing analysis of p16 exon 1 and 2 and p15 exon 1 and 2 failed to demonstrate mutations in either p16 or p15 in any of these specimens. No aberrant 5' CpG island hypermethylation of p16 or p15 was found in any of the primary tumours by Southern blot. These data suggest that the p16/MTS1 gene has a limited role in HCC. However, deletions of the p15/MTS2 gene are found in 13% HCC and might be involved in a subset of HCC.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and various epithelial cells. Unexpectedly, it has been reported to inhibit the growth of hepatoma cells in vitro. To clarify this phenomenon, we examined the effects of recombinant baculovirus-expressed HGF on the growth of 6 human hepatoma cell lines. The growth of Hep3B and HepG2 cells was markedly stimulated to 1.8- and 1.7-fold, respectively, PLC/PRF/5 to 1.4-fold, and SK-Hep-1 to 1.2-fold in a dose-dependent manner under HGF concentrations below 20 ng/ml. Neither HuH-7 nor HCC36 were affected. None of these cells were inhibited. All these cells expressed c-Met, the membrane receptor for HGF, and their c-Met would be activated to be phosphorylated upon addition of HGF. They also contained the ERK2 subgroup of mitogen-activated protein kinases (MAPKs). When HGF was added, their ERK2 would also be phosphorylated. The extent of ERK2 phosphorylation was partially correlated to their growth response to HGF. In conclusion, HGF could stimulate the growth of certain human hepatoma cells, probably through activation of c-Met and MAPKs.

Human hepatoma cell lines are commonly used as alternatives to primary hepatocytes for the study of drug metabolism in vitro. However, the phase I cytochrome P450 (CYP) enzyme activities in these cell lines occur at a much lower level than their corresponding activities in primary hepatocytes, and thus these cell lines may not accurately predict drug metabolism. In the present study, we selected hepatocyte nuclear factor-1 alpha (HNF1α) from six transcriptional regulators for lentiviral transfection into Hep G2 cells to optimally increase their expression of the CYP3A4 enzyme, which is the major CYP enzyme in the human body. We subsequently found that HNF1α-transfected Hep G2 enhanced the CYP3A4 expression in a time- and dose-dependent manner and the activity was noted to increase with time and peaked 7 days. With a multiplicity of infection (MOI) of 100, CYP3A4 expression increased 19-fold and enzyme activity more than doubled at day 7. With higher MOI (1,000 to 3,000), the activity increased 8- to 10-fold; however, it was noted the higher MOI, the higher cell death rate and lower cell survival. Furthermore, the CYP3A4 activity in the HNF1α-transfected cells could be induced by CYP3A4-specific inducer, rifampicin, and metabolized nifedipine in a dose-dependent manner. With an MOI of 3,000, nifedipine-metabolizing activity was 6-fold of control and as high as 66% of primary hepatocytes. In conclusion, forceful delivery of selected transcriptional regulators into human hepatoma cells might be a valuable method to enhance the CYP activity for a more accurate determination of drug metabolism in vitro.

Bile duct hamartomas (von Meyenburg's complexes) of the liver are usually detected at laparotomy or autopsy as an incidental finding, and usually they are multiple. We report two cases of proved bile duct hamartomas of the liver. The first was in a 65-year-old man whose initial sepsis and many hepatic lesions were interpreted as microabscess of the liver. The second patient was a 39-year-old man, a hepatitis B surface antigen carrier, in whom an incidental hepatic tumor was found. We suggest that liver biopsy be done in hepatic lesions with uncertain clinical features, because the histologic findings may change the treatment plan.

BackgroundBecause of the increased adoption rate of electronic medical record (EMR) systems, more health care records have been increasingly accumulating in clinical data repositories. Therefore, querying the data stored in these repositories is crucial for retrieving the knowledge from such large volumes of clinical data. ObjectiveThe aim of this study is to develop a Web-based approach for enriching the capabilities of the data-querying system along the three following considerations: (1) the interface design used for query formulation, (2) the representation of query results, and (3) the models used for formulating query criteria. MethodsThe Guideline Interchange Format version 3.5 (GLIF3.5), an ontology-driven clinical guideline representation language, was used for formulating the query tasks based on the GLIF3.5 flowchart in the Protégé environment. The flowchart-based data-querying model (FBDQM) query execution engine was developed and implemented for executing queries and presenting the results through a visual and graphical interface. To examine a broad variety of patient data, the clinical data generator was implemented to automatically generate the clinical data in the repository, and the generated data, thereby, were employed to evaluate the system. The accuracy and time performance of the system for three medical query tasks relevant to liver cancer were evaluated based on the clinical data generator in the experiments with varying numbers of patients. ResultsIn this study, a prototype system was developed to test the feasibility of applying a methodology for building a query execution engine using FBDQMs by formulating query tasks using the existing GLIF. The FBDQM-based query execution engine was used to successfully retrieve the clinical data based on the query tasks formatted using the GLIF3.5 in the experiments with varying numbers of patients. The accuracy of the three queries (ie, “degree of liver damage,” “degree of liver damage when applying a mutually exclusive setting,” and “treatments for liver cancer”) was 100% for all four experiments (10 patients, 100 patients, 1000 patients, and 10,000 patients). Among the three measured query phases, (1) structured query language operations, (2) criteria verification, and (3) other, the first two had the longest execution time. ConclusionsThe ontology-driven FBDQM-based approach enriched the capabilities of the data-querying system. The adoption of the GLIF3.5 increased the potential for interoperability, shareability, and reusability of the query tasks.

To provide an efficient way for tracking patients' condition over long periods of time and to facilitate the collection of clinical data from different types of narrative reports, it is critical to develop an efficient method for smoothly analyzing the clinical data accumulated in narrative reports.To facilitate liver cancer clinical research, a method was developed for extracting clinical factors from various types of narrative clinical reports, including ultrasound reports, radiology reports, pathology reports, operation notes, admission notes, and discharge summaries. An information extraction (IE) module was developed for tracking disease progression in liver cancer patients over time, and a rule-based classifier was developed for answering whether patients met the clinical research eligibility criteria. The classifier provided the answers and direct/indirect evidence (evidence sentences) for the clinical questions. To evaluate the implemented IE module and the classifier, the gold-standard annotations and answers were developed manually, and the results of the implemented system were compared with the gold standard.The IE model achieved an F-score from 92.40% to 99.59%, and the classifier achieved accuracy from 96.15% to 100%.The application was successfully applied to the various types of narrative clinical reports. It might be applied to the key extraction for other types of cancer patients.

To investigate the carcinogenic effect of environmental aflatoxin exposure, 56 cases of hepatocellular carcinoma (HCC) diagnosed between 1991 and 1995 were identified and individually matched by age, sex, residence and date of recruitment to 220 healthy controls from the same large cohort in Taiwan. Blood samples were analyzed for hepatitis B and C viral markers and for aflatoxin-albumin adducts; urine was tested for aflatoxin metabolites. We obtained information about sociodemographic characteristics, habitual alcohol drinking, cigarette smoking and diet in a structured interview. Hepatitis B virus surface antigen (HBsAg) carriers had a significantly increased risk for HCC. After adjustment for HBsAg serostatus, the matched odds ratio (ORm) was significantly elevated for subjects with high levels of urinary aflatoxin metabolites. When stratified into tertiles, a dose-response relationship with HCC was observed. The ORm for detectable aflatoxin-albumin adducts was not significant after adjustment for HBsAg serostatus. HBsAg-seropositive subjects with high aflatoxin exposure had a higher risk than subjects with high aflatoxin exposure only or HBsAg seropositivity only. In male HBsAg-seropositive subjects, adjusted ORs were 2.8 (95% confidence interval [CI] = 0.9-9.1) for detectable compared with non-detectable aflatoxin-albumin adducts and 5.5 (CI = 1.3-23.4) for high compared with low urinary aflatoxin metabolite levels. Our results suggest that environmental aflatoxin exposure may enhance the hepatic carcinogenic potential of hepatitis B virus. A large-scale study will be needed to evaluate the effect of aflatoxin exposure on HBsAg non-carriers.

Hepatocyte growth factor (HGF) is a cytokine with pleiotropic effects on many different cell types. Its biological activities depend on the disulfide-linked alpha beta heterodimeric molecule. To study the functions of the beta-chain of HGF, insect cell-expressed HGFs prepared from nested deletions from C-terminus of beta-chain were studied for their biological activities and ligand-binding functions. The results demonstrated that the functions of HGF are dependent on the intactness of its beta-chain. The loss of HGF activities is correlated with the progressive deletion of beta-chain. There is not a single critical domain in the beta-chain that is essential for the functions of HGF. We propose that beta-chain may act to stabilize the ligand-receptor binding or contribute to the proper conformation of HGF to interact with its receptor.

A reliable, reproducible, and convenient in vitro platform for drug metabolism determination and toxicity prediction is of tremendous value but still lacking. In the present study, a collection of 24 hepatic transcription factors and nuclear receptors in different combinations were surveyed, and 10 among them were finally selected to induce the expression and enzyme activities of cytochrome P450 (CYP) 3A4, 1B1, and 2C9 in human dermal fibroblasts (HDFs). The expression and activities of these CYPs in the induced HDFs were higher than those in commonly used hepatoma cell lines. High CYP expression and activities could be further enhanced by culturing the induced HDFs either as spheroids or into several kinds of scaffolds, particularly the tri-copolymer scaffold composed of gelatin, chondroitin and hyaluronan. More strikingly, there showed a synergistic effect of seeding and culturing the spheroids into the tri-copolymer scaffold. Scanning electron microscopy and confocal microscopy disclosed well accommodation of these spheroids inside the scaffolds and displayed a high survival rate. Moreover, the spheroid/scaffold constructs could metabolize an anti-hypertension drug nifedipine into oxidized nifedipine, showing their applicability in studying drug metabolism. This study presents a strategy to induce the expression and enzyme activities of critical CYPs in HDFs, and may have potential to establish an in vitro platform to study drug metabolism and to predict the possible human risk of drug toxicity.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths in Taiwan. HCC with duodenal involvement are rare and are associated with a poor prognosis. The purpose of this retrospective study was to collect clinical information and data regarding survival following various treatments.Between 1996 and 2009, 21 cases (17 men) were diagnosed with HCC and duodenal invasion and metastases by diagnostic imaging, endoscopy with biopsy, or surgically collected specimens sent to pathology. The clinical course was analyzed from the patients' medical records.Gastrointestinal bleeding was reported in 18/21 patients. Diagnostic imaging showed that the majority of cases involved direct tumor invasion (predominantly from the right liver lobe) and six cases from metastasis. Tumor mass and ulcerations were the most common features noted on endoscopy. In addition to the component therapy and medication treatment, panendoscopic hemostasis, surgery, transcatheter arterial embolization, and radiotherapy were performed for the management of duodenal involvement and gastrointestinal bleeding. Survival duration after duodenal involvement ranged from 0.2 to 57.8 months (mean 10.5 months).Gastrointestinal bleeding in advanced HCC should raise suspicions of duodenal involvement. HCC can involve the duodenum by direct invasion (from either the left or right liver lobes) or metastasis. The prognosis for HCC patients with duodenal involvement is poor, but is improved by supportive care and application of various treatment modalities.

A 66-year-old woman with hepatitis C related liver cirrhosis presented to our hospital for dyspnoea and cyanosis. Tachypnoea, low grade fever, clubbed fingers, palmar erythaema, spider angioma, wheezing and severe hypoxaemia were found. Chronic obstructive pulmonary disease (COPD) with acute exacerbation was diagnosed and she recovered after bronchodilator and antibiotic treatment. However, dyspnoea and hypoxaemia recurred with widened alveolar–arterial gradient, which was unusual in COPD. A pulmonary function test showed moderate obstructive ventilatory defect and chest high resolution CT scan disclosed some dilated vessels over the left lower lung. Interestingly, platypnoea and orthodeoxia were observed, therefore hepatopulmonary syndrome was suspected and was confirmed by contrast echocardiography, lung perfusion scan and 100% oxygen administration. Her dyspnoea improved gradually after oxygen use. She was followed in our clinic for 3 years and her respiratory condition on home oxygen remained stable, as did the liver cirrhosis.

Background: The clonality of hepatocellular carcinoma is still controversial. To evaluate the clonality of this cancer, mostly small multiple hepatocellular carcinomas of 18 patients were analyzed by DNA finger-printing. Methods: DNA from the liver tissues was digested with Hinfl. After gel electrophoresis, in gel-hybridization with a synthetic oligonucleotide probe GTGGTGGTGGTGGTG was performed. The band patterns were compared among the tumors and the nontumor part. In patients positive for hepatitis B surface antigen (HBsAg), the Integration pattern of hepatitis B viral DNA in liver tissues was also analyzed. Results: Seven of the 9 HBsAg-positive patients had tumors with different clonalitles by both methods. In the remaining 9 HBsAg-negative patients, 4 had different band patterns in their tumors by DNA fingerprinting. Conclusions: Multiple hepatocellular carcinomas frequently have different clonalitles; this finding reiterates the importance of eliminating the underlying cause and the contributing factors of hepatocarcinogenesis.

The prevalence of antibodies to hepatitis C virus (anti-HCV) in a hepatitis B virus hyperendemic region was evaluated with a second-generation enzyme immunoassay. A general population of 1500 from Northern Taiwan was tested with this immunoassay which includes synthetic peptides from the capsid and non-structural protein region as the solid-phase antigen. A total of 37 subjects out of 1500 (2.5%) were positive, with no significant difference in distribution between hepatitis B antigenemic and non-antigenemic samples. The prevalence of anti-HCV was higher in subjects with abnormal than those with normal liver function tests (8.2% vs. 1.5%). Prevalence increased with age. By polymerase chain reaction, 43% of anti-HCV-positive samples were HCV-RNA-positive. However, only 1 (0.6%) of 150 anti-HCV-negative subjects had HCV-RNA. Comparison of the results with those from a conventional recombinant C100-3-based assay showed that the capsid/non-structural region synthetic antigen system provided more accurate sensitivity and specificity. The more sensitive assay revealed a high prevalence for HCV infection of 2.5% among this general population in Taiwan.

Conventionally, the diagnosis of hepatocellular carcinoma (HCC) is performed by qualitative examination of histopathological specimens, which takes times for sample preparation in fixation, section and stain. Our objective is to demonstrate an effective and efficient approach to apply multiphoton microscopy imaging the HCC specimens, with the advantages of being optical section, label-free, subcellular resolution, minimal invasiveness, and the acquisition of quantitative information at the same time. The imaging modality of multiphoton autofluorescence (MAF) was used for the qualitative imaging and quantitative analysis of HCC of different grades under ex-vivo, label-free conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, and obtained quantitative parameters in characterizing the disease. Our results demonstrates the capability of using tissue quantitative parameters of multiphoton autofluorescence (MAF), the nuclear number density (NND), and nuclear-cytoplasmic ratio (NCR) for tumor discrimination and that this technology has the potential in clinical diagnosis of HCC and the in-vivo investigation of liver tumor development in animal models.

An adult rat liver progenitor cell line Lig-8 was established. In the induction by sodium butyrate, these cells were shown to be able to differentiate into both hepatocytes and bile duct cells expressing albumin and cytokeratin-19, the markers of respective cell types. In order to generate Lig-8 specific antibody for further studies, we produced a monoclonal antibody using the whole Lig-8 cells as immunogen. The yielded monoclonal antibody, named Ligab, belongs to IgG subclass G1 and kappa light chain. It specifically stained on Lig-8 cells in the cytoplasm but not on a rat hepatoma cell line H4IIE. Its immunoreaction against Lig-8 cell lysate on dot blots diminished as the concentration of sodium dodecyl sulfate (SDS) in the lysate increased to 2%, a level in the sample buffer of standard SDS-polyacrylamide gel electrophoresis (PAGE). Not surprisingly, Ligab failed to detect its reacting antigen in Lig-8 cell lysate by standard SDS-PAGE-based immunoblotting. It could detect this antigen only by native PAGE-based immunoblotting. These characteristics suggested that the antigenic epitope for Ligab is likely a molecular structure instead of a peptide sequence. More interestingly, expression of Ligab-reacting antigen in Lig-8 cells declined as the cells were induced to differentiate by sodium butyrate. This antigen is very likely a differentiation-related marker for these cells, and this monoclonal antibody may help study the molecular mechanisms of liver progenitor cell differentiation.

Background and Aim: Antemortem diagnosis of hepatocellular carcinoma (HCC) with cardiac metastasis is uncommon. To clarify the clinical manifestation and survival of HCC patients with cardiac metastases, we initiated the present study. Methods: We retrospectively analyzed 48 HCC patients with metastases into cardiac cavity diagnosed antemortem. The baseline clinical characteristics, echocardiogram, treatment modality and the outcome data were collected. Results: The most common symptoms of cardiac metastasis included asymptomatic in 19 cases (39.5%), bilateral lower leg edema in 18 cases (37.5%) and exertional dyspnea in 15 cases (31.3%). The median and mean survival times from the time of diagnosis of cardiac metastasis were 102 days and 161 days, respectively. Compared with another cohort of 48 patients with age-, gender-, and stage-matched HCC patients without cardiac metastasis, the median survival in the cardiac metastasis group was similar to the control group (68 days) (P = 0.67). The cause of death was HCC in 29, hepatic failure in seven, multiple organ failure in four, gastrointestinal bleeding in three, sepsis in two, pulmonary embolism in one, respiratory failure in one, and acute myocardial infarction in one. Conclusions: Hepatocellular carcinoma patients with cardiac metastases were in the advanced stages. These patients had limited survival from the diagnosis of cardiac metastases. The most common cause of death was related to HCC per se or the underlying liver disease. Only a few patients expired because of cardiac metastases.

Liver progenitors, so-called oval cells, proliferate remarkably from periportal areas after severe liver injury when hepatocyte regeneration is compromised. These cells invade far into the liver parenchyma. Molecular mechanisms underlying these behaviors of oval cells remain poorly understood. In this study, we treated rats with 2-acetylaminofluorene/carbon tetrachloride to induce hepatic oval cells. By expression microarray analysis, we investigated global gene expression profiles in liver tissue, with an emphasis on adhesion molecules, extracellular matrix proteins, matrix metalloproteinases (MMPs), growth factors/cytokines, and receptors that might contribute to the distinct behaviors of oval cells. Genes upregulated at least twofold were selected. We then performed immunostaining to verify the microarray results and identified expression of MMP-7 and CD44 in oval cells. Staining of cytokeratin (CK)-19, an oval-cell marker, was similar between oval cells located next to periportal areas and those located far within the parenchyma. In contrast, CD44 staining was more intense in the parenchyma than in periportal areas, suggesting a role of CD44 in oval-cell invasion. Moreover, newly differentiated CK-19+ hepatocytes within foci did not show CD44 staining, suggesting that CD44 is related to the undifferentiated oval-cell phenotype. We then investigated oval-cell reactivity in CD44-deficient mice fed an oval cell-inducing diet of 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Results showed significantly reduced oval-cell reactivity in CD44-deficient mice. Thus, oval cells express MMP-7 and CD44, and CD44 appears to play critical roles in the proliferation, invasion, and differentiation of hepatic oval cells in rodents.

The aim of this study was to compare six prognostic staging systems (Okuda stage, TNM stage, CLIP score, BCLC stage, JIS score and Tokyo score) in predicting survival in patients with hepatocellular carcinoma (HCC). A total of 2010 Taiwanese HCC patients were included. Demographic, laboratory and tumour characteristics were determined at diagnosis. Predictors of survival included serum levels of albumin, total bilirubin, alkaline phosphatase, alpha-fetoprotein, ascites, tumour size and portal vein invasion. The Tokyo score was the most informative one for predicting the survival of HCC patients as a whole, receiving surgical resection, or receiving transarterial chemoembolisation. CLIP score was the best fit system for HCC patients receiving chemotherapy or supportive care. Each staging system showed a significant difference in predicting the probability of survival across different stages. The applicability of staging systems for patients with HCC was dependent on treatment methods.

Hepatitis B virus (HBV) DNA was assayed using the polymerase chain reaction in serum samples of 116 hepatitis B surface antigen (HBsAg) carriers, including 30 positive for hepatitis B e antigen (HBeAg) and 86 negative for HBeAg. In the HBeAg-positive group, all were positive for HBV DNA. In the HBeAg-negative group, 80.2% were positive for HBV DNA (80.0% in the healthy carrier group, 90.0% in the chronic active liver disease group, and 69.2% in patients with cirrhosis). This study indicated that every HBeAg-positive carrier as well as the majority of HBeAg-negative carriers were infectious and, in the latter group, that viral replication is most active in patients with chronic active liver disease.

Tumor may induce local immunosuppression and make the tumor-infiltrating lymphocytes (TILs) functionally inhibited and lose the antitumor effects. Therefore, we did the phenotypic analysis of TILs in hepatocellular carcinoma (HCC) tissues.Lymphocytes were isolated from paired HCC tissues (TILs) and the corresponding non-tumor liver tissues (non-tumor-liver infiltrating lymphocytes, NILs) of 28 patients and were subjected to flow cytometric analysis.Compared with the non-tumor portion, HCC tissues had less intensity of lymphocyte infiltration. TILs had higher CD3+/CD56+ ratio than NILs. Around 70%-90% NILs or TILs expressed the antigen-experienced or memory phenotypes. Around 60%-70% CD4+ or CD8+ NILs and TILs expressed the activation markers CD69 and HLA-DR. However, we found that CD25 is under-expressed in both the CD8+ NILs and TILs. In addition, more percentage of CD4+ CD25+ T cells was detected in the TILs than in the NILs.HCC tissues had less lymphocytes infiltration. Most of infiltrating lymphocytes from the HCC tissues and the corresponding non-tumor liver tissues were activated and expressed antigen-experienced phenotypes. However, the CD25 was underexpressed in the CD8+ TILs and the CD4+ CD25+ T cells were increased in the TILs. These factors might impair the antitumor immunity in HCCs.

Gene inactivation through DNA hypermethylation plays a pivotal role in carcinogenesis. This study aimed to profile aberrant DNA methylation in different stages of liver disease, namely noncirrhosis, cirrhosis and hepatocellular carcinoma (HCC), and also to clarify the influence of hepatitis B virus (HBV) infection on the aberrant DNA methylation in HCCs. Promoter methylation in p14(ARF), p16(INK4a), O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione S-transferase pi (GSTP1) and E-cadherin (E-Cad) genes of 58 HCCs paired with adjacent nontumorous tissues was assayed by methylation-specific PCR. HBV infection was determined using a hepatitis B virus surface antigen (HBsAg) serological assay. The frequency of p16(INK4a) promoter methylation increased from noncirrhotic, cirrhotic, to HCC tissues (noncirrhotic vs. HCC, p < 0.001), while that of GSTP1 promoter methylation increased in cirrhotic tissues compared to noncirrhotic ones (p = 0.029). The frequency of GSTP1 promoter hypermethylation is significantly higher in HCC than in nontumorous tissues (p = 0.022) from HBsAg-positive patients, but not the HBsAg-negative controls (p = 0.289). While the frequency of E-Cad promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg-positive patients (p = 0.438), it was lower in HCCs than in nontumorous tissues from HBsAg-negative patients (p = 0.002). In contrast, the frequency of p16(INK4a), MGMT and p14(ARF) promoter hypermethylation in HCCs was unrelated to HBsAg status. In conclusion, aberrant DNA methylation may begin at different stages of liver disease in a gene-dependent manner. Moreover, HBV infection may enhance or maintain GSTP1 and E-Cad promoter methylation and thereby affect hepatocarcinogenesis.

It is not rare to find hepatic hemangiomas in people in Taiwan where hepatitis B is endemic during ultrasonographic examinations for the screening of hepatocellular carcinoma, and we can therefore monitor these hemangiomas longitudinally since detected.Totally 180 hepatic hemangiomas in 130 adult patients were followed up for more than 5 years with at least twice ultrasound examinations in our hospital. The diagnosis of hemangiomas was made by typical ultrasonographic findings as well as compatible findings in other imaging modalities. The largest diameter of the same tumor at various examinations was compared in each patient. The doubling time of tumor volume was calculated for the enlarging hepatic hemangiomas.The diameters of the 180 hemangiomas ranged from8 cm -4 cm in 11 (6.1%),or =4 cm -2 cm in 55 (30.6%), andor =2 cm in 114 (63.3%). Among them, the diameter of 14 (7.7%) hemangiomas in 13 patients increased. The tumor volume doubling time of these hemangiomas ranged from 17.3 to 178.1 months.Although hepatic hemangiomas are benign, some of them indeed grow slowly. The growth rates of these hemangiomas varied and were definitely slower as compared with those of malignant hepatic tumors.

Intravital imaging of hepatobiliary excretion is vital for elucidating liver metabolism. In this work, we describe a novel method to observe the intravital dynamics of the uptake, processing, and excretion of an organic anion, 6-carboxyfluorescein diacetate (6-CFDA) in the hepatobiliary system. This is achieved by the use of multiphoton microscopy and an intravital hepatic imaging chamber. The high-quality images show sequential uptake and processing of 6-CFDA from the hepatocytes and the subsequent excretion into bile canaliculi within approximately 50 min. This is a promising technique to study intravital hepatic physiology and metabolism.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Metastasis, the spread process of cancer cells from a primary tumor to seed secondary tumors in distant sites, is one of the big challenges in cancer treatment today. For most patients, when the cancer is detected, metastasis has already occurred. The growing evidences implied that miRNAs play an important role in developmental lineage and different states of cancers as well as cancer invasion. In this study, we would like to establish a metastasis-based prognostic miRNA signature. First, 16 lung adenocarcinoma cells were divided into the high or low-invasive group. The miRNAs whose expression correlated with invasion were identified. Next, we investigated whether the expressions of identified miRNAs are correlated to patients’ survival. Quantitative RT-PCR results showed that the expression of miR-10a* is significantly correlated to the five-year survival in 98 lung adenocarcinoma patients. The inhibition of miR-10a* attenuated the invasion abilities of the highly invasive lung cancer cells. Moreover, we investigated the underlying regulation of miR-10a*. We found that miR-10a* negatively regulated Histone deacetylase 5 (HDAC5) was assayed by Western Blot, RT-PCR and luciferase reporter assay. These evidences suggested that HDAC5 is a direct target of miR-10a*. Besides, inhibition of HDAC5 promoted invasive ability. RNAs derived from two paired groups (pre-miR-10a* and pre-NC; sh-HDAC5 and sh-LacZ) were assayed by microarray and pathway analysis. The analysis result of MetaCore implied that miR-10a* trigger TGF-beta signalings. RT-PCR and Western Blot were utilized to validate the differentially altered expression of certain genes in miR-10a*-mediated pathways. The rescue effect of HDAC5 expression on miR-10a*-mediated phenotypic changes is also evaluated to elucidate the importance of HDAC5 in miR-10a*-mediated invasive ability. Our findings indicated that miR-10a* may play an oncogenic miRNA role in lung cancer. Citation Format: Pin-Yen Hsu, Bing-Ching Ho, Chiou-Ling Cheng, Hsuan-Yu Chen, Sung-Liang Yu, Guan-Tarn Huang. miRNA-10a* enhances invasiveness via suppression of HDAC5 in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3075. doi:10.1158/1538-7445.AM2015-3075

The purpose of this study was to compare the clinical features and survival in patients with hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) and hepatitis C virus-related hepatocellular carcinoma (HCV-HCC). A total of 2820 HCC patients were included. The mean age of HBV-HCC patients was 52.5+/-13.4 years, while it was 64.8+/-8.7 years in HCV-HCC patients. The male/female ratio was 7.8 in HBV-HCC, while it was 1.7 in HCV-HCC. The mean alpha-fetoprotein level in HBV-HCC was 11,661+/-22,805 ng/mL, while it was 5079+/-15,005 ng/mL in HCV-HCC. The mean tumour size was 6.4+/-4.1 cm in HBV-HCC, while it was 4.6+/-3.1 cm in HCV-HCC. The median survival was 11.1 months in HBV-HCC, while it was 23.9 months in HCV-HCC. Compared with HBV-HCC patients, HCV-HCC patients were older, had a lower male/female ratio, lower white blood cell count, lower serum albumin level, higher serum ALT level, lower serum alpha-fetoprotein level, smaller tumour size and survived longer.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death throughout the world. Although hepatitis B or C viral infections are main risk factors for HCC, the molecular mechanisms leading to HCC formation have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. In this study, we employed two-dimensional difference gel electrophoresis (2D-DIGE) combined with nano flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) to investigate differentially expressed proteins in HCC. For each of eight HCC patients, Cy3-labeled proteins isolated from tumor tissue were combined with Cy5-labeled proteins isolated from the surrounding nontumor tissue and separated by 2D gel electrophoresis along with a Cy2-labeled mixture of all tumor and nontumor samples as an internal standard. Thirty-four protein spots corresponding to 30 different proteins were identified by nanoLC-MS/MS as showing significant change (paired t-test, p0.05) in the level of expression between tumor and nontumor tissues. Sixteen proteins were up-regulated and 14 were down-regulated in HCC; they seem to play important roles in a variety of pathways including glycolysis, fatty acid transport and trafficking, amino acid metabolism, iron and xenobiotic metabolism, ethanol metabolism, cell cycle regulation, cytoskeleton, and stress. A remarkable finding is the up-regulation of 14-3-3gamma protein in HCC. 14-3-3 isoforms had been linked to carcinogenesis because they are involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. In conclusion, 2D-DIGE is an efficient strategy that enables us to identify differentially expressed proteins in HCC. Identification of potential biomarkers, such as the pinpointing of 14-3-3gamma in our findings, may provide further useful insights into the pathogenesis of HCC.

Hepatocellular carcinoma (HCC) is prevalent in Southeast Asia and sub-Saharan Africa.1 With the widespread use of alpha fetoprotein (AFP), ultrasound, and other diagnostic modalities as screening tools, many small HCC tumors are detected before symptoms develop and are treated effectively.2,3 Surgical resection is considered the standard treatment of small HCCs, and results have been promising.4 Initially, percutaneous ethanol injection therapy (PEIT) was considered an alternative treatment of patients with small HCC and poor liver reserve for whom resection posed the risk of liver failure. However, several studies have shown similar or even better results with PEIT than with surgical resection.5–7 In Taiwan, HCC is the leading cause of cancer mortality. Many small HCCs are detected by surveillance, and routine surveillance of at-risk groups has been common in daily practice for at least a decade.2,3 Resection and PEIT are both widely used for the treatment of small HCCs,4,8 and the Taiwan National Health Insurance covers both treatments. Because the existing literature does not contain objective comparisons of these 2 treatment modalities9 and a prospective randomized comparative trial has been suggested repeatedly,9–11 we performed a randomized study comparing surgicalresection with PEIT for the treatment of small HCC.

Needle liver biopsy is essential for the diagnosis of liver fibrosis and cirrhosis. However, sampling variability has been reported by semiquantitative methods. The aim of the present study was to investigate quantitatively the sampling variability of needle liver biopsy on the assessment of liver fibrosis/cirrhosis.Liver samples of 12 patients undergoing autopsy, liver transplantation, or liver tumor resection were investigated. Nine to 10 liver biopsies at least 5 mm apart were taken from each of 12 removed human livers using Trucut needles. Hydroxyproline level of each liver core was determined. Sections1 cm(2) and stained by Sirius red were used for the reference staging of liver fibrosis in each patient according to the METAVIR scoring system.Hydroxyproline levels for the three groups, F0-1, F2-3, and F4, were significantly elevated as the fibrosis score increased. There was a wide range of hydroxyproline contents for the 9-10 biopsy samples from each liver, the coefficient of variation being between 13% and 36% (mean, 23%). Several differences were noted between the amount of collagen present according to hydroxyproline measurement and the stages of fibrosis according to histology.Although increases in collagen content (by hydroxyproline) approximately paralleled increases in fibrosis stage (by histology), there was considerable overlap and some discrepancies. Much work remains to be done to clarify the significance of the apparent huge variations in fibrosis within diseased livers. It is advisable for clinicians to interpret staging diagnoses by histological staging with some caution.

Background: An accurate staging system is required to assess hepatocellular carcinoma (HCC) patients in order to benefit from hepatic resection before surgery. Cancer of the Liver Italian Program (CLIP) score was considered to be better than the Okuda staging system to predict survival. Japan Integrated Staging Score (JIS score) includes tumor, nodes, metastases (TNM) stage and Child–Pugh grade as a new staging system for HCC. The purpose of the present paper was to compare the CLIP, Okuda, TNM and JIS staging systems for HCC patients undergoing surgery. Methods: From 1991 to 1995, 599 patients undergoing hepatic resection for HCC from four medical centers in Taiwan were evaluated. All patients were classified by Okuda, CLIP, TNM and JIS systems. Factors associated survivals were analyzed. Results: There was no statistical difference in survival between CLIP 0 and 1 patients, or among CLIP 2–4 patients. The prognostic validation of the Okuda and CLIP scoring systems in discriminating survival in HCC patients undergoing surgery was not satisfied. The TNM system was successful in predicting survival for HCC patients undergoing surgery. The JIS score could also differentiate survivals for those patients except for JIS 3. By multivariate analysis, age ≥60 years old, serum albumin 5 cm and TNM stage were associated with survival. Conclusion: Both the Okuda and CLIP systems are not superior to TNM staging for HCC patients who undergo surgical resection. Whether JIS score is feasible for those patients with advanced HCC needs further evaluation.

Multiphoton microscopy is to a powerful imaging modality in the life sciences. The improved image contrast, greater imaging depths, and focus limited photodamage enable multiphoton microscopy to be especially useful for in vivo bioimaging applications. For multiphoton microscopy to be widely applicable in for in vivo biopsy applications, characterization of the tissues of interest must first be performed ex vivo. This presentation is an overview of the microscopic and spectroscopic characterization of the tissue types being pursued by my group. Both multiphoton images and spectroscopic information of a diverse range of normal, damaged, or diseased tissues will be presented and the implications of our results discussed.

In this work, we apply multiphoton microscopy in diagnosing toxin (CCl/sub 4/) induced liver fibrosis in mice. We found that multiphoton induced spectroscopic signatures may be used to identify stellate cell proliferation and necrotic sites. Our study suggests that multiphoton imaging may be developed into an effective diagnosis tool for liver fibrosis in vivo.

Diseases associated with the liver, a major internal organ, can lead to serious health problems. In this work, we present multiphoton images of normal and diseased liver specimens and we will characterize the changes to pathological liver specimens. In particular, we will focus on the physiological changes associated with liver fibrosis. Our results show that multiphoton microscopy is a useful technique for distinguishing normal and diseased liver tissues and that it has potential applications for in vivo diagnosis of liver diseases.

Summary Background: Transcatheter arterial embolization is a major palliative treatment for unresectable hepatocellular carcinoma, but the survival benefit of transcatheter arterial embolization is controversial. Aim: To evaluate the role of transcatheter arterial embolization in different stage of unresectable hepatocellular carcinoma and to select patients who can get the best benefit from the treatment. Methods: From 1991 to 1995, 476 patients who had unresectable hepatocellular carcinoma from four medical centres in Taiwan were enrolled. Among them, 425 underwent transcatheter arterial embolization, and 51 received supportive treatment alone. The survivals between the two groups were compared. Results: Among the 476 patients, transcatheter arterial embolization can significantly prolong survival. The 1-, 2-, and 5-year survival rates for patients who underwent transcatheter arterial embolization were 60.2%, 39.3%, and 11.5%; and the rates for patients who underwent supportive treatment were 37.3%, 17.6%, and 2%, respectively (P = 0.0002). The survival benefit of transcatheter arterial embolization was observed in patients between Cancer and the Liver Italian Program 0 and Cancer and the Liver Italian Program 4. In multivariate analysis, transcatheter arterial embolization, tumour size

Percutaneous microwave coagulation therapy (PMCT) can effectively treat hepatocellular carcinomas (HCCs) smaller than 2 cm. However, for tumors 2 to 3 cm in size, combination of transarterial chemoembolization (TACE) or multiple insertions of electrodes may be more effective. This study investigated the treatment efficacy of PMCT for tumors 2 to 3 cm in size.Nineteen HCCs smaller than 3 cm in diameter (2 cm in 11, and 2-3 cm in 8) in 18 patients (including 14 previously treated patients) were treated by PMCT under ultrasound guidance. One or 2 PMCT electrodes were consecutively inserted either into the left and right portion, or into the distal and proximal portion of the tumor, according to the size, shape, and margin of tumors and puncture direction. Liver function tests and contrast-enhanced computed tomography were used to examine preoperative status and response to PMCT.After an average of 1.6 emissions of PMCT, 18 tumors (95%) were completely ablated. The only case of treatment failure was due to a tumor location which made the approach of the electrode difficult. Bacteremia developed after the procedure in 1 patient (5%) and local inflammatory reaction of the puncture wound in another (5%). During follow-up ranging from 5 to 19 months, no recurrence was noted at the site of the original tumors. Tumor recurrence was detected at another site 2-9 months after PMCT in 9 of the 14 previously treated patients.PMCT can effectively and safely treat HCCs smaller than 3 cm in size without combination of TACE or multiple insertions of electrodes.

Purpose: The role of transcatheter arterial embolization (TAE) for patients with resectable hepatocellular carcinoma (HCC) is controversial. Analyzing a cohort of nation-wide data can delineate the beneficial effect of TAE for those patients. Methods: From 1991 to 1995, 818 patients who had potentially resectable HCC from four medical centers in Taiwan were enrolled. Among them, 599 underwent curative resection, 157 received TAE and 62 received supportive treatment alone. The survivals among the three groups were compared. Results: The 5-year survival rates for patients who underwent surgery, TAE and supportive treatment were 43.6%, 25.6% and 3.7%, respectively. Surgery offered the best survival for those patients. TAE could also prolong survival as compared with supportive treatment (P=0.0001). However, among patients who were in advanced tumor stage (Cancer and the Liver Italian Program (CLIP) score ≥2), no statistical difference in survival was noted between patients who underwent TAE or supportive treatment. In multivariate analysis, single tumor, serum albumin ≥3.5 g/dl, tumor size less than 5 cm, early-stage tumor (CLIP score=0–1) and aggressive treatment including surgery and TAE were independent factors associated with a better survival. Conclusions: Surgery is superior to TAE for patients with resectable HCC. In patients who refuse surgery, TAE can be considered for selected patients whose tumors are in early stage.

Due to modern diagnostic imaging and the sensitive alpha-fetoprotein test, small hepatocellular carcinoma can now be detected at an early stage. Studies have shown that surgical resection of the tumors is a valuable treatment. Local treatment under ultrasound guidance was initially considered as an alternative when patients' liver reserves were not good enough for surgical resection; however, this technique has been improved and the results indicate that its survival rate can compete with that of surgical resection. In follow-up studies of patients with small hepatocellular carcinoma, a 5-year survival of 60% has been achieved after percutaneous ethanol injection therapy. Percutaneous microwave coagulation therapy and percutaneous radiofrequency ablation therapy have been shown to have some advantages over percutaneous ethanol injection therapy, although the follow-up durations of these studies were not long enough. Percutaneous ethanol injection therapy, percutaneous microwave coagulation therapy and percutaneous radiofrequency ablation therapy have become the 3 most widely used techniques for the treatment of hepatocellular carcinomas that are less than 5 cm in diameter and have a tumor number less than 3. In general, a tumor size of 3 to 5 cm is a good candidate for radiofrequency ablation and a tumor size of 2 to 3 cm is suitable for radiofrequency ablation or microwave coagulation. If the tumor size is around 2 cm or less, microwave coagulation or ethanol injection is often chosen due to the relatively low cost and similar efficacy. Ethanol injection also has the advantage of needing only a fine needle for injection. Informed selection of the appropriate technique, or combining a technique with transcatheter hepatic arterial embolization according to the tumor size and number, might provide the most effective treatment and achieve better results for hepatocellular carcinoma, even if the liver reserve is not good. However, large-scale, randomized, controlled trials are required before a definitive conclusion can be reached.

Although 2-dimensional (2D) ultrasonography (US) with conventional color and power Doppler US has been applied widely in the differential diagnosis of hepatic tumors and in the follow-up of treatment of hepatocellular carcinomas (HCCs), it has limited capabilities for demonstrating associated arteries of HCCs. Three-dimensional (3D) US can provide more accurate depiction of stereotypic vascular patterns than 2D US. This study investigated the efficacy of 3D power Doppler US versus angiography for demonstrating associated arteries of HCCs.A total of 24 HCCs with surrounding or intratumoral arteries detected by 2D power Doppler US were studied using a US machine equipped with power Doppler software and a built-in stereo image processing workstation. Serial 2D images were reconstructed into 3D images to analyze the distribution and routes of arteries associated with HCCs. These results were compared with angiographic findings.Three-dimensional US imaging of vasculature associated with HCCs in the 24 patients was categorized into 3 patterns: peripheral arteries around the tumor (pattern I, n = 9); no more than 4 intratumoral arteries with or without originating peripheral arteries (pattern II, n = 9); and prominent (more than 4) intratumoral arteries (pattern III, n = 6). Most feeding arteries shown in angiograms that could be demonstrated by 3D power Doppler US were in HCCs with pattern I and II vasculature. In HCCs with pattern III, the basket vascular pattern shown on angiograms could also be demonstrated on 3D power Doppler US. The vascular proficiency, including major feeding arteries and branches of feeding arteries,could be shown to significantly increase from pattern I to III on 3D power Doppler, but this method had a lower detection rate of smaller branches and neovascularization than angiographic study.Three-dimensional power Doppler US can effectively depict the associated arteries of HCCs, especially the major feeding arteries. Three vascularization patterns of HCCs were categorized in 3D images. There is a good correlation of the findings of 3D power Doppler US with angiographic study. These findings demonstrate the usefulness of 3D power Doppler as a non-invasive alternative to angiographic examination.

Stem cell-based therapies for degenerative disorders and injuries are promising in the new era. Multipotential mesenchymal stem cells (MSCs) from bone marrow (BM) are on the leading edge because they are easy to expand in culture while maintaining their multilineage potential. In vitro assessment of the chondrogenic and osteogenic potentials of cultured MSCs has been established, and the BM used in those experiments was exclusively from healthy donors via iliac crest aspiration. It is unknown whether human marrow obtained from femurs also contains these multipotential MSCs. We collected marrow from proximal femurs of two patients undergoing total hip replacement surgery for femoral head osteonecrosis and isolated and culture expanded MSCs to about 20 population doublings. These cells were homogeneously positive for beta1-integrin. When pelleted into aggregates and cultured in a medium containing transforming growth factor-beta3 for 14 days, the cells began to express mRNA for aggrecan and collagen type II and to deposit immunoreactive collagen type II and sulfated proteoglycans in the matrix, hallmarks of chondrogenic differentiation. These MSCs could also be differentiated into osteocytic lineage in vitro, as shown by increased expression of alkaline phosphatase activity and deposition of mineral content onto culture plates. These results indicate that femoral BM obtained during hip surgeries also contained multipotential MSCs. These data imply that direct replacement therapy using MSCs from in situ marrow may be possible in the future and that an MSC bank may be established by using marrow from this approach, bypassing the necessity for iliac marrow aspiration from healthy donors.

It has been frequently reported that culture-expanded mesenchymal stem cells from bone marrow of healthy donors can be induced to differentiate to osteocytic lineage. This study examined the potential for osteogenic differentiation of mesenchymal stem cells obtained from two patients with myeloproliferative disorders.Mesenchymal stem cells were derived from bone marrow aspirates obtained in an outpatient clinic from two patients, one with polycythemia vera and the other with essential thrombocythemia. Nucleated bone marrow cells were directly cultured in flasks. Adherent fibroblastic cells in monolayers were isolated by removing nonadherent cells during medium changes. Osteogenic differentiation was induced in expanded adherent cells for 2 weeks in osteogenic medium containing 100 nmol/L dexamethasone, 10 mmol/L beta-glycerophosphate, and 0.05 mmol/L L-ascorbic acid-2-phosphate. Osteogenic differentiation was evaluated by alkaline phosphatase staining and determination of calcium in deposited minerals on culture plates. The expression of osteopontin mRNA was determined by reverse transcription-polymerase chain reaction.After induction in osteogenic medium, the expression of alkaline phosphatase in mesenchymal stem cells became more intense. Induced alkaline phosphatase-positive cells assumed an irregular shape with multiple spiculate projections, while uninduced alkaline phosphatase-positive cells had a flattened polygonal shape or were elongated. Calcium deposition on the plates of induced cells was 0.39 +/- 0.03 mumol/well in cells from the patient with polycythemia vera and 0.54 +/- 0.03 mumol/well in cells from the patient with essential thrombocythemia, but was not detectable in uninduced cells from either patient. Induction by osteogenic medium markedly increased the expression of osteopontin mRNA in stem cells derived from both patients.In this study, mesenchymal stem cells obtained from aspiration of bone marrow in patients with myeloproliferative disorders were expanded by culture. After osteogenic induction, these cells were shown to be able to differentiate into osteocytic lineage in vitro.

We sought to evaluate whether serum HBV DNA levels correlates with the liver free HBV DNA levels in chronic hepatitis B.Thirty-three consecutive chronic hepatitis B patients with cirrhosis were included in this study. Twenty cases had detectable serum HBV DNA (1.8 pg/ml). All had detectable free liver HBV DNA.There was a strong correlation between the serum and liver HBV DNA levels (P = 0.0018, r = 0.717). Thirteen cases had undetectable serum HBV DNA. Among them, six cases still had detectable liver free HBV DNA. Eight cases were HBeAg-positive. Among them, seven cases were positive for both serum and liver HBV DNA, and one case was negative for both serum and liver HBV DNA. Twenty-five cases were HBeAg-negative and anti-HBe-positive. Among them, 13 cases were positive for serum HBV DNA, and 19 cases were positive for liver HBV DNA. No significant difference was noted for positivity of serum HBV DNA or liver free HBV DNA between HBeAg-positive and HBeAg-negative groups. The level of serum HBV DNA(491 +/- 772 pg/ml versus 203 +/- 447 pg/ml, p = 0.07) and liver free HBV DNA (33 +/- 81 pg/microg versus 6 +/- 15 pg/microg, p = 0.13) was also not statistically different.In conclusion, serum HBV DNA levels is strongly correlated with liver HBV DNA levels in chronic hepatitis B with cirrhosis. Liver free HBV DNA can still be detected in about half of the cirrhotic patients with undetectable serum HBV DNA. Serum HBeAg is not a good predictor of serum or liver HBV DNA levels in cirrhotic patients.

Human chromosome band 16q24 commonly undergoes loss of heterozygosity (LOH) in human hepatocellular carcinoma (HCC). To further localize the region of deletion on 16q24 and to evaluate the genetic role of 17-beta-HSD, which is near 16q24, in HCC, we examined the pattern of loss of heterozygosity in 88 HCC patients. DNAs from 88 pairs of HCCs and corresponding non-tumor parts were prepared. Loss of heterozygosity on chromosomes 16q24 was investigated by 11 sets of microsatellite markers. Mutation analysis of type II 17-beta-HSD was performed by automatic sequencing. LOH on 16q24 for at least 1 locus was found in 43 of the 88 tumor DNAs (49%). Three non-overlapping regions of frequent LOH were defined in these 43 tumors with partial deletions. The first region was between D16S516 loci and D16S507, encompassed by a 1-cM region, defined by the D16S504. The second region was defined by the 17HSDB2 locus between D16S505 and D16S422, encompassed approximately by a 1-cM region. The third region was between D16S520 and D16S413, defined by D16S3048, encompassed approximately by a 4-cM region. Homozygous deletions of any exons in 17HSDB2 gene were identified in 7 of 27 cases (26%). Automated sequencing analysis of 17HSDB2 failed to demonstrate mutations in any of these specimens. Our data suggest that the 17HSDB2 locus is a frequent target of deletion in HCC but the inactivation of 17HSDB2 may not involve sequence mutations. Furthermore, the presence of the other 2 frequent LOH regions suggest that the putative tumor suppressor genes at these locations might be involved in the development of HCC.

Though regular sonographic examination can early detect small hepatocellular carcinoma, the therapeutic results remains unsatisfactory. Antigen-specific immunotherapy is an alternative approach for controlling tumors. The prerequisite for antigen-specific cancer immunotherapy is the identification of appropriate tumor antigens. Recently, a new category of tumor-specific shared antigens, called cancer-testis antigens, has been identified. The cancer-testis antigens have been found in a variety of cancers. However, the expression of cancer-testis antigens in human hepatocellular carcinomas is unknown. The aim of this current study is to investigate the expression of cancer-testis antigens in human hepatocellular carcinomas. Reverse-transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of the SSX-1,-2,-4,-5, SCP-1, NY-ESO-1 genes in tumorous and corresponding non-tumorous liver tissues. In the 30 hepatocellular carcinomas studied, SSX-1,-2,-4,-5, SCP-1, and NY-ESO-1 mRNA expressions were detected in 24 (80%), 14 (46.7%), 22 (73.3%), 10 (33.3%), 2 (6.7%), and 11 (36.7%), respectively. Expressions of these genes were detected in few non-tumor liver tissues. The cancer-testis antigens are expressed in a high percentage of hepatocellular carcinomas. These cancer-testis antigen gene products are potential targets for antigen-specific immunotherapy of hepatocellular carcinoma.

Loss of heterozygosity (LOH) on chromosome 13q is one of the most common genetic alterations in hepatocellular carcinoma (HCC) and might be involved in liver cancer development through inactivation of tumour suppressor genes. In order to narrow down the region of 13q loss, we examined the pattern of loss of heterozygosity (LOH) in tumours from 88 HCC patients, using 18 microsatellite markers on 13q. Thirty-eight of the 88 tumours (43%) showed LOH for at least one marker. Of these, two tumours (5%) showed 13q whole arm allelic loss, while the remaining 36 tumours (95%) had partial allelic loss. The LOH pattern defined by the 36 tumours suggested the existence of at least three different smallest common deleted regions which might be involved in the carcinogenesis of HCC. The first, the most centromeric in the 13q12.3 is, close to the BRCA2 gene, defined by D13S171; the second, the most telomeric region in the 13q31-32 band, is defined by D13S154 and D13S157; the third, the intermediate region at 13q14.3, which is near the RB gene, is defined by loci D13S268. The rate of LOH at 13q31-32 was significantly higher in Hepatitis B-surface antigen (HBsAg)-positive patients than HBsAg-negative HCC patients, pointing to a candidate gene related to the development of HBsAg-positive HCCs.

The elucidation of the genetic changes of hepatocellular carcinoma (HCC) is very important for understanding the molecular mechanism of liver carcinogenesis. In order to identify the gains or losses in DNA sequence copy number in HCC, we used comparative genomic hybridisation to study 40 cases (44 tumours) of HCC. Tumour DNA and DNA from non-neoplastic liver tissue were labelled with different fluorochromes and then simultaneously hybridised to normal metaphase spread chromosomes. An image acquisition system was used to quantitate signal intensities contributed by tumour and reference DNA along the entire length of each chromosome. Regions of amplification and deletion were demonstrated as quantitative alterations. Losses were prevalent on chromosome regions 16q (43%), 17p (20%), 13q (20%), 4q (15%) and 8p (15%). Gains frequently occurred on 8q (30%), 1q (20%), 6p (20%) and 17q (18%). Hepatitis B virus carriers had a significantly higher frequency of losses on chromosome 16q. Furthermore, the minimal region of losses was narrowed down to 16q11-q22. This study confirms the presence of previously known chromosomal aberrations in HCC and highlights a new significant correlation between losses on chromosome 16q and hepatitis B virus carriers.

Sepsis and liver abscess are serious complications following transarterial embolization (TAE) for hepatocellular carcinoma (HCC). However, the exact incidence and the necessity of antibiotic prophylaxis remain undetermined. Between November 1996 and November 1997, we prospectively studied bacterial infections in 231 HCC patients who underwent 287 angiographic procedures without antibiotic prophylaxis, including 176 TAEs and 111 hepatic arteriographies (HAs). Four of the 111 HAs were complicated by transient asymptomatic bacteremia. Of the 176 TAEs, 2 were associated with asymptomatic bacteremia, and 7 (4%) were associated with symptomatic bacterial infection, including 3 cases of sepsis, 2 of liver abscess, and 2 of infected biloma. For patients with HCC, TAE was associated with a higher risk of developing symptomatic bacterial infections than was HA (4% vs. 0, respectively; P = .03). Previous gastrectomy was the only possible risk factor for liver abscess. Finally, early diagnosis and treatment of these infectious complications usually result in successful outcome.

The objective of this study was to quantity the extent of emergency department (ED) overcrowding in Taiwan and to identify possible solutions. The ED log was reviewed for all patients who presented to the National Taiwan University Hospital's ED from January 16, 1996 through February 15, 1996. Charts from patients held longer than 72 hours were reviewed. Among 5,810 patients, 213 (3.6%) were held in the ED for more than 72 hours (7.1 patients per day). In 149 (70.0%) of them, admission was indicated but delayed (42 because more than one subspecialty were involved, 57 because of unavailability of bed, and 50 because of the disparity in admission priority between the emergency physicians and house staffs). Eighteen (8.4%) patients did not meet admission criteria (13 could have been treated in outpatient clinics, 3 needed placement in nursing homes, 2 because of personal problems). The others (22%) recovered while waiting. Significant overcrowding exists in EDs in Taiwan. Four solutions are proposed: (1) creation of a holding unit; (2) flexible ward assignment; (3) pre-established rules for admission priority-setting; and (4) active interfacility transfer. Only through these efforts can EDs in Taiwan guarantee an optimal level of care in the face of a growing patient demand.