Optimization of variable number tandem repeat typing set for differentiating Mycobacterium tuberculosis strains in the Beijing family.

Variable number tandem repeat (VNTR) typing of Mycobacterium tuberculosis, as presently used, has often failed to differentiate clonal strains especially in areas where the Beijing family genotype is predominant. We have evaluated mycobacterial interspersed repetitive units (MIRUs), Queen's University of Belfast (QUB) loci and exact tandem repeat (ETR) loci individually for their abilities to differentiate the Beijing and non-Beijing genotype families of M. tuberculosis. The best locus was QUB 3232 followed by QUB 11b. Available MIRU, QUB and ETR loci were then consecutively arranged in declining order of discriminatory power, and combined to form an optimal set of 12-loci VNTR typing (MIRU 10, 26, 39, 40; QUB 11a, 11b, 15, 18, 26, 3232, 3336; ETR A) that possessed comparable discriminatory ability to IS6110 restriction fragment length polymorphism. This optimized 12-loci VNTR typing set can become an important tool for tracking M. tuberculosis of the Beijing family.

Transcript AA454543 is a novel prognostic marker for hepatocellular carcinoma after curative partial hepatectomy.

BACKGROUND: We have previously reported on the cDNA microarray gene expression profiles of hepatocellular carcinomas (HCCs). Among the genes that show prognostic significance and are overexpressed in tumor compared with adjacent nontumorous liver, transcript AA454543 may have potential for practical use. Our aim is to validate the prognostic significance of transcript AA454543 by alternative research methods and in a separate group of HCC patients. METHODS AND RESULTS: The data of transcript AA454543 derived from microarray analysis of 48 patients having curative partial hepatectomy (group 1) were verified by quantitative reverse transcription polymerase chain reaction (r = 0.618, P < .001). A separate sample set of HCCs obtained from 53 patients (group 2) was examined and the association of AA454543 expression level with overall survival was again validated (P = .027). By Cox regression analysis, transcript AA454543 [hazard ratio (HR) = 3.0, P = .017] and pathologic tumor node metastasis (pTNM) stage (HR = 3.3, P = .010) were independent prognostic factors for overall survival. The accuracy of prediction for 3-year overall survival for transcript AA454543 (74.2%, P = .001) and pTNM stage (76.4%, P = .001) was comparable as measured by the area under the receiver operating characteristic curve. CONCLUSION: Transcript AA454543 is potentially useful molecular prognostic marker for overall survival after curative partial hepatectomy for HCC.

Claudin-10 expression level is associated with recurrence of primary hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) patients with the same clinicopathologic features can have remarkably different disease outcomes after curative hepatectomy. To address this issue, we evaluated the cDNA microarray gene expression profiles of HCCs and identified claudin-10 expression level was associated with disease recurrence. The aim of the current study is to validate the microarray data by an alternative research method applicable for routine practice. EXPERIMENTAL DESIGN: Quantitative reverse transcription-PCR (RT-PCR) was used to validate the microarray data on claudin-10 expression level. The assay was repeated on a separate HCC sample set to consolidate the prognostic significance of claudin-10. RESULTS: Claudin-10 expression level by quantitative RT-PCR and by microarray measurement showed a high concordance (r = 0.602, P < 0.001). Quantitative RT-PCR was repeated on a separate HCC sample set and the association of claudin-10 expression with recurrence was again confirmed (hazard ratio, 1.2; 95% confidence interval, 1.0-1.4; P = 0.011). By multivariable Cox regression analysis, claudin-10 expression and pathologic tumor-node-metastasis stage were independent factors for prediction of disease recurrence. CONCLUSION: Claudin-10 expression of HCC can be used as a molecular marker for disease recurrence after curative hepatectomy.

Mechanism of metastasis by membrane type 1-matrix metalloproteinase in hepatocellular carcinoma.

AIM: To investigate the precise role of membrane type 1-matrix metalloproteinase (MT1-MMP) in hepatocellular carcinoma (HCC) metastasis. METHODS: Human HCC cells Hep3B with overexpression of MT1-MMP were established by stable transfection, and compared with control cells carrying the empty vector. Cells were examined in vivo for their differences in the metastatic ability of athymic nude mice, and analyzed in vitro for their differences in invasion ability by invasion chamber coated with Matrigel, adhesion towards collagen I and migration through culture chamber. Cell proliferation and apoptosis in adherent and suspension status were evaluated by MTT and flow cytometry analysis. RESULTS: We found that overexpression of MT1-MMP could increase intrahepatic metastasis in nude mice with orthotopic implantation of HCC cells (incidence of 100% [MT1-MMP transfectants] vs 40% [vector control transfectants], P<0.05). MT1-MMP could also enhance cell invasion through Matrigel (107.7 vs 39.3 cells/field, P<0.001), adhesion towards matrix (0.30 vs 0.12 absorbance unit at 540 nm, P<0.001), cell migration (89.3 vs 39.0 cells/field, P<0.001), and cell proliferation (24.3 vs 40.5 h/doubling, P<0.001). We also observed that MT1-MMP supported cell survival (71.4% vs 23.9%, P<0.001) with reduced apoptosis (43.7% vs 51.0%, P<0.05) in an attachment-free environment. CONCLUSION: MT1-MMP overexpression could enhance metastasis. In addition to its active role in matrix degradation during tumor invasion, MT1-MMP enhances tumor cell survival upon challenge of detachment, which is important during metastasis when cells enter the circulation.

Granulin-epithelin precursor overexpression promotes growth and invasion of hepatocellular carcinoma.

PURPOSE: Granulin-epithelin precursor (GEP) is a novel growth factor. Our earlier cDNA microarray study indicated that GEP was overexpressed in hepatocellular carcinoma (HCC). The aim of this study was to investigate the clinical significance of GEP expression and its potential as a therapeutic target in HCC. EXPERIMENTAL DESIGN: A total of 110 pairs of HCCs and adjacent nontumor liver tissues, and 22 normal liver tissues were examined. The GEP RNA level was examined by quantitative reverse transcription-PCR, and protein localization by immunohistochemistry. The GEP function was examined by transfection experiments. RESULTS: The RNA levels of the HCCs were significantly higher than those of the nontumor liver tissues and normal livers (P <0.001). GEP protein staining was observed in tumor cytoplasm, and the GEP protein levels of the HCCs were also significantly higher than those of the nontumor liver tissues and normal livers (P <0.001). The majority of HCCs demonstrated up-regulation of GEP protein compared with their adjacent liver tissues [79 (71.8%) of 110]. Positive correlation of GEP RNA with protein levels was observed in HCCs (P <0.01). Strong GEP expression was associated with large HCCs, venous infiltration, and early intrahepatic recurrence (P <0.05). Functional studies on the HCC cell line Hep3B demonstrated that reduction of GEP protein levels resulted in decreased cell proliferation rates, tumor invasion ability, anchorage-independent growth in soft agar, and tumorigenicity in nude mice (P <0.05). CONCLUSION: GEP is an important factor for HCC growth, invasion, and metastasis. GEP has the potential to serve as a tumor marker and therapeutic target.

Decreased expression of cytochrome P450 2E1 is associated with poor prognosis of hepatocellular carcinoma.

Cytochrome P450-2E1 (CYP2E1) is one of the major hepatic enzymes involved in the metabolism of procarcinogen. Our study aimed to investigate the differential expression level of CYP2E1 and its clinicopathological significance in hepatocellular carcinoma (HCC). CYP2E1 revealed low level of expression in 70% of the tumor tissues, when compared to the adjacent nontumor tissues, at both mRNA and protein levels. The low expression of CYP2E1 was significantly correlated with the aggressive tumor phenotype, including poor differentiation status (by the Edmondson grading system) (p=0.038), absence of tumor capsule (p=0.030) and younger age of the patients (p=0.002). Multivariate analysis indicated that CYP2E1 expression level and pTNM stage were independent prognostic factors for disease-free survival. CYP2E1 was also shown to have a differential expression level in different liver tissues. The level of CYP2E1 was significantly higher in nontumor tissues from HCC patients compared to the intermediate level in cirrhosis livers from noncancer patients and normal livers from healthy persons. Tumor tissues were shown to have the lowest expression level. In conclusion, our results have shown that CYP2E1 is upregulated in the nontumor tissue and downregulated in tumor tissue, which is associated with aggressive tumor type and poor prognosis of the patients. It suggested that the differential expression of CYP2E1 may play an important role in HCC tumorigenesis.

Clinical significance of thrombospondin 1 expression in hepatocellular carcinoma.

PURPOSE: Thrombospondin 1 (THBS 1) is a matricellular protein capable of modulating angiogenesis. However, the actual role of THBS 1 in angiogenesis and tumor progression remains controversial. Hepatocellular carcinoma (HCC) is a hypervascular tumor characterized by neovascularization. The significance of THBS 1 in HCC remains unknown. In this study, the significance of THBS 1 in HCC was evaluated by correlating its expression with clinicopathological data. The possible role of THBS 1 in the angiogenesis of HCC was also studied by correlating its expression with vascular endothelial growth factor (VEGF) expression. EXPERIMENTAL DESIGN: Sixty HCC patients were recruited in this study. THBS 1 and VEGF protein expression in tumorous livers were localized by immunohistochemical staining and quantified by ELISA. THBS 1 mRNA was quantified by quantitative reverse transcription-PCR. RESULTS: Immunohistochemical staining of THBS 1 was positive in HCC cells in 51.7% of patients and in stromal cells in 65% of patients. Tumor THBS 1 protein level was significantly correlated with its mRNA expression (P = 0.001) and was significantly correlated with tumor VEGF protein levels (P = 0.001). Its expression was significantly associated with the presence of venous invasion (P = 0.008) and advanced tumor stage (P = 0.049). High THBS 1 expression was also a prognostic marker of poor survival in HCC patients. CONCLUSIONS: This study shows that high expression of THBS 1 is associated with tumor invasiveness and progression in HCC. THBS 1 appears to be a proangiogenic factor that stimulates angiogenesis in HCC in view of its positive correlation with VEGF expression.