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Purpose: We applied a unique method to identify genes expressed in whole blood that can serve as biomarkers to detect colorectal cancer (CRC)., Experimental Design: Total RNA was isolated from 211 blood samples (110 non-CRC, 101 CRC). Microarray and quantitative real-time PCR were used for biomarker screening and validation, respectively., Results: From a set of 31 RNA samples (16 CRC, 15 controls), we selected 37 genes from analyzed microarray data that differed significantly between CRC samples and controls (P < 0.05). We tested these genes with a second set of 115 samples (58 CRC, 57 controls) using quantitative real-time PCR, validating 17 genes as differentially expressed. Five of these genes were selected for logistic regression analysis, of which two were the most up-regulated (CDA and MGC20553) and three were the most down-regulated (BANK1, BCNP1, and MS4A1) in CRC patients. Logit (P) of the five-gene panel had an area under the curve of 0.88 (95% confidence interval, 0.81-0.94). At a cutoff of logit (P) >+0.5 as disease (high risk), <-0.5 as control (low risk), and in between as an intermediate zone, the five-gene biomarker combination yielded a sensitivity of 94% (47 of 50) and a specificity of 77% (33 of 43). The intermediate zone contained 22 samples. We validated the predictive power of these five genes with a novel third set of 92 samples, correctly identifying 88% (30 of 34) of CRC samples and 64% (27 of 42) of non-CRC samples. The intermediate zone contained 16 samples., Conclusion: Our results indicate that the five-gene biomarker panel can be used as a novel blood-based test for CRC.

Background: Promoter hypermethylation of tumor suppressor genes (TSGs) is a common phenomenon in liver carcinogenesis, although the controlling mechanism remains unclear., Materials and Methods: The mRNA expression of DNA methyltransferases (DNMT1, 2, 3a, 3b and splice variants 3b3 and 3b4) and methyl-CpG binding protein (MBD2) were quantitated in 51 liver specimens (41 hepatocellular carcinoma (HCC), 1 cholangiocarcinoma, 1 macroregenerative nodule and 8 HCC cell lines) and the expression levels were correlated with the promoter methylation status of 14 TSG, including APC, RASSF1A, SOCS-1, GSTP1, E-cadherin, p14, p15, p16, DAP-kinase, HIC1, MGMT, TIMP-3, hMLH1 and HLTF., Results: Up-regulations of DNMT1, DNMT2, DNMT3a, DNMT3b4 and MBD2 were suggested in more than 40% of the cases. In particular, the overexpression of DNMT3b and the splice variant DNMT3b3 were identified in as many as 91% and 97.8% of cases, respectively. Using methylation-specific PCR, the most frequently methylated TSGs were APC (90.2%), RASSF1A (86.3%), SOC-1 (74.5%), GSTP1 (72.5%), E-cadherin (64.7%) and p16 (58%). Statistical correlations did not suggest the DNMTs and MBD2 expressions in association with cumulative methylated index in individual cases, but increased expression levels of DNMT2 and DNMT3a showed significant association with the hypermethylation of GSTP1 (p=0.014) and DAP-kinase (p=0.006), respectively. Furthermore, the analysis with clinicopathological data indicated aberrant DAP-kinase methylation was significantly associated with advanced stage T3/T4 HCC tumors (p=0.032) and that p16 hypermethylation was distinct more prevalent in tumors arising from a cirrhotic background (p=0.005)., Conclusion: Our study indicated that DNMT deregulations are common in liver cancers and the existence of a relationship between DNMT2 and DNMT3a overexpression and promoter hypermethylation of candidate tumor suppressor genes in HCC., (Copyright© 2006 International Institute of Anticaner Research (Dr. John G. Delinassios), All rights reserved.)

Objective: To evaluate the status of promoter hypermethylation of Ras association domain family protein 1A (RASSF1A), hypermethylated in cancer 1 (HIC1) and p73 genes in hepatocellular carcinoma (HCC) and to explore the correlation with clinicopathological features., Methods: Forty cases of HCC and their corresponding non-tumor liver tissues, other 2 cases of healthy donor livers were detected using methylation specific polymorphism chain reaction (MSP) method., Results: The frequency of promoter hypermethylation of RASSF1A showed 90.0% and 72.5% in tumor and corresponding non-tumor tissues respectively, and there was significant difference between them (P < 0.05). The frequency of promoter hypermethylation of HIC1 showed 77.5% and 70.0% in tumor and corresponding non-tumor tissues respectively. The frequency of hypermethylation of HIC1 in non-tumor liver tissues showed significant correlation between younger and older patients. The frequency of promoter hypermethylation of p73 showed 5.0% in tumor tissues. However, none of hypermethylation of the gene was detected in corresponding non-tumor liver tissues. There was none of hypermethylation of the three genes showed in two cases of healthy donor livers., Conclusion: Promoter hypermethylation of RASSF1A and HIC1 genes are common event in HCC and play an important role in the pathogenesis and may be used to develop novel diagnostic and therapeutic approaches for HCC in the future.

Background: Functional tests provide diagnostic and prognostic information in patients with suspected coronary disease and are recommended in investigating and guiding management of these patients. There is little data on their utilization, especially in patients with low to intermediate pre-test probability of disease., Methods: From 6053 consecutive patients who underwent 6830 coronary angiograms for suspected coronary disease, 758 patients were subsequently found to have normal coronary arteries. Clinical data, functional tests performed prior to angiography and referring physicians were analyzed., Results: The 758 patients had mean pre-test probability of disease of 42+/-30%. Only 483 patients had undergone functional tests before angiography. There were no differences in gender, age, and pre-test probability between patients who underwent functional tests and those who did not. Three hundred thirteen patients underwent angiography as inpatients while 445 were day-only patients. Inpatients were less likely to have undergone functional tests prior to angiography. Inpatient status was the only independent predictor of not undergoing functional tests (OR 5.9, p<0.001). Functional tests revealed inducible ischaemia in only 241 of the 483 patients. Patients referred by cardiologists were more likely to have undergone functional tests compared with those referred by other physicians. Procedural cardiologists and non-procedural cardiologists had similar rate of use of functional tests., Conclusions: In our patients with normal coronary arteries, utilization of functional tests was low, particularly for inpatients. A significant proportion proceeded to angiography despite negative functional tests. Referrer characteristics and inpatient status, rather than pre-test probability, appeared to have greater impact on utilization of functional tests.

Background & Aims: This study aimed to determine whether intrahepatic hepatitis B virus (HBV) covalently closed circular (ccc) DNA and total HBV DNA levels at the end of therapy would predict sustained response to therapy., Methods: Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients receiving either lamivudine monotherapy or combination of peginterferon and lamivudine had liver biopsy at the end of 1 year therapy and were followed for 52 more weeks after cessation of therapy. Serum HBV DNA, intrahepatic HBV ccc DNA, and total HBV DNA levels were determined., Results: Forty-seven patients, including 34 males and 13 females, were studied. Twenty-seven patients received combination therapy, and 20 patients received lamivudine monotherapy. Twenty-nine patients had end-of-treatment virologic response, and 15 patients had sustained response 52 weeks after therapy. At the end of treatment, log serum HBV DNA levels correlated well with log intrahepatic HBV cccDNA and log intrahepatic total HBV DNA levels. Log intrahepatic cccDNA and log intrahepatic total DNA levels were significantly lower among patients with sustained virologic response. The adjusted odds ratio for log cccDNA was 5.3 (95% CI: 1.5-18.2, P = .009) and, for log intrahepatic HBV DNA, was 4.4 (95% CI: 1.3-14.7, P = .015) to predict sustained virologic response. Using log cccDNA at -0.80 copies/genome equivalent as cutoff, the sensitivity, specificity, and positive and negative predictive values and accuracy of predicting sustained virologic response were 73%, 78%, 56%, 86%, and 77% respectively., Conclusions: Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.

Objective: The FHIT (fragile histidine triad) is a candidate tumor suppressor gene (TSG) located on chromosome 3p14.2. Hypermethylation and loss of heterozygosity (LOH) are major mechanisms in the inactivation of tumor suppressor genes. In this study, the methylation status of FHIT and LOH of 3p14 in 61 cases of human sporadic ovary carcinomas were investigated., Methods: Sixty-one primary ovary carcinomas and 10 borderline ovarian tumors were analyzed with methylation specific PCR (MSP) to detect the CpG island methylation status in the FHIT promoter region. In addition, 45 cases of ovary carcinomas and their corresponding non-tumor ovary tissues were investigated with D3S1287 microsatellite polymorphic marker for LOH., Results: Hypermethylation of FHIT gene was observed in 39.3% (24/61) of ovarian carcinomas. The frequencies of hypermethylation in serous ovarian carcinoma, mucinous ovarian carcinoma, endometrioid ovarian carcinoma and ovary borderline tumor were 45.2% (19/42), 14.3% (1/7), 33.3% (4/12) and 60.0% (6/10), respectively. Ten of twenty-three (43.5%) informative tumors showed LOH and 6 of 18 (33.3%) informative cases showed homozygous deletions. The status of FHIT methylation was not associated with clinical stage and differentiation grade, there was no significant difference between the malignant and borderline tumors., Conclusion: Hypermethylation and allelic deletion of FHIT are frequent events in ovarian carcinomas and are important mechanisms for the loss of expression of this gene.

Human cyotsolic malate dehydrogenase (MDH1) is important in transporting NADH equivalents across the mitochondrial membrane, controlling tricarboxylic acid (TCA) cycle pool size and providing contractile function. Cellular localization studies indicate that MDH1 mRNA expression has a strong tissue-specific distribution, being expressed primarily in cardiac and skeletal muscle and in the brain, at intermediate levels in the spleen, kidney, intestine, liver, and testes and at low levels in lung and bone marrow. The observed MDH1 localizations reflect the role of NADH in the support of a variety of functions in different organs. These functions are primarily related to aerobic energy production for muscle contraction, neuronal signal transmission, absorption/resorption functions, collagen-supporting functions, phagocytosis of dead cells, and processes related to gas exchange and cell division. During neonatal development, MDH1 is expressed in human embryonic heart as early as the 3rd month and then is over-expressed from the 5th month until the birth. The expression of MDH1 is maintained in the adult heart but is not present in levels as high as in the fetus. Finally, over-expression of MDH1 is found in left ventricular cardiac muscle of dilated cardiomyopathy (DCM) patients when contrasted to the diseased non-DCM and normal heart muscle by in situ hybridization and Western blot. These observations are compatible with the activation of glucose oxidation in relatively hypoxic environments of fetal and hypertrophied myocardium.

Objective: Histological assessment of liver fibrosis is important in the management of chronic hepatitis B (CHB) infection but poorly accepted by patients because of its invasiveness. The aim of this study was to develop a noninvasive model to assess liver fibrosis in CHB patients using clinical and routine laboratory data., Patients and Methods: This was a retrospective study on 235 treatment-naive viremic CHB patients. Univariate analysis of data from the training cohort (n = 150) followed by multivariate logistic regression were performed to identify independent predictors of significant fibrosis and generate predictive models. The models were validated with the remaining patients or validation cohort (n = 85) and by receiver operating characteristics (ROC) analysis., Results: Body mass index (BMI), platelet count, serum albumin, and total bilirubin levels were identified as independent predictors of bridging fibrosis or cirrhosis (Ishak stage 3-6). ROC analysis was performed using the predictive probabilities derived from the regression models. The area under the ROC curve of the best model was 0.803 (95% CI: 0.729-0.878) for the training cohort, 0.765 (95% CI: 0.644-0.885) for the validation cohort, and 0.791 (95% CI: 0.728-0.854) for the entire cohort. Using the low cut-off probability of 0.15, significant fibrosis could be excluded in 83 patients of the total patient population (negative predictive value 0.92)., Conclusions: Our noninvasive model comprising BMI and three routine laboratory tests was accurate in predicting absence of significant fibrosis. Application of this model could provide useful additional information on the stage of disease, guide future management decisions, and potentially decrease the need for liver biopsy in some CHB patients.

Background: Non-alcoholic fatty liver disease is an important cause of chronic hepatitis and cryptogenic cirrhosis. The natural history of non-alcoholic fatty liver disease is not well understood especially in Asian populations., Aim: To investigate the histological progression in Chinese patients with biopsy-proven non-alcoholic fatty liver disease., Methods: Chinese patients who had liver biopsy at least 3 years ago and confirmed to have non-alcoholic fatty liver disease were invited for a second liver biopsy. Clinical and laboratory parameters related to their liver function and metabolic syndrome were recorded and analysed. Liver biopsies were scored for the degree of steatosis, necroinflammation and fibrosis. Correlation coefficients were calculated to assess the association between changes in histological scores and metabolic parameters., Results: Seventeen patients who had been followed up for a median period of 6.1 (range: 3.8-8.0) years underwent a second liver biopsy. Nine (53%) patients had progressive disease with worsening of fibrosis score. No statistically significant correlation was found between the changes in histological scores and metabolic parameters. Seven patients developed hypertension or diabetes mellitus during the period of follow-up., Conclusions: Non-alcoholic fatty liver disease is a progressive disease in Chinese patients as in their Caucasian counterparts. Diagnosis of non-alcoholic fatty liver disease may predate development of new components of metabolic syndrome.

Background: Conventional interferon and lamivudine monotherapy are unsatisfactory in treating hepatitis B virus (HBV) infection., Objective: To evaluate the efficacy and safety of pegylated interferon-alpha2b and lamivudine combination therapy for chronic hepatitis B., Design: Randomized, controlled, open-label trial., Setting: Outpatient clinic in a referral center., Participants: 100 treatment-naive patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B and moderately elevated alanine aminotransferase levels., Measurement: The primary end point was sustained virologic response (HBeAg seroconversion and HBV DNA level < 500,000 copies/mL) at 24 weeks after cessation of treatment., Intervention: A staggered regimen of combination therapy with pegylated interferon-alpha2b (1.5 microg/kg of body weight per week; maximum, 100 microg) given for 32 weeks plus lamivudine (100 mg daily) given for 52 weeks versus lamivudine (100 mg daily) monotherapy given for 52 weeks. Of the 100 participants, 96% completed treatment and 80% completed post-treatment follow-up., Results: The rate of sustained virologic response was 36% for the combination treatment group and 14% for the lamivudine monotherapy group (absolute difference, 22 percentage points [95% CI, 6 to 38 percentage points]). End-of-treatment outcomes showed that, compared with monotherapy, patients receiving combination therapy more often had virologic response (60% vs. 28% [absolute difference, 32 percentage points (CI, 14 to 50 percentage points)]); had more substantial reductions of HBV DNA (3.91 log10 copies/mL vs. 2.83 log10 copies/mL); and less often had lamivudine-resistant mutants (21% vs. 40%). The percentages of patients with normalization of alanine aminotransferase levels and histologic improvement did not differ. Adverse effects, such as transient influenza-like symptoms, alopecia, and local erythematous reactions, were more common with combination therapy., Limitations: This study lacked a double-blind design and was conducted at 1 institution. Because of the staggered pegylated interferon-lamivudine regimen, patients assigned to combination therapy received treatment for 8 weeks longer than those assigned to monotherapy., Conclusions: In patients with HBeAg-positive chronic hepatitis B, staggered combination treatment with pegylated interferon-alpha2b and lamivudine may lead to a higher rate of virologic response than lamivudine monotherapy.

Human BRE, a death receptor-associating intracellular protein, attenuates apoptotic response of human and mouse tumor cell lines to death receptor stimuli in vitro. In this report, we addressed whether the in vitro antiapoptotic effect of BRE could impact on tumor growth in vivo. We have shown that the mouse Lewis lung carcinoma D122 stable transfectants of human BRE expression vector developed into local tumor significantly faster than the stable transfectants of empty vector and parental D122, in both the syngeneic C57BL/6 host and nude mice. In vitro growth of the BRE stable transfectants was, however, not accelerated. No significant difference in metastasis between the transfectants and the parental D122 was detected. Thus, overexpression of BRE promotes local tumor growth but not metastasis. We conclude that the enhanced tumor growth is more likely due to the antiapoptotic activity of BRE than any direct effect of the protein on cell proliferation.

Telomerase is an important tumor marker but few antibodies to the enzyme have been described or used without difficulty in histochemical detection. Here we report specific detection of the enzyme in cell and tissue preparations using a new monoclonal antibody (mAb 476) and a new antigen-retrieval buffer (Enhancing buffer). When used to detect telomerase under normal immunostaining conditions in HL-60 cells or tissue sections of hepatocellular carcinoma or metastatic choriocarcinoma, unexpectedly, the antibody stained the cytoplasm rather than the nucleus. Nuclear staining, however, was revealed using the Enhancing buffer. Since other nuclear antigens in the HL-60 cell could be stained both ordinarily and in the Enhancing buffer, nuclear telomerase appears to be shrouded by the nuclear matrix or blocked by accessory proteins. The cytoplasmic activity seen in normal buffer but absent largely from the Enhancing buffer may be an artifact or the nascent, "naked" enzyme. With a known cytoplasmic antigen (proteinase-3) chosen arbitrarily for comparison, the antigenicity was found enhanced, instead, by the Enhancing buffer. The mode of action of the Enhancing buffer differs from that of microwave irradiation or the signal amplification (CSA) used by some investigators. The latter was found to enhance the cytoplasmic reactivity rather than the nuclear reactivity of mAb 476.

Background/aim: Accurate histological assessment of liver fibrosis is essential in the management of chronic hepatitis B (CHB). Although semi-quantitative scoring systems describe well the pathological patterns of hepatic structure, they produce fibrosis evaluation that is not very precise. Image analysis or morphometry has the theoretical advantage of providing truly quantitative data., Patients and Methods: The present study aimed at validating a new image analysis system, Bioquant Nova Prime, in estimating collagen content in liver biopsy samples from patients with CHB. The biopsies were stained with picrosirius red and the areas of collagen were measured. The results were correlated with laboratory parameters and Ishak modified histological scores. Discriminative reliability of morphometry was determined using receiver operating characteristics (ROC) analysis., Results: There was excellent interobserver agreement (r=0.84-0.94, P<0.01) in the morphometric analysis. Significant correlations between the quantitative morphometric data and the semi-quantitative score (Spearman's r=0.68-0.78, P<0.001) were also demonstrated. Excellent discriminative power of morphometry in differentiating mild from advanced fibrosis and cirrhosis from absence of cirrhosis was shown by the ROC analysis., Conclusions: Our results validated the use of Bioquant Nova Prime in estimating collagen content in liver biopsies. We showed that morphometry is a sensitive method of liver fibrosis quantification in CHB and complements semi-quantitative histological scoring system. This tool, with its reliable intraassay variability, could be of special value in assessing histological response to treatment after anti-viral or anti-fibrotic therapy.

Hepatitis B virus is a major etiological factor of hepatocellular carcinoma, but the underlying mechanisms remain unclear. We have previously demonstrated that upregulation of cyclooxygenase (COX)-2 in chronic hepatitis B persisted despite successful antiviral therapy. In this study, we investigated the relationship between the transactivator HBx and COX-2 in hepatitis B virus-associated chronic liver diseases. Expressions of HBx and COX-2 in tissue specimens were determined by single and double immunohistochemistry. The effects of HBx on COX-2 and prostaglandin E2 production were studied by transfection. HBx was expressed in 11/11 (100%) of chronic hepatitis B, 23/23 (100%) of cirrhosis, and 18/23 (78%) of hepatocellular carcinoma, whereas no immunoreactivity was found in four nonalcoholic steato-hepatitis controls. COX-2 expression was also detected in all specimens of liver lesions except in only 29% of poorly differentiated hepatocellular carcinoma. Significant correlation between HBx and COX-2 immunoreactivity scores was found in different types of chronic liver diseases (chronic hepatitis B, rs = 0.68; cirrhosis, rs = 0.57; hepatocellular carcinoma, rs = 0.45). Double immunohistochemistry showed colocalization of HBx and COX-2 in hepatic parenchymal cells. Similar to COX-2, there was no significant change in HBx expression in patients with chronic hepatitis B after interferon and lamivudine therapy when hepatitis B virus DNA became undetectable and inflammation subsided. Transfection of Hep3B hepatocellular carcinoma cells with HBx increased COX-2 expression and prostaglandin E2 production. HBx was localized mainly in the cytoplasm and less in nucleus, as found in the liver lesions. In conclusion, our results strongly suggested that there was a close relationship between HBx and COX-2. COX-2 might represent an important cellular effector of HBx that contributes to hepatitis B virus-associated hepatocarcinogenesis.

Objective: To assess the role of methylated mismatch repair (MMR) genes (hMLH1, hMSH2 and hMSH3) in the carcinogenesis and progression of hepatocellular carcinoma (HCC)., Methods: Samples of 38 cases of HCC along with their corresponding noncancerous tissues, 2 samples of donated normal tissue and 6 cell lines were collected and subject to the methylation-specific PCR (MSP) to examine promoter methylation status of MLH1, MSH2 and MSH3. Six tumor cell lines were analyzed before and after 5-aza-2'-deoxycytidine treatment. In addition, alterations of mRNA expression of MMRs were investigated by quantitative reverse transcription-PCR., Results: CpG island methylation of hMLH1 and hMSH2 was observed in 13.2% (5 of 38 samples) and 68.4% (26 of 38 samples) respectively in HCC, 2.6% (1 of 38 samples) and 55.3% (21 of 38) respectively in corresponding noncancerous tissues, but not in normal control tissues. Promoter methylation of the hMSH2 gene was present in 83.3% of cell lines tested (5/6), but none were observed for the hMLH1 gene. Promoter methylation of the hMSH3 gene was not identified in any tissue samples or cell lines. After 5-aza-2'-deoxycytidine treatment, hMSH2 methylation was induced or completely reversed, and its mRNA expression was increased in most cell lines., Conclusions: Our results suggest that promoter hypermethylation of hMLH1 and hMSH2 genes is common in HCC. Particularly, there is a high frequency of methylation of hMSH2 in both cancer and noncancerous tissues, but not in normal control tissue. Therefore, hypermethylation of MMR genes, especially hMSH2, may be involved in the carcinogenesis of HCC and may serve as an early diagnostic marker for HCC. The close correlation between hMSH2 methylation and low expression of its mRNA suggests that hMSH2 methylation is an important pathway in the regulation of gene expression.

Objective: Our purpose was to find out whether percutaneous biopsy of hepatocellular carcinoma will cause significant dissemination of tumor into the circulation by quantitative analysis of circulating tumor DNA., Subjects and Methods: In this prospective study of 32 patients with suspected hepatocellular carcinoma who underwent sonographically guided liver biopsy, a peripheral venous blood sample was obtained before and 5 min after the procedure. Biopsy was performed using an 18-gauge biopsy gun. DNA was extracted from the plasma of the blood samples for methylation-specific polymerase chain reaction. Quantitative measures of the plasma tumor DNA were determined with real-time quantitative polymerase chain reaction, and the amount was expressed as a methylation index (%) in plasma., Results: Nineteen (59.4%) of 32 patients did not have detectable p16 tumor suppressor gene marker (p16M) in plasma before biopsy, and they showed no detectable plasma p16M after biopsy. Thirteen (65%) of 20 patients had p16M identified in the plasma before liver biopsy. Quantitative analysis of the plasma tumor DNA in these 13 patients showed no statistically significant difference in the methylation index before and after biopsy (p = 0.345, Wilcoxon's signed rank test)., Conclusion: No evidence exists that percutaneous liver biopsy results in hematogenous dissemination of hepatocellular carcinoma as shown by quantitative analysis of circulating tumor DNA (p16M) using methylation-specific real-time polymerase chain reaction.

Objective: We reported here a series of 49 patients with unresectable hepatocellular carcinoma (HCC) who underwent nonsurgical treatment to downstage the disease followed by salvage surgery, their long-term outcome, and pattern of recurrence., Summary Background Data: Most HCC patients present with unresectable disease and are treated with chemotherapy or intra-arterial therapy with a palliative intent. Occasionally, there are good responses to treatment so that salvage surgery becomes feasible afterward. However, long-term outcomes of these patients are seldom reported., Methods: Patients with unresectable hepatocellular carcinoma, from September 1993 to June 2002, who received salvage surgery after downstaging by systemic chemotherapy, intra-arterial yttrium-90 microspheres, or sequential treatment were included in this study. Systemic chemotherapy consisted of combination doxorubicin, cisplatin, interferon-alpha and 5-fluorouracil (5-FU), or single-agent doxorubicin. The choice of treatment was according to stage of disease and contemporary clinical trial protocol. Survival, recurrence pattern, and surgical outcome were studied., Results: There were 49 patients in this study with 40 males and 9 females, age ranged from 12 to 69 years. Forty patients (81.6%) were hepatitis B positive. Thirty-two patients had combination chemotherapy alone (65.3%), 8 patients had single agent chemotherapy alone (16.3%), 4 patients received intra-arterial yttrium-90 microspheres alone (8.2%), and 5 patients received sequential therapy (10.2%). Twenty-eight (57.1%) patients received major hepatic resection. Thirteen patients (26.5%) had complete necrosis of the tumor after treatment. Twenty-one patients (42.9%) had recurrence after surgery, and 14 of them were intrahepatic recurrence. The median survival was 85.9 months. The 1-year, 3-year, and 5-year survival rates were 98%, 64%, and 57%, respectively., Conclusions: Salvage surgery after successful downstaging can provide long-term control of disease in a small proportion of patients with unresectable hepatocellular carcinoma.

Background: Non-alcoholic fatty liver disease is prevalent in affluent countries and is a cause of cirrhosis and possibly hepatocellular carcinoma., Aim: To examine the clinical and histological features of biopsy-proven non-alcoholic fatty liver disease and investigate the predictors of severe histological disease in Chinese patients., Methods: Electronic records of all patients (n = 247) who underwent liver biopsy between 1996 and 2003 in our hospital were retrieved. Patients who had histological features of non-alcoholic fatty liver disease were identified. The demographic, clinical, laboratory and histological (Brunt's criteria) parameters of these patients were analysed., Results: Forty-two patients had histology-proven non-alcoholic fatty liver disease. The median age was 47 years (range 23-69). All except one patient had features of metabolic syndrome. The median alanine aminotransferase was 93 (range 24-270) IU/L. Thirty-six (85.7%) patients had steatohepatitis and 11 (26.1%) also had fibrosis. Only one patient had stage 3 fibrosis. The presence of diabetes mellitus predicted higher grade steatohepatitis and fibrosis (P = 0.019) whereas alanine aminotransferase level had no correlation with histological severity of steatohepatitis. After a median follow-up of 42 months, no patient developed hepatic decompensation., Conclusions: Most Chinese patients with non-alcoholic fatty liver disease had features of the metabolic syndrome. Histological activity was generally mild. Diabetes mellitus was the most important predictor of severe histological disease.

We explored the feasibility of studying nuclear matrix protein (NMP) expressions of the hepatocytes in normal and cirrhotic rat livers with liver regeneration after partial hepatectomy. Sixteen Wistar healthy rats were studied with experimental liver regeneration and/or liver cirrhosis. Two-dimensional (2-D) gel electrophoresis was used to generate these NMP compositions from these rat liver samples. Several antibodies against cytokeratin, vimentin, actin, B23, HNF4alpha, and heat shock protein 70 were used for identification by Western blot. Totally, 41 strongly stained protein spots were characterized on the 2-D gels. Thirty-four protein spots were detected in all of these rat livers, of which, cytokeratin, vimentin, actin, HNF4alpha, and heat shock protein 70 were identified. B23 was detected in the regenerated livers. Three protein spots (s33, s34, and s35) were detectable only in NMP preparation extracted from the regenerating rat livers after hepatectomy. Another three protein spots (s36, s37, and s38) were detectable only in NMP preparation extracted from thioacetamide-induced cirrhotic rat livers. Under these conditions including experimental liver regeneration and/or liver cirrhosis, Over thirty higher abundance NMPs of hepatocytes were consistently expressed and considered as common and basic NMPs. Some of the NMPs are specific for liver regeneration and may play a critical role in cell proliferation and cell cycle, and some are specific for liver cirrhosis., (Copyright 2004 Wiley-Liss, Inc.)

Evidence indicates that cyclooxygenase (COX)-2-derived prostaglandins (PGs) contribute to tumor growth by inducing angiogenesis. We investigated the role of COX-2 in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). COX-2 and vascular endothelial growth factor (VEGF) expressions were examined by immunohistochemistry in 24 HBV-associated HCC. Tumor micro-vessel density (MVD) was assessed using CD34 immunohistochemistry. Hep3B HCC cell line, which carries integrated HBV genome, was stably transfected with human COX-2 cDNA. COX-2 and VEGF expressions were determined by Western blot while PG level was determined by ELISA. The effects of PGs on VEGF expression were also investigated. Expression of COX-2 and VEGF in HCC cells were observed in 19 (79%) and 16 (67%) cases, respectively. Well-differentiated HCC expressed COX-2 more strongly than less-differentiated HCC (p<0.001). COX-2 expression was found to correlate with VEGF expression and MVD (p=0.003 and 0.004, respectively). COX-2 overexpressing Hep3B clone had higher VEGF expression as compared to non-COX-2 expressing clone and parental cells. Treatment of the COX-2 overexpressing cells with a COX-2-selective inhibitor, NS-398 (10 microM), decreased PGE2 level and attenuated VEGF expression. Addition of PGE2 (10 microM) and the stable analog of PGI2, carbaprostacyclin (5 microM), to Hep3B cells also increased VEGF expression. Up-regulation of COX-2 correlates with VEGF expression and tumor angiogenesis in HBV-associated HCC. Moreover, COX-2 up-regulates VEGF expression in HCC cells, possibly via PGs production. Selective inhibition of COX-2 may block HCC associated angiogenesis and thus provides a rational approach for treatment of this malignancy.

Acticoat is a polyethylene mesh coated with nanocrystalline silver. It has been used widely as a dressing for chronic wounds, acute partial-thickness burn wounds and donor sites. In this study, the in vitro cytotoxicity of Acticoat on cultured keratinocytes is tested. Human keratinocytes are cultivated on a pliable hyaluronate-derived membrane (Laserskin) using dermal fibroblasts as the feeder layer. When the cultured Laserskin (CLS) is subconfluent it is covered by Acticoat, which is exposed to water (Group 1), phosphate-buffered saline (Group 2) or culture medium (Group 3). The control group is not exposed to the Acticoat. After 30 minutes incubation at 37 degrees C, the inhibitory effect of the nanocrystalline silver on keratinocyte growth is measured by an MTT assay. Compared with the control, the relative viability of the CLS dropped to 0%, 0% and 9.3%, respectively. Thus, Acticoat is cytotoxic to cultured keratinocytes and should not be applied as a topical dressing on cultured skin grafts.

It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix., (Copyright 2003 Wiley-Liss, Inc.)

Background/aims: Comparative genomic hybridization analysis on hepatocellular carcinoma (HCC) indicated frequent gains of 1q and an amplicon at 1q21-q22. Current cytogenetic evidences confer much importance on 1q21-q22, where a role in drug resistance, tumor metastasis and shorter patient survival had been implicated., Methods: Using positional mapping by interphase cytogenetics, we investigated the amplicon 1q21-q22 in five HCC cases. Three amplification maxima represented by yeast artificial chromosomes (YACs) 955E11, 876B11 and 945D5 that mapped to regions 1q21.1, 1q21.2 and 1q22, respectively, were indicated. We further investigated candidate genes expression in the mapped YACs by quantitative reverse-transcription-polymerase chain reaction. A panel of genes encoding protein transcripts involved in apoptosis, cell cycle progression, calcium binding and jumping translocation was studied., Results: Among ten HCC cases with the amplicon 1q21-q22 examined, we found a significant gene expression level of JTB, SHC1, CCT3 and COPA in the tumors than the paired adjacent non-malignant liver tissues (P< or =0.04)., Conclusions: Our interphase findings on 1q21-q22 pinpointed three affected loci between D1S305 and D1S2369. Up-regulation of candidate genes identified within these over-represented regions may represent targets in the progression of HCC and may carry prognostic significance.

This report describes a case of cytomegalovirus (CMV) infection of the nasopharynx. A 47 year old man presented with a nasopharyngeal mass of one month's duration. The patient had a history of pneumonia one month previously. Sinus computed tomography incidentally picked up a nasopharyngeal mass. The initial biopsy showed lymphoid hyperplasia. Repeated nasopharyngoscopy showed a prominent central nasopharyngeal mass without ulceration. Histology of the nasopharyngeal biopsy revealed several enlarged epithelial cells with characteristic CMV cytopathic changes. An immunohistochemical study, using a monoclonal IgG antibody against a CMV antigen, confirmed CMV infection. The patient's nasopharyngeal mass decreased in size gradually on follow up. To the best of our knowledge, this is the first reported case of CMV infection of the nasopharynx in the English literature. This disease entity should be considered in those patients presenting with nasopharyngeal mass, biopsy negative for malignancy, and no underlying immunosuppression or immunodeficiency.

Frequent chromosome 1 abnormalities detected in human hepatocellular carcinoma have been implicated in early genetic events of liver carcinogenesis. Recurrent loss of 1p with a common deleted region 1p36-p34 has been reported from microsatellite analysis, whereas common gain of the whole chromosome q-arm was described from several comparative genomic hybridization studies. The relationships between copy number changes and allelic status however remains unclear. In this study, we have conducted a simultaneous comparative genomic hybridization and microsatellite analysis study on chromosome 1 in 31 hepatocellular carcinoma cases. Microsatellite analysis revealed frequent loss of heterozygosity on 1p at loci D1S468 (74%), D1S450 (67%), D1S2667 (65%), D1S2697 (75%), D1S199 (52%), and D1S234 (67%) corresponded to the distal 1p36 region and coincided with 12 cases (86%) that presented losses on 1p by comparative genomic hybridization analysis. Although comparative genomic hybridization indicated a common deleted region of 1p36-p35 in the current series, microsatellite analysis has refined the smallest overlapping region (SOR) to 1p36.13-p36.22. Gain of 1q as revealed by comparative genomic hybridization suggested low and high-level gains, and cases that displayed an amplicon below the heterochromatic region 1q21-q25. Common allelic imbalances of polymorphic markers D1S2635 (64%), D1S484 (67%), D1S2878 (65%), D1S196 (70%), D1S249 (64%) D1S2785 (75%), D1S2842 (73%) and D1S2836 (74%) that corresponded to the regions 1q23.1-q24.2, 1q32.1 and 1q43-q44 were detected. Three distinct regions of allelic imbalances were thus suggested on recurring 1q gain found in hepatocellular carcinoma. Furthermore, microsatellite analysis has enabled a mapping of common overrepresented regions and suggested SOR on 1q23.1-q23.3 (D1S2635-D1S2878), 1q25.1-q31.1 (D1S452-D1S238), and 1q43 (D1S2785-D1S2842). Our current study has refined chromosome 1 aberrations in hepatocellular carcinoma to four regions of allelic imbalances. The SORs delineated should provide basis for further molecular investigation in hepatocarcinogenesis on genes residing on these chromosomal regions.

Objective: To study the B23 expression and relocation in the process of liver regeneration at different time periods after partial hepatectomy (PH) on healthy rats, and to study the feasibility of using B23 as a proliferation marker., Methods: Eighteen rats underwent partial hepatectomy (PH). The regenerative liver tissues were obtained at day 1, 2, 3, 4,and 7 after PH. The expression of B23 in hepatocytes of the regenerative tissues was measured using monoclonal antibody against nucleophosmin/B23 by Western blot and immunohistochemistry. The expression of proliferate cell nuclear antigen (PCNA) in these cells were measured using monoclonal antibody against PCNA by both methods for comparison with that of B23., Results: Expression of B23 in the steady state of liver cells before PH was hardly detected by both methods. The increased expression of B23 in the regenerative liver cells was quantitatively changed like a parabolic curve at day 1 to days 7 after PH, with the peak expression of B23 at day 3 after PH. This quantitative variation of B23 expression was similar to that of PCNA expression. The variation of location of B23 in the regenerative liver cells was observed from nucleolus (G1-S phase) to nucleoplasm (S-G2 phase) and to cytoplasm and mitotic chromosome (M phase)., Conclusion: B23 expression is quantitatively increased and moves in location in the process of liver regeneration after PH. B23 may be regarded as a proliferation marker in liver regeneration and liver cell hyperplasia.

Introduction: There have been very limited and inconsistent attempts at combining the cultured epidermal autograft (CEA) with the neodermis of artificial skin (Integra). The reasons for this remain unknown. The basement membrane proteins of conventional CEA sheets are easily damaged by the dispase treatment during the harvesting of the CEA from the culture flask. The damage of the basement membrane proteins may affect the anchorage of CEA onto the neodermis of Integra. A new Composite Biocompatible Skin Graft (CBSG) was recently developed., Methods: Composite biocompatible skin graft consists of autologous keratinocytes cultivated on a pliable hyaluronate-derived membrane (Laserskin)which has been pre-seeded with allogenic dermal fibroblasts. Basement membrane proteins of CBSG are protected from the dispase treatment because the keratinocytes are directly seeded onto Laserskin. The engraftment of CBSG was evaluated on 20 wounds of 10 rats. Integrawas grafted on two freshly excised full-thickness wounds (3cm in diameter) in the dorsum of each animal. A polypropylene ring was applied to each wound to prevent the migration of epithelium from the edges. Composite Biocompatible Skin Graft was used to cover the neodermis of Integra after the silicone membrane was removed 14-21 days postgrafting., Results: Fourteen (70%) of 20 skin biopsies taken at day 21 from the centre of the grafted wounds revealed regenerated epithelium., Conclusion: A feasible delivery system of cultured keratinocytes onto theneodermis of Integra is demonstrated in this animal -experiment.

Background: Cyclooxygenase-2 may play a role in the development of hepatocellular carcinoma, but the relationship between cyclooxygenase-2 and chronic hepatitis B is unknown., Aim: To investigate the expression and cellular localization of cyclooxygenase-2 in chronic hepatitis B patients and the effects of anti-viral therapy., Methods: Using immunohistochemistry, in situ hybridization, Western blot and reverse transcription polymerase chain reaction, protein and messenger RNA expression and cellular localization of cyclooxygenase-2 in 35 chronic hepatitis B patients were assessed. Fourteen histologically normal and non-viral-infected livers were used as controls. The cyclooxygenase-2 immunoreactivities of paired liver biopsies from 12 patients receiving anti-viral therapy were compared., Results: Immunohistochemistry and in situ hybridization revealed that cyclooxygenase-2 expression was confined to hepatocytes. Patients with chronic hepatitis B had significantly higher cyclooxygenase-2 expression compared with controls. The cyclooxygenase-2 expression of hepatitis B e antigen-positive and -negative chronic hepatitis B patients was not significantly different, although the necro-inflammatory activity of the latter group was significantly lower. Over-expression of cyclooxygenase-2 in patients with chronic hepatitis B was further confirmed by Western blot and reverse transcription polymerase chain reaction. Twelve hepatitis B e antigen-positive chronic hepatitis B patients received anti-viral therapy: lamivudine in seven and interferon in five. Despite hepatitis B e antigen seroconversion, disappearance of hepatitis B virus DNA in serum, normalization of liver enzymes and a significant reduction in necro-inflammatory activity in all 12 patients, no significant change in cyclooxygenase-2 expression was found., Conclusions: Chronic hepatitis B is associated with elevated cyclooxygenase-2 levels in hepatocytes, and the over-expression of this enzyme does not reflect inflammatory activity. Up-regulation of cyclooxygenase-2 persists after successful anti-viral therapy.

Objectives: We aimed to study the relationship between the hepatitis B virus (HBV) genotypes, core promoter/precore stop codon mutations, and histological liver damage among hepatitis B e antigen (HBeAg)-negative patients., Methods: Liver biopsy specimens of 55 HBeAg-negative chronic HBV-infected patients were studied. A histological activity index was scored for degree of necroinflammation (HAI-NI) and fibrosis (HAI-F) as described by Knodell et al. HBV DNA was determined by a cross-linking assay and polymerase chain reaction (PCR) at the core promoter/precore region and the S region. PCR-positive samples were directly sequenced for core promoter and precore mutations and examined by restriction fragment length polymorphism for genotyping., Results: Forty-one males and 14 females at a median age of 43 were studied. HBV DNA was detectable in 32 (58%) and 37 (67%) patients by the cross-linking assay and PCR, respectively, at the time of liver biopsy. The median (range) HAI-NI and HAI-F scores were 5 (1-10) and 2 (0-4), respectively. HBV DNA detectable by either the cross-linking assay or PCR was associated with a higher HAI-NI score. Eleven and 31 patients had genotypes B and C HBV, respectively. Genotype C HBV was associated with higher HAI-NI than genotype B HBV. Core promoter mutations and precore stop codon mutation were detected in 74% and 40% patients, respectively, but they were not associated with higher HAI-NI or HAI-F scores., Conclusions: Detectable HBV DNA and genotype C HBV, but not core promoter or precore stop codon mutations, are associated with more severe liver damage in HBeAg-negative patients.

Dermal fibroblasts are known to play an important role in wound healing. In this study, cultured autologous keratinocyte suspension was applied with fibrin glue to the full-thickness wounds in rats (N = 20). Histological analysis on day 14 showed regenerated epithelium in 10 wounds (50%). Keratinocytes were also premixed with allogeneic dermal fibroblasts in a ratio of 3:1 and 5:1 before application to other full-thickness wounds (N = 20) with fibrin glue. Regeneration of epithelium was observed in 10 (50%) and 9 (45%) wounds respectively. Acute inflammatory reaction and mild to moderate proliferation of fibroblasts in the subepithelial layer of the allogeneic fibroblasts were noted. The addition of dermal fibroblasts to keratinocytes/fibrin glue does not enhance the take rate of the cultured keratinocyte suspension.

Objective: To examine the surgical and pathologic findings of 15 patients who had initially unresectable hepatocellular carcinoma (HCC) and received preoperative systemic chemoimmunotherapy and sequential resection., Summary Background Data: More than 80% of patients with HCC present for treatment at an unresectable stage. Conventional treatment has produced a low tumor response rate in this group of patients. Recently, new systemic chemoimmunotherapy has been found to be effective and able to make previously unresectable HCC resectable. Sequential resection after response to chemoimmunotherapy could therefore induce complete clinical remission., Methods: From July 1996 to February 1999, 150 patients with unresectable HCC were treated with systemic chemoimmunotherapy consisting of cisplatin, alpha-interferon, doxorubicin, and 5-fluorouracil for a maximum of six cycles. The residual tumors were reassessed for resectability after treatment aiming at complete remission in the patients after combined modality treatment. Twenty-seven patients had a more than 50% regression in tumor size (2 complete remissions, 25 partial remissions). Fifteen patients had resectable disease after treatment, and all underwent sequential resection with curative intent. Treatment outcome and the surgical and pathologic features of these 15 patients were studied., Results: Fifteen of 150 patients responded to chemoimmunotherapy and underwent sequential resection. They were considered to have unresectable disease as a result of extensive local disease (with and without major vascular involvement) in 10 patients and the presence of extrahepatic or metastatic disease in 5 patients. All patients except two were hepatitis B carriers. Surgical resection of the residual lesion after chemoimmunotherapy was successful for all patients. Eight of the patients had complete pathologic remission. The rest had minimal residual disease (<5%) only. All 15 patients entered complete clinical remission after surgery. Thirteen patients were still alive as of this writing and two had died of recurrent disease. The 1-, 2-, and 3-year survival rates were 100%, 100%, and 53%, respectively. The mean follow-up period was 27 months (range 15-37). Neither the median disease-free nor overall survival had been reached. Ten patients remained in complete remission as of this writing., Conclusion: Combined modalities with systemic chemoimmunotherapy and surgical resection can achieve complete clinical remission and long-term control of disease in patients with unresectable HCC.

Purpose: To determine the accuracy of ultrasonography (US)-guided percutaneous biopsy in diagnosing malignant neoplasms for hepatic lesions 1 cm or smaller., Materials and Methods: In this prospective study, 64 consecutive patients with 74 discrete focal hepatic lesions depicted at US were referred for liver biopsy to confirm the exact nature of the lesions. Mean lesion size was 0.84 cm +/- 0.13 (range, 0.5-1.0 cm). Biopsy was performed with an 18-gauge automated biopsy gun in 46 lesions (once [n = 37], twice [n = 7], three times [n = 2]) or a 22-gauge needle in 28 lesions (once [n = 23], twice [n = 4], three times [n = 1]). Measures were taken to ensure accurate and effective lesion sampling. The histologic diagnosis of malignant tumor and findings on follow-up US images of "benign" nodules for 15-39 months were the criterion standard., Results: No complications occurred. All specimens obtained were sufficient for diagnosis. Histologic examination revealed various types of primary and secondary malignant tumors (n = 44), hemangioma (n = 5), cirrhosis (n = 13), focal fatty change (n = 8), focal fatty sparing (n = 2), and abscess (n = 2). The diagnostic discrimination of US-guided biopsy in diagnosing malignant tumors in these small lesions was sensitivity, 98%; specificity, 100%; positive predictive value, 100%; negative predictive value, 97%; and accuracy, 99%., Conclusion: Percutaneous biopsy under US control is highly accurate in providing a definitive histologic diagnosis of malignant neoplasms for small hepatic lesions if measures for ensuring precise and effective lesion sampling are taken.

Three nasopharyngeal carcinoma (NPC) cell lines (CNE-1, CNE-2 and NPC/HK-1), two squamous cell carcinoma (SCC) cell lines (T2/CUHK and PWH-S1) and six head and neck cancer specimens (NPC [n = 4], SCC tongue [n = 1] and a thyroid cancer [n = 1]) were incubated with interferon (IFN)-alpha (5 x 10(4) iu/mL) and/or 13-cis retinoic acid (13RA; 10(-5) mol/L) for two days at 37 degrees C. In vitro chemosensitivity was measured using MTT assay. Mild growth inhibition of the five cell lines by IFN-alpha ranged from 7.1% to 51.8% (mean: 18.5%), whereas with 13RA it was zero to 19.7% (mean: 7%). Greater inhibition (14.8-51.0%, mean: 31.8%) was achieved when the two drugs were used in combination. Growth inhibition of the six surgical specimens ranged from 6.9% to 21% (mean: 13.6%) with IFN-alpha; zero to 10.3% (mean: 6.0%) with 13RA; and 6.6-26.5% (mean: 17.7%) when the two agents were combined. Four of the 11 samples showed synergistic antitumour effect when IFN-alpha and 13RA were combined, and six showed subadditive effect. The results show that IFN-alpha and 13RA have a mild in vitro antitumour effect on head and neck cancer cells, and the drug synergistic effect demonstrated in this study suggests that the two agents should be used in combination in clinical application.

A cDNA microarray technique, which allows simultaneous analysis of differential expression of mRNA of over 4000 known human genes, was utilized to study the gene expression in 10 pairs of HCC and nontumorous tissues from ethnic Chinese patients in Hong Kong. A total of 211 genes were found to be highly expressed and 147 genes were downregulated in more than 1 out of 10 of the HCC pairs. The results were significant by two-tailed Wilcoxon test (P < or = 0.05 with 95% confidence) on the intensity of each DNA spot of the 10 HCC pairs. Six genes were highly expressed and 10 genes were downregulated in more than 30% of HCC pairs. Results are consistent with other published reports using traditional differential display, subtractive hybridization, or immunohistochemical staining methods. We also detected that beta-actin and glyceraldehyde 3-phosphate dehydrogenase (G3PDH), which have been commonly used as an internal standard control in mRNA expression studies, were highly expressed in HCC when compared with nontumorous tissue. It is concluded that cDNA microarray analysis is an effective method in the detection of differential gene expression in HCC.