Clinical impact of plasma TGF-beta1 and circulating TGF-beta1 mRNA in diagnosis of hepatocellular carcinoma.

BACKGROUND: Transforming growth factor-beta (TGF-beta) plays an important role in the regulation of cell growth and differentiation, angiogenesis, extracellular matrix formation, immunosuppression and cancer development. In this study, we investigated the levels of TGF-beta1 and TGF-beta1 mRNA expression, their relationship with HBV replication, and their diagnostic value for hepatocellular carcinoma (HCC). METHODS: Total RNAs were extracted from HCC samples and matched non-tumor tissues, and from peripheral blood mononuclear cells in HCC patients. TGF-beta1 mRNA was amplified by RT-PCR and confirmed by DNA sequencing. The distribution of TGF-beta1 expression was assessed by immunohistochemistry. The clinical characteristics were analyzed between TGF-beta1 and HBV replication. The diagnostic value of circulating TGF-beta1 and TGF-beta1 mRNA levels were investigated in HCC patients. RESULTS: The incidence of hepatic TGF-beta1 expression was 83.3% in HCC samples, 43.3% in the surrounding tissues, 94.7% in the HBV DNA-positive group, and 63.6% in the HBV DNA-negative group. Liver TGF-beta1 expression was associated with the degree of HCC differentiation and the status of HBV replication, but not with the size or number of tumors. Circulating TGF-beta1 level and incidence of TGF-beta1 mRNA were significantly higher in the HCC group than in any group of patients with benign liver disease, with a higher sensitivity of 89.5% and a specificity of 94.0% for HCC diagnosis when circulating TGF-beta1 levels were >1.2 microg/L. No significant correlation was found between TGF-beta1 expression and AFP level or tumor size. Combining TGF-beta1 level and serum AFP raised the detection rate to 97.4%. CONCLUSIONS: Abnormal expression of hepatic TGF-beta1 is associated with the degree of HCC differentiation and HBV replication. Both circulating TGF-beta1 and TGF-beta1 mRNA can be used as sensitive biomarkers for the diagnosis and prognosis of HBV-induced HCC.

Dynamic alteration of telomerase expression and its diagnostic significance in liver or peripheral blood for hepatocellular carcinoma.

AIM: To investigate the dynamic alteration of telomerase expression during development of hepatocellular carcinoma (HCC) and its diagnostic implications in liver tissues or peripheral blood mononuclear cells for HCC. METHODS: Dynamic expressions of liver telomerase during malignant transformation of hepatocytes were observed in Sprague-Dawly (SD) rats fed with 0.05% of 2-fluoenyacetamide (2-FAA). Total RNA and telomerase were extracted from rat or human liver tissues. The telomerase activities in livers and in circulating blood were detected by a telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA), and its diagnostic value was investigated in patients with benign or malignant liver diseases. RESULTS: The hepatoma model displayed the dynamic expression of hepatic telomerase during HCC development. The telomerase activities were consistent with liver total RNA levels (r = 0.83, P<0.01) at the stages of degeneration, precancerosis, and cancerization of hepatocytes. In HCC patients, the telomerase levels in HCC tissues were significantly higher than in their adjacent non-cancerous tissues, but liver total RNA levels were lower in the former than in the latter. Although the circulating telomerase of HCC patients was abnormally expressed among patients with chronic liver diseases, the telomerase activity was a non-specific marker for HCC diagnosis, because the incidence was 15.7% in normal control, 25% in chronic hepatitis, 45.9% in liver cirrhosis, and 85.2% in HCC, respectively when absorbance value of telomerase activity was more than 0.2. If the value was over 0.6, the incidence was 60% in HCC group and 0% in any of the others (P<0.01) except in two cases with liver cirrhosis. However, the combination of circulating telomerase with serum alpha-fetoprotein level could increase the positive rate and the accuracy (92.6%, 125 of 135) of HCC diagnosis. CONCLUSION: The overexpression of telomerase is associated with HCC development, and its abnormality in liver tissues or in peripheral blood could be a useful marker for diagnosis and prognosis of HCC.

Abnormal expression of HSP gp96 associated with HBV replication in human hepatocellular carcinoma.

BACKGROUND: Heat shock protein (HSP) gp96 is a member of the HSP90 family and presumably overexpresses as a result of stimulation by mutated or abnormal proteins. Its abnormal expression correlates with carcinogenesis, progression and prognosis of hepatocellular carcinoma (HCC). In this study, we investigated the pathological characteristics of liver gp96 expression and its relationship with hepatitis B virus (HBV) replication in HCC patients. METHODS: Tumor specimens were prospectively collected from 30 HCC patients undergoing liver resection. Total RNAs were extracted from HCC or their non-cancerous tissues. The distribution of gp96 expression in hepatocytes was investigated by streptavidin peroxidase (S-P) immunohistochemistry and tissue HBV-DNA was detected by the in situ molecular hybridization technique. The association of gp96 expression with HBV replication, and the histopathological characteristics of HCC were analyzed. RESULTS: The gp96 was strongly expressed in HCC (73.3%, 22 of 30) and weakly (46.7%, 14 of 30) in non-cancerous tissues. The gp96 expression in HCC tissues was correlated with degree of tumor differentiation and tumor size, but not with tumor number (P>0.05). Immunohistochemical analysis showed that 17 of 19 HCC patients with HBV-DNA-positive were strongly expressed for gp96, whereas only 5 of 11 patients with HBV-DNA-negative were positive for gp96. A significant difference was found between the two groups (89.5% vs. 45.5%, P<0.05). CONCLUSIONS: The abnormal expressions of HSP gp96 in HCC tissues are associated with HBV replication. This finding indicates that HBV infection plays an important role in the development of HCC.

Expression and alteration of insulin-like growth factor II-messenger RNA in hepatoma tissues and peripheral blood of patients with hepatocellular carcinoma.

AIM: To investigate the clinical values of serum free insulin-like growth factor II (IGF-II) levels and IGF-II mRNA in hepatocellular carcinoma (HCC) tissues and peripheral blood for diagnosis of HCC and monitoring of extrahepatic metastasis. METHODS: Total RNAs were extracted from HCC tissues or peripheral blood mononuclear cells from patients with HCC, liver diseases devoid of cancer, non-hepatic tumors, and healthy controls, respectively. IGF-II cDNAs were synthesized through random primers and reverse-transcriptase, amplified by polymerase chain reaction (PCR), and confirmed by DNA sequencing analysis. Serum free IGF-II levels in patients with different liver diseases were analyzed by an enzyme-linked immunosorbent assay. RESULTS: The amplified fragments of IGF-II mRNA by RT-PCR were identical to originally designed ones with a size of 170 bp and confirmed by sequencing analysis. The dilution experiments revealed that the lowest sensitivity of our system was 2 ng/L of total RNA. The positive frequencies of IGF-II mRNA were 100% in HCC tissues, 53.3% in para-cancerous tissues, and 0% in non-cancerous tissues, respectively. The serum free IGF-II levels were significantly higher in HCC than those in chronic hepatitis or liver cirrhosis. The positive frequency of circulating IGF-II mRNA was 34.2% in HCC, no amplified fragment was found in other liver diseases, extrahepatic tumors, and normal controls, respectively. The circulating IGF-II mRNA correlated with the stage of HCC, and its positive rate was 100% in HCC with extrahepatic metastasis and 35.5% in HCC with AFP-negative. No significant correlation was found between tumor sizes and circulating IGF-II mRNA fragment. CONCLUSION: The abnormal expressions of free IGF-II and IGF-II mRNA are useful tumor markers for HCC diagnosis, differentiation of extrahepatic metastasis and monitoring postoperative recurrence.

Quantitative analysis of vascular endothelial growth factor, microvascular density and their clinicopathologic features in human hepatocellular carcinoma.

BACKGROUND: Angiogenesis is known to be essential to the survival, growth, invasion, and metastasis of tumor cells. Vascular endothelial growth factor (VEGF) are an important angiogenic factor regulating tumor angiogenesis, but its significance and tumor pathologic features are unclear in hepatocellular carcinoma (HCC). In the present study, we analyzed expression of tissue VEGF, alteration of microvascular density (MVD) in microvessel angiogenesis, development and metastasis of HCC, and level of serum VEGF in differential diagnosis of benign and malignant liver diseases. METHODS: Tumor specimens were prospectively collected from HCC patients undergoing resection. Total RNAs were extracted and the expression levels were detected from different parts of HCC tissues. The cellular distributions of VEGF and MVD of liver tumors and their paracancerous and distal cancerous tissues were investigated by streptavidin peroxidase (S-P) immunohistochemistry, respectively. The VEGF levels of circulating blood and hepatoma tissues were measured by enzyme-linked immunosorbent assay. RESULTS: The incidence of VEGF expression was 63.9% in HCCs (23/36 cases), 78.3% in non-encapsulated HCCs (18/23), and 90.9% in HCCs with extrahepatic metastasis (10/11), respectively. The VEGF expression was tightly correlated with MVD (P<0.01). The MVD in HCC with metastasis, low differentiation or non-encapsulation was significantly higher than that in HCC with intact capsule, high differentiation, or no metastasis. No significant difference was found between VEGF, MVD, tumor size, and hepatitis virus infection. The level of total RNA in HCC tissues was significantly lower but the VEGF level significantly higher than those in paracancerous or distal cancerous ones (P<0.01). The abnormal expression levels of VEGF in sera of HCC patients were directly correlated with the metastasis and recurrence of tumors. CONCLUSION: The high expression of VEGF and abnormality of tissue MVD are useful predictors for vascular invasion and metastasis of liver tumors.

Glycylproline dipeptidyl aminopeptidase isoenzyme in diagnosis of primary hepatocellular carcinoma.

AIM: To investigate the role of glycylproline dipeptidyl aminopeptidase (GPDA) isoenzyme in the diagnosis of primary hepatocellular carcinoma (PHC), especially in patients with negative alpha-fetoprotein (AFP). METHODS: A stage gradient polyacrylamide gel discontinuous electrophoresis system was developed to separate serum GPDA isoenzymes, which were determined in 102 patients with PHC, 45 cases with liver cirrhosis, 24 cases with chronic hepatitis, 35 cases with benign liver space-occupying lesions, 20 cases with metastatic liver cancer and 50 cases with extra-hepatic cancer, as well as 80 healthy subjects. The relationships between GPDA isoenzymes and AFP, the sizes of tumors, as well as alanine aminotransferase (ALT) were also analyzed. RESULTS: Serum GPDA was separated into two isoenzymes, GPDA-S and GPDA-F. The former was positive in all subjects, while the latter was found mainly in majority of PHC (85.3 %) and a few cases with liver cirrhosis (11.1 %), chronic hepatitis (33.3 %), metastatic liver cancer (15.0 %) and non-hepatic cancer (16.0 %). GPDA-F was negative in all healthy subjects and patients with benign liver space-occupying lesions, including abscess, cysts and angioma. There was no correlation between GPDA-F and AFP concentration or tumor size. GPDA-F was consistently positive and not correlated with ALT in PHC, but GPDA-F often converted to negative as decline of ALT in benign liver diseases. The electrophoretic migration of GPDA-F became sluggish after the treatment of neuraminidase. CONCLUSION: GPDA-F is a new useful serum marker for PHC. Measurement of serum GPDA-F is helpful in diagnosis of PHC, especially in patients with negative AFP. GPDA-F is one kind of glycoproteins rich in sialic acid.

Abnormal expression of hepatoma-derived gamma-glutamyltransferase subtyping and its early alteration for carcinogenesis of hepatocytes.

BACKGROUND: Although the hepatoma-specific band of gamma-glutamyltransferase (GGT) is a highly sensitive marker in diagnosis of hepatocellular carcinoma, the kinetic expression and the early alterations of GGT in the development of hepatoma remain unclear. In this study, we investigated the expression and the alterations of GGT multiple molecular forms in hepatotumorigenesis. METHODS: The expression of GGT in a chemically induced hepatocarcinogenesis model was examined by giving 0.05% of 2-fluoenylacetamide in diet for 12 weeks. The expression levels of total RNA and GGT, and the changes of liver pathology, GGT multiple molecular forms and sugar-chain heterogeneity were investigated at the different stages of rat hepatoma development. RESULTS: Pathological examination and biochemical analysis found that liver GGT was over-expressed and secreted into blood during canceration. Serum total GGT and liver GGT specific activities (IU/g) including soluble and membrane-combined GGT were significantly higher (P<0.05) in experimental groups than those in control group, respectively. A highly positive correlation was found between total GGT activities and total RNA levels (r=0.90, P<0.05) of the liver. Both were higher six weeks later than before. Con A-non-reactive-GGT was increased consistantly during the development of rat hepatoma. GGT multiple molecular forms in the liver and sera of experimental rats showed that fetal liver-type GGT bands were associated with the development of hepatoma. CONCLUSIONS: Fetal liver-type GGT in sera and the liver of rats is closely related to hepatotumorigenesis. It can be used as a sensitive enzymatic marker for the early diagnosis of liver cancer.

[Feasibility and clinical application of design about the hollowed titanium stick supporting the femoral head and preventing it from collapsing].

OBJECTIVE: To evaluate the feasibility and the clinical practicality of the design about the hollowed titanium stick supporting the femoral head and preventing it from collapsing. METHODS: From Jan.2003 to Jun.2007, 36 patients (46 hips) diagnosed as cystis degeneration of the femoral head were treated by surgical operation, including 20 males and 16 females with an average age of 40 years ranging from 18 to 56 years old, and the course of the disease was from 10 to 24 months (16 months on average). According to ARCO staging,there were 24 patients (34 hips) in NFH II, of which 11 hips were II a stage, 13 hips were II b stage and 10 hips were II c stage; there were 6 patients diagnosed as osteofibrous,4 patients as simple bone cyst and 2 patients as chondromyxoid fibroma. Under X-ray the percutaneous narrow core decompression and focus infection elimination were performed and supported the sclerotin under the cartilage with titanium stick. The patients were followed-up at the first, third, sixth, twelfth, twenty-fourth and thirty-sixth month after the operation. The clinical evaluation was done by X-ray and the indexes included stable, unstable and abortive. The data was analyzed by Fisher exact probility and the suviaval rate was analyzed by Kaplan-Meier suviaval curve using statistical soft ware SPSS13.5. RESULTS: There were no unstable or failure cases on each period from the 1st month to the 12th month after the operation, indicating that the supporting effect of the titanium stick was exact during 12 months after the operation. There were unstable and failure cases from the 12th month to the 24th month after the operation, which were mainly in stage NFH II c but the comparision of the stable rate in this period and 12 months after the operation had no obvious statistical differences (P>0.05) indicating that the supporting effect of the titanium stick was feasible during the stage. One of the unstable cases deteriorated and failed but there were no new unstable cases, both the stable rate and the unstable rate had no change and the failure rate rose on the 36th month after the operation. The compar- ision of the stable rate on each period after the operation had no obvious difference (P=0.197>0.05), which indicated that the supporting effect of the titanium stick was persistent. By the difference of the etiology the three-year survival rate of the relevant NFH II c pathological changes was the lowest-70% and the survival rate of the pathological changes induced by other etiological factors was 90.2%. CONCLUSION: The design about the hollowed titanium stick supporting the collapsed femoral head is feasible. Using the hollowed titanium stick to support the femoral head and prevent it from collapsing is pragmatic in the clinical and the effect is positive, however, when it comes to the NFH II c pathological changes, the choice should be made discreetly.

[Detection of GGT-II by dot-ELISA with monoclonal antibody in the diagnosis of hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Hepatoma-specific gamma- glutamyltransferase isoenzyme II(GGT-II) is considered as the best hepatoma marker except alpha-fetoprotein (AFP), but there is no simple and easy method to determine it now. The purpose of this study was to explore the value of detection of GGT-II by dot-ELISA with monoclonal antibody in the diagnosis of hepatocellular carcinoma (HCC). METHODS: GGT-II was purified and then BALB/c mouse was immunized. The monoclonal antibody against GGT-II was raised by the hybridoma technique. Serum GGT-II was detected in 123 cases with hepatocellular carcinoma and 164 cases with various benign liver diseases using both dot-ELISA and electrophoresis simultaneously. RESULTS: The positive rate of serum GGT-II in HCC by dot-ELISA was 71.5%, which was not significantly different from that by electrophoresis (76.4%). However, the false positive rates of GGT-II by dot-ELISA in liver cirrhosis (23.7%) and chronic hepatitis (27.1%) were significantly higher than those by electrophoresis (10.0% and 8.4% for liver cirrhosis and chronic hepatitis, respectively). CONCLUSION: Detection of GGT-II by dot-ELISA with monoclonal antibody is helpful for the diagnosis of HCC, but its diagnostic specificity deserves to be improved.

[Factors related to acute upper gastrointestinal bleeding after transcatheter arterial chemoembolization in patients with hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Acute upper gastrointestinal bleeding (UGIB) often occurs after transcatheter arterial chemoembolization (TACE) in the patients with hepatocellular carcinoma (HCC). The authors studied the factors associated with UGIB for better prevention and management of the complication. METHODS: Epirubicin, cisplatin, mitomycin, 5-fluorouracil, lipidol and/or gelfoam were infused via catheters inserted in ciliac artery, common hepatic artery, arteria hepatica propria, or left or right hepatic artery by Seidinger method in 208 cases of advanced HCC confirmed by image techniques, alpha-fetoprotein (AFP) and/or pathology. Factors related to UGIB (vomiting of blood and/or melena, or positive fecal occult blood) were analyzed with reference to endoscopy, biochemical parameters of liver function, selection of blood vessels, and the amount of drugs. RESULTS: Of 208 patients, 31 cases were complicated with UGIB. Acute gastric mucosal lesion was confirmed in 18 cases; acute ulcer in 3 cases; Mallory-Weiss syndrome in 3 cases; and esophageal varices bleeding in 2 cases. Positive correlation was found between B grade of Child-Pugh hepatic functional reserve and bleeding (r = 0.59, P < 0.005). The incidence of UGIB in patients in whom drugs were infused via ciliac artery (7/18, 38.9%); or common hepatic artery (18/38, 47.4%) was significantly higher than in those via arteria hepatica propria, left, or right hepatic artery (5/146, 3.4%; P < 0.005). Patients with larger amount of chemotherapy drug and embolization agent had higher bleeding rate. CONCLUSION: Many factors may be associated with UGIB after TACE in patients with HCC, such as higher scores of hepatic functional reserve in Child-Pugh grading, selection of blood vessels, and amount of drugs. In order to reduce the incidence of UGIB, these factors should be necessarily considered in improvement of TACE procedure, in inspection and management after TACE.