Use of liver stiffness measurement for liver resection surgery: correlation with indocyanine green clearance testing and post-operative outcome.

BACKGROUND: Liver stiffness measurement (LSM) using transient elastography has recently become available for the assessment of liver fibrosis. Whether LSM can predict the functional liver reserve in patients undergoing liver resection is not certain. AIM: To correlate liver stiffness measurement (LSM) with indocyanine green (ICG) clearance test and liver biochemistry, and to determine its usefulness in predicting postoperative outcomes in patients undergoing liver resection. PATIENTS AND METHODS: Transient elastography and ICG clearance test were performed pre-operatively in 44 patients with hepatocellular carcinoma. The LSM and ICG retention rate at 15 minutes (R15) were correlated with pre-operative factors and post-operative outcomes. RESULTS: There was significant correlation between ICG R15 and LSM. In patients with LSM ≥11 kPa vs <11 kPa, there was significantly higher ICG R15 (17.1% vs 10.0% respectively, p = 0.025). For patients with ICG R15≥10% compared to those <10%, there was significantly higher LSM (12.0 vs 7.6 kPa respectively, p = 0.015). Twenty-eight patients proceeded to resection. There was a significant correlation between LSM and the peak INR after liver resection (r = 0.426, p = 0.024). There was a significant correlation between ICG R15 and the post-operative peak AST level (r = -0.414, p = 0.029) and peak ALT level (r = -0.568, p = 0.002). The operative time was a significant independent factor associated with post-operative complications and peak INR. CONCLUSION: LSM correlated well with ICG R15 in patients undergoing liver resection, and predicted early post-operative complications. Addition of LSM to ICG R15 testing may provide better prognostic information for patients undergoing resection.

Rapid measurement of indocyanine green retention by pulse spectrophotometry: a validation study in 70 patients with Child-Pugh A cirrhosis before hepatectomy for hepatocellular carcinoma.

BACKGROUND: The indocyanine green (ICG) retention test is the most popular liver function test for selecting patients for major hepatectomy. Traditionally, it is done using spectrophotometry with serial blood sampling. The newly-developed pulse spectrophotometry is a faster alternative, but its accuracy on Child-Pugh A cirrhotic patients undergoing hepatectomy for hepatocellular carcinoma has not been well documented. This study aimed to assess the accuracy of the LiMON(®), one of the pulse spectrophotometry systems, in measuring preoperative ICG retention in these patients and to devise an easy formula for conversion of the results so that they can be compared with classical literature records where ICG retention was measured by the traditional method. METHODS: We measured the liver function of 70 Child-Pugh A cirrhotic patients before hepatectomy for hepatocellular carcinoma from September 2008 to January 2009. ICG retention at 15 minutes measured by traditional spectrophotometry (ICGR15) was compared with ICG retention at 15 minutes measured by the LiMON (ICGR15(L)). RESULTS: The median ICGR15 was 14.7% (5.6%-32%) and the median ICGR15(L) was 10.4% (1.2%-28%). The mean difference between them was -4.3606. There was a strong correlation between ICGR15 and ICGR15(L) (correlation coefficient, 0.844; 95% confidence interval, 0.762-0.899). The following formula was devised: ICGR15=1.16XICGR15(L)+2.73. CONCLUSIONS: The LiMON provides a fast and repeatable way to measure ICG retention at 15 minutes, but with constant underestimation of the real value. Therefore, when comparing results obtained by traditional spectrophotometry and the LiMON, adjustment of results from the latter is necessary, and this can be done with a simple mathematical calculation using the above formula.

Gene expression profiling of liver cancer stem cells by RNA-sequencing.

BACKGROUND: Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90(+) liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90(+) cells sorted from tumor (CD90(+)CSCs) with parallel non-tumorous liver tissues (CD90(+)NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: CD90(+) cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90(+) cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90(+)CSCs and CD90(+)NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90(+)CSCs and CD90(+)NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90(+)CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90(+)CSCs compared to CD90(+)NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90(+)CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90(+)CSCs in liver tumor tissues. CONCLUSIONS/SIGNIFICANCE: The identified genes, such as GPC3 that are distinctly expressed in liver CD90(+)CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells.

Hematopoietic chimerism in liver transplantation patients and hematopoietic stem/progenitor cells in adult human liver.

UNLABELLED: Liver transplantation (LT) is a cure for many liver diseases. Blood chimerism of donor origin can develop after LT, which raises the possibility of the existence of hematopoietic stem/progenitor cells (HSPCs) in the liver. We characterized the blood chimerism in a large cohort of 249 LT patients and analyzed putative HSPCs in adult human livers. The overall incidence of chimerism was 6.43%, of which 11.11% was among short-term (1 day to 6 months) and 3.77% was among long-term (6 months to 8 years) LT patients. Hematopoietic Lin(-) CD34(+) CD38(-) CD90(+) populations have been demonstrated to generate long-term lymphomyeloid grafts in transplantations. In human adult livers, we detected Lin(-) CD34(+) CD38(-) CD90(+) populations accounting for 0.03% ± 0.017% of the total single liver cells and for 0.05% ± 0.012% of CD45(+) liver cells. Both Lin(-) CD34(+) and Lin(-) CD45(+) liver cells, from extensively perfused human liver grafts, were capable of forming hematopoietic myeloid-lineage and erythroid-lineage methylcellulose colonies. More importantly, Lin(-) CD45(+) or CD45(+) liver cells could be engrafted into hematopoietic cells in an immunodeficient mouse model. These results are the first evidence of the presence of putative HSPC populations in the adult human liver, where the liver is a good ectopic niche. The discovery of the existence of HSPCs in the adult liver have implications for the understanding of extramarrow hematopoiesis, liver regeneration, mechanisms of tolerance in organ transplantation, and de novo cancer recurrence in LT patients. CONCLUSION: The human adult liver contains a small population of HSPCs. In LT patients, there are two types of chimerisms: transient chimerism, resulting from mature leucocytes, and long-term chimerism, derived from putative HSPCs in the liver graft.

Prediction of posthepatectomy recurrence of hepatocellular carcinoma by circulating cancer stem cells: a prospective study.

OBJECTIVE: To investigate whether circulating cancer stem cells (CSCs) of hepatocellular carcinoma (HCC) can predict its recurrence after hepatectomy. BACKGROUND: HCC recurrence frequently occurs within the first year after hepatectomy, probably due to circulating tumor cells that have been shed from the primary tumor before hepatectomy. Because CSCs are more likely to initiate tumor growth than mature cancer cells, a high level of circulating CSCs may be a hint for HCC recurrence. METHODS: Multicolor flow cytometry was used to detect the number of circulating CSCs (CD45CD90CD44) in the peripheral circulation of 82 HCC patients 1 day before hepatectomy. The patients were monitored by CT or MRI for recurrence every 3 months. RESULTS: Forty-one (50%) patients had recurrence after a median follow-up period of 13.2 months (range, 1.3-57.1 months). Patients with recurrence had a higher median level of circulating CSCs than patients without recurrence (0.02% vs. 0.01%; P < 0.0001). Circulating CSCs > 0.01% predicted intrahepatic recurrence (relative risk 3.54; 95% CI, 1.41-8.88; P = 0.007) and extrahepatic recurrence (relative risk 10.15; 95% CI, 3-34.4; P = 0.0002). Patients with >0.01% circulating CSCs had a lower 2-year recurrence-free survival rate (22.7% vs. 64.2%; P < 0.0001) and overall survival rate (58.5% vs. 94.1%; P = 0.0005) than patients with ≤0.01% circulating CSCs. On multivariable analysis, circulating CSCs > 0.01%, tumor stage and tumor size were independent factors predicting recurrence-free survival. CONCLUSIONS: Circulating CSCs predicted posthepatectomy HCC recurrence with high accuracy. They may be the target of eradication in the prevention of posthepatectomy HCC metastasis and recurrence.

High-intensity focused ultrasound for hepatocellular carcinoma: a single-center experience.

OBJECTIVE: This study aims to evaluate the outcome of patients with hepatocellular carcinoma (HCC) treated by high-intensity focused ultrasound (HIFU) in a single tertiary referral center. BACKGROUND: HIFU is the latest developed local ablation technique for unresectable HCC. The initial experience on its efficacy is promising, but the survival benefit of patients undergoing HIFU for HCC is poorly defined. METHODS: From October 2006 to December 2008, 49 patients received HIFU for unresectable HCC. Each patient underwent a single session of HIFU with a curative intent. Treatment efficacy and survival outcome were evaluated. Clinicopathologic factors affecting the primary technique effectiveness and overall survival rates were investigated by univariate analysis. RESULTS: The median size of the treated tumors was 2.2 cm, ranging from 0.9 to 8 cm. The majority of patients had single tumors (n = 41, 83.6%). Thirty-one patients (63.2%) had artificial right pleural effusion during HIFU treatment to reduce damage to the lung and diaphragm. The hospital mortality rate was 2% (n = 1) and the complication rate was 8.1% (n = 4). The primary technique effectiveness rate was 79.5% (39 of 49 patients). It increased from 66.6% in the initial series to 89.2% in the last 28 patients. Tumor size (≥3.0 cm) was the significant risk factor affecting the complete ablation rate. The 1- and 3-year overall survival rates were 87.7% and 62.4%, respectively. Child-Pugh liver function grading was the significant prognostic factor influencing the overall survival rate. CONCLUSIONS: HIFU is an effective treatment modality for unresectable HCC with a high technique effectiveness rate and favorable survival outcome.

Identification of local and circulating cancer stem cells in human liver cancer.

UNLABELLED: Increasing evidence has revealed the importance of cancer stem cells (CSCs) in carcinogenesis. Although liver CSCs have been identified in hepatocellular carcinoma (HCC) cell lines, no data have shown the presence of these cells in human settings. The present study was designed to delineate CSCs serially from HCC cell lines, human liver cancer specimens to blood samples, using CD90 as a potential marker. The number of CD90(+) cells increased with the tumorigenicity of HCC cell lines. CD45(-)CD90(+) cells were detected in all the tumor specimens, but not in the normal, cirrhotic, and parallel nontumorous livers. In addition, CD45(-)CD90(+) cells were detectable in 90% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis. A significant positive correlation between the number of CD45(-)CD90(+) cells in the tumor tissues and the number of CD45(-)CD90(+) cells in the blood samples was identified. CD90(+) cells sorted from cell lines and CD45(-)CD90(+) cells from the tumor tissues and blood samples of liver cancer patients generated tumor nodules in immunodeficient mice. Serial transplantation of CD90(+) cells from tumor xenografts generated tumor nodules in a second and subsequently third batch of immunodeficient mice. Treatment of CD90(+) CSCs with anti-human CD44 antibody induced cell apoptosis in a dose-dependent manner. CONCLUSION: Identification of CD45(-)CD90(+) CSCs in both tumor tissues and circulation suggests that CD45(-)CD90(+) could be used as a marker for human liver cancer and as a target for the diagnosis and therapy of this malignancy.

Significance of CD90+ cancer stem cells in human liver cancer.

This study characterized cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) cell lines, tumor specimens, and blood samples. The CD90+ cells, but not the CD90(-) cells, from HCC cell lines displayed tumorigenic capacity. All the tumor specimens and 91.6% of blood samples from liver cancer patients bore the CD45(-)CD90+ population, which could generate tumor nodules in immunodeficient mice. The CD90+CD44+ cells demonstrated a more aggressive phenotype than the CD90+CD44(-) counterpart and formed metastatic lesions in the lung of immunodeficient mice. CD44 blockade prevented the formation of local and metastatic tumor nodules by the CD90+ cells. Differential gene expression profiles were identified in the CD45(-)CD90+ and CD45(-)CD90(-) cells isolated from tissue and blood samples from liver cancer patients and controls.

Significance of the serum brain-derived neurotrophic factor and platelets in hepatocellular carcinoma.

The present study investigated the significance of the serum brain-derived neurotrophic factor (BDNF) and platelets in relation to the clinicopathological features of hepatocellular carcinoma (HCC) patients. Localization of the BDNF expression in human HCC tissues was performed by immunohistochemistry. The measurement of soluble BDNF in the serum was performed by enzyme-linked immunosorbent assay. BDNF was expressed in the cytoplasm of the tumor cells. A positive correlation between the tissue and serum levels of BDNF was identified in the HCC patients. The serum levels of BDNF were positively correlated with the platelet counts in the HCC patients. A higher level of serum BDNF was significantly correlated with a tumor size >5 cm, poorly differentiated HCC, the presence of microsatellite tumor nodules, and the absence of cirrhosis in the non-tumorous tissues. A higher level of the serum BDNF/platelet ratio was associated with a poorer disease-free survival after hepatic resection. This study suggested that the tumor cell was a source of serum BDNF in HCC. A higher serum BDNF level was associated with a more advanced tumor status in the HCC patients. The interaction between serum BDNF and platelets might play an important role in HCC tumor progression.

Liver intestine-cadherin (CDH17) haplotype is associated with increased risk of hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC), the most common form of liver cancer, is a leading cause of cancer death worldwide. We previously showed that aberrant mRNA splicing of the liver intestine-cadherin gene CDH17 in liver tissues was triggered by the specific constellation of two CDH17 single nucleotide polymorphisms (651T and IVS6+35G). CDH17 aberrant splicing was highly associated with tumor dissemination and shorter survival of HCC patients. Consequently, it is highly relevant to assess whether the presence of these single nucleotide polymorphisms in the general population represents a risk to the development of HCC. EXPERIMENTAL DESIGN: We conducted a case-control study including 164 HCC and 99 cirrhosis patients and 293 healthy controls. Genotyping was done by PCR and direct sequencing. Odds ratio (OR) and chi2 analysis were used to analyze genotypes and haplotypes. RESULTS: Genotypes 651TT [OR, 2.62; 95% confidence interval (95% CI), 1.34-5.03] and IVS6+35 GG (OR, 1.95; 95% CI, 1.04-3.62) were highly associated with HCC disease. The 651T (C>T) and IVS6+35G (A>G) alleles were also overrepresented in HCC patients and, in particular, the T-G haplotype was the most prevalent in HCC patients when compared with healthy controls (OR, 1.57; 95% CI, 1.167-2.109; P=0.004), which was in agreement with the aberrant splicing observed in tumor tissues. There was no significant difference in genotype and allele frequencies between cirrhosis patients and controls. CONCLUSION: The functional T-G haplotype of CDH17 (651 C>T and IVS6+35A>G) is a genetic susceptibility factor for the development of HCC in a Chinese population.

Significance of circulating endothelial progenitor cells in hepatocellular carcinoma.

This study evaluated the significance of circulating bone marrow-derived endothelial progenitor cells (EPCs) in patients with hepatocellular carcinoma (HCC), a solid tumor with rich neovasculature. Eighty patients with HCC were recruited for the study, and 16 patients with liver cirrhosis and 14 healthy subjects were also included for comparison. Blood samples were taken before treatment. Total mononuclear cells were isolated from peripheral blood, preplated to eliminate mature circulating endothelial cells, and colony-forming units (CFUs) formed by circulating EPCs were counted. To validate the CFU scores, FACS quantification of EPCs using CD133, VEGFR2, and CD34 as markers was performed in 30 cases. Our study showed significantly higher mean CFU scores in patients with HCC compared to patients with cirrhosis and healthy controls (P = .001 and .009, respectively). Furthermore, the CFU scores of patients with HCC positively correlated with levels of serum alpha-fetoprotein (r = .303, P = .017), plasma VEGF (r = .242, P = .035), and plasma interleukin-8 (IL-8) (r = .258, P = .025). Patients with unresectable HCC had higher CFU scores than patients with resectable tumors (P = .027). Furthermore, for those who underwent curative surgery, higher preoperative CFU scores were observed in patients with recurrence within 1 year compared with those who were disease-free after 1 year (P = .013). In conclusion, higher circulating levels of EPCs are seen in patients with advanced unresectable HCC as compared to patients with resectable HCC or those with liver cirrhosis. Our evidence supports the potential use of circulating level of EPCs as a prognostic marker in patients with HCC.

Porcine liver: morphologic characteristics and cell viability at experimental radiofrequency ablation with internally cooled electrodes.

PURPOSE: To evaluate morphologic characteristics and cell viability of radiofrequency ablation zones in porcine liver. MATERIALS AND METHODS: Approval of the study protocol was obtained from the Ethics Committee on Use of Live Animals for Teaching and Research at University of Hong Kong. Internally cooled electrodes were used to produce 120 ablated zones ex vivo and 60 ablated zones in vivo with single electrodes (1-, 2-, and 3-cm exposed lengths) or clustered electrodes (1.0-, 2.0-, and 2.5-cm exposed lengths) at 4, 8, 12, and 16 minutes of ablation (ex vivo) and 8 and 12 minutes of ablation (in vivo). Morphologic measurements of each ablated zone were performed. Cell viability in each ablated zone was assessed qualitatively with histochemical staining and quantitatively with measurement of intracellular adenosine 5'-triphosphate (ATP) concentration. RESULTS: Exposed length of electrode (coefficient = 0.79, standard error = 0.04, P < .001), duration of ablation (coefficient = 0.14, standard error = 0.01, P < .001), and clustered electrode design (coefficient = 1.21, standard error = 0.05, P < .001) were independent factors that affected minimal transverse diameter and volume of ablated zone in ex vivo study. Similar morphologic characteristics existed among ablated zones in in vivo study. Mean distance of ablation beyond the electrode tip remained constant (ex vivo, 1.0 cm +/- 0.08 [standard deviation]; in vivo, 0.5 cm +/- 0.05) regardless of different ablation conditions. Histochemical staining revealed no viable hepatocytes from center to margins of white zone in each ablated area. Mean intracellular ATP concentration in margins of white zone (9.5 x 10(-12) mol/microg DNA +/- 1.43) was lower than that in red zone (4088 x 10(-12) mol/microg DNA +/- 65.97, P < .001) and in adjacent normal liver (4528 x 10(-12) mol/microg DNA +/- 52.74, P < .001). CONCLUSION: Distance of ablation beyond the tip of the electrode remained constant (ex vivo, 1.0 cm; in vivo, 0.5 cm) with different conditions of ablation. Complete and uniform cellular destruction was achieved in the white zone of ablated area.

Identification of brain-derived neurotrophic factor as a novel functional protein in hepatocellular carcinoma.

This study aims to identify a novel molecule that may contribute to hepatocarcinogenesis in a rat orthotopic hepatocellular carcinoma model. The hepatocellular carcinoma model was generated by injection of tumor cells into the left lobe of the liver. Proteomic approaches, including ProteinChip and two-dimensional electrophoresis, were used to identify proteins from serially collected rat serum samples. By both ProteinChip and two-dimensional electrophoresis techniques, the level of a 27-kDa protein was found to be augmented in serum samples during tumor development, decreased after left lobectomy, and reincreased at the time of tumor recurrence. The protein was identified to be brain-derived neurotrophic factor (BDNF). By using specific primers and monoclonal antibody, the expression pattern of BDNF was confirmed in tumor tissue but not in the adjacent nontumorous liver tissue. In addition, the truncated isoform of BDNF receptor-tyrosine protein kinase receptor B was only found in tumor tissue. An in vitro study showed that exogenous BDNF could induce tumor cell proliferation predominantly in relatively small numbers of inoculated cells. Administration of BDNF to tumor cell lines induced significantly increased expression of heat shock protein 90 (Hsp90) and cyclin D1, and blocking the activity of Hsp90 could reverse the up-regulation of cyclin D1 induced by BDNF. The present study revealed that BDNF and its receptor were uniquely expressed in tumor tissue and cell lines of hepatocellular carcimona but not in nontumorous liver tissue and normal cell line. BDNF could stimulate tumor cell proliferation in a Hsp90-dependent manner.

Chemokine receptors support infiltration of lymphocyte subpopulations in human hepatocellular carcinoma.

The presence of lymphocyte infiltration in human hepatocellular carcinoma (HCC) is evident. However, immune regulation of lymphocytes in HCC is poorly characterized. In this study, we investigated the chemokine receptor and memory, activation and adhesion markers of major leukocyte subsets present in tumor, nontumor liver, and peripheral blood. T cells from both tumor and peritumor liver displayed high levels of activation and homing markers. CCR5 and CXCR3 were expressed in a large proportion of CD45RO+, CD69+, CD27+, and CD11a+ T cells from tumor compared with T cells from circulation. The proportion of CCR6- and CXCR3-expressing natural killer cells (NK) and natural killer T cells (NKT) was significantly increased in the tumor and nontumor liver compared with peripheral blood. This study demonstrates the role of chemokine receptors in the recruitment of specific lymphocyte subsets to the liver in HCC and suggests the importance of these receptors in regulation of immune defense against HCC.

Desensitization of T lymphocyte function by CXCR3 ligands in human hepatocellular carcinoma.

AIM: Despite the presence of lymphocyte infiltration, human hepatocellular carcinoma (HCC) is typically a rapidly progressive disease. The mechanism of regulation of lymphocyte migration is poorly understood. In this study, we investigated various factors regulating T cell migration in HCC patients. We examined serum CXC chemokine levels in HCC patients and demonstrated the production of CXC chemokines by HCC cell lines. We determined the effect of both HCC patient serum and tumor cell conditioned supernatant upon lymphocyte expression of chemokine receptor CXCR3 as well as lymphocyte migration. Lastly, we examined the chemotactic responses of lymphocytes derived from HCC patients. METHODS: The serum chemokines IP-10 (CXCL10) and Mig (CXCL9) levels were measured by cytometric bead array (CBA) and the tumor tissue IP-10 concentration was measured by ELISA. The surface expression of CXCR3 on lymphocytes was determined by flow cytometry. The migratory function of lymphocytes to the corresponding chemokines was assessed using an in vitro chemotactic assay. Phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot analysis. RESULTS: Increased levels of IP-10 and Mig were detected in HCC patient serum and culture supernatants of HCC cell lines. The IP-10 concentration in the tumor was significantly higher than that in the non-involved adjacent liver tissues. HCC cell lines secreted functional chemokines that induced a CXCR3-specific chemotactic response of lymphocytes. Furthermore, tumor-cell-derived chemokines induced initial rapid phosphorylation of lymphocyte ERK followed by later inhibition of ERK phosphorylation. The culture of normal lymphocytes with HCC cell line supernatants or medium containing serum from HCC patients resulted in a significant reduction in the proportion of lymphocytes exhibiting surface expression of CXCR3. The reduction in T cell expression of CXCR3 resulted in reduced migration toward the ligand IP-10, and both CD4+ and CD8+ T cells from HCC patients exhibited diminished chemotactic responses to IP-10 in vitro compared to T cells from healthy control subjects. CONCLUSION: This study demonstrates functional desensitization of the chemokine receptor CXCR3 in lymphocytes from HCC patients by CXCR3 ligands secreted by tumor cells. This may cause lymphocyte dysfunction and subsequently impaired immune defense against the tumor.

Reduced expression of chemokine receptors on peripheral blood lymphocytes in patients with hepatocellular carcinoma.

OBJECTIVES: Hepatocellular carcinoma (HCC) is a rapidly progressive malignancy. Chemokine receptors are important mediators of lymphocyte migration in cancer. This study evaluated expression of chemokine receptors on lymphocytes of HCC patients. METHODS: Chemokine receptor expression on peripheral blood lymphocytes (PBL) was determined by flow cytometry and RT-PCR. Tumor infiltrating lymphocytes (TIL) and adjacent nontumor liver infiltrating lymphocytes (NIL) were also studied. RESULTS: The expressions of CCR5, CCR6, and CXCR3 on PBL were significantly reduced in HCC patients compared with normal controls, which occurred concurrently with increased expression of the chemokine receptors in TIL and NIL. Reduced expression of CXCR3 on PBL correlated with large tumor size and advanced tumor stage. The reduced chemokine receptor expression was consistent with the reduced mRNA levels and intracellular protein levels in PBL. HCC patients exhibited lower proportions of CD4(+) and CD8(+) T cells with CCR5, CCR6, and CXCR3 expression on PBL, which occurred concurrently with the increased expression of these chemokine receptors on TIL and NIL. The reduced CCR6 and CXCR3 expression on PBL correlated with the reduced memory phenotype in circulation and increased memory phenotype in liver. Furthermore, CCR5-expressing memory T cells were increased in liver compartment compared with circulation. CONCLUSION: This study demonstrated that reduced chemokine receptor expression on PBL was concurrent with increased chemokine receptor expression on both TIL and NIL in HCC. The results demonstrated the role of chemokine receptors in recruitment of lymphocytes from peripheral blood to HCC. The findings have important implications in understanding of immunopathogenesis of HCC.

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.

High serum levels of vascular endothelial growth factor predict poor response to transarterial chemoembolization in hepatocellular carcinoma: a prospective study.

Vascular endothelial growth factor (VEGF) is an important mediator of tumor angiogenesis. A high serum VEGF level has been shown to predict poor response to chemotherapy and poor survival in several cancers, but its prognostic value in hepatocellular carcinoma (HCC) remains unknown. We conducted a prospective study to evaluate the prognostic significance of pretreatment serum VEGF levels on tumor response to treatment and survival of patients with HCC undergoing transarterial chemoembolization (TACE). Pretreatment serum VEGF levels were measured by an enzyme-linked immunosorbent assay in 80 patients with inoperable HCC undergoing TACE. Serum VEGF levels were correlated with clinical data, tumor response to TACE and survival results. The median serum VEGF level was 240 pg/ml (range 9-1730). Serum VEGF levels were positively correlated with the presence of venous tumor thrombus (P=0.011). Pretreatment serum VEGF levels were significantly higher in patients with progressive disease (median 434 pg/ml) than those with stable (median 176 pg/ml, P=0.010) or responsive disease (median 142 pg/ml, P<0.001) after TACE. Patients with serum VEGF >240 pg/ml had significantly worse survival than those with serum VEGF <240 pg/ml (median survival 6.8 vs. 19.2 months, P=0.007). In a Cox multivariate analysis, serum VEGF >240 pg/ml was an independent prognostic factor of survival. In conclusion, the results of this study suggest that serum VEGF level may be useful as a novel prognostic predictor of tumor response and survival of patients with inoperable HCC undergoing TACE treatment.

Tissue factor expression correlates with tumor angiogenesis and invasiveness in human hepatocellular carcinoma.

PURPOSE: Recent studies have shown that tissue factor (TF) may be involved in tumor angiogenesis and metastasis. The role of TF in hepatocellular carcinoma (HCC) was unknown. This study evaluated whether TF expression correlates with microvessel density (MVD), vascular endothelial growth factor (VEGF) expression, tumor invasiveness, and prognosis in human HCC. EXPERIMENTAL DESIGN: Tissue samples were obtained from 58 specimens of resected HCC. Immunohistochemical expression of TF was examined, and tumor MVD was evaluated using CD34 as the endothelial marker. TF and VEGF protein levels in the tumor cytosol were quantified by ELISA. Clinicopathologic and follow-up data of patients were prospectively collected. RESULTS: The immunohistochemical expression of TF in the tumors correlated significantly with tumor MVD (P = 0.002). The median cytosolic TF protein level in the tumors was 720 pg/mg total protein (range, 67-2406 pg/mg total protein). A significant positive correlation was found between TF and VEGF levels in the tumor cytosol (r = 0.475, P < 0.001). High tumor cytosolic TF level was associated with venous invasion (P = 0.004), microsatellite nodules (P = 0.024), unencapsulated tumor (P = 0.007), and advanced tumor stage (P = 0.010). A higher than median tumor cytosolic TF level was an independent predictor of poor survival (risk ratio, 1.836; 95% confidence interval 1.130-5.312, P = 0.023). CONCLUSIONS: This study shows that TF is related to tumor angiogenesis and invasiveness in HCC. Evaluation of tumor TF expression may be useful as a prognostic indicator in patients with HCC.

Hepatic stress gene expression and ultrastructural features under intermittent Pringle manoeuvre.

BACKGROUND: Intermittent Pringle manoeuvre resulted in less blood loss and better preservation of liver function when it was applied for fewer than 120 minutes. The mechanism of better preservation of liver function under intermittent Pringle manoeuvre at molecular level remains unclear. Furthermore, the ultrastructural features in the liver with chronic diseases under intermittent Pringle manoeuvre have not been studied. The aim of the study is to investigate the expression of stress genes and ultrastructural change of the liver under intermittent Pringle manoeuvre. METHODS: From July 1995 to February 1998, 131 patients underwent hepatectomy for liver tumours (61 patients without Pringle manoeuvre and 70 patients with intermittent Pringle manoeuvre). Twenty-five patients (15 with Pringle manoeuvre and 10 without Pringle manoeuvre) were included in the study of hepatic stress gene expression during hepatectomy. Twenty-two patients (18 patients with intermittent Pringle manoeuvre and four patients without Pringle manoeuvre) were randomly assigned for electron microscopic examination. RESULTS: For the expression of stress genes, both the heat shock genes (HSP 70A and HSC 70) and acute phase genes (TNF-alpha and interleukin-6) were detected simultaneously in the patients with or without intermittent Pringle manoeuvre. The patients under intermittent Pringle manoeuvre had relatively higher mRNA levels of heat shock gene 70 family, which is related to intracellular repair and protection. Induction of TNF-alpha and interleukin-6 genes, which contributed to ischaemia-reperfusion injury and postoperative complication, was not found in the patients under intermittent Pringle manoeuvre. Under the electron microscopy, the hepatic ultrastructure was well maintained under intermittent Pringle manoeuvre whatever the liver status, even when the accumulated ischaemic duration was extended to 120 min. CONCLUSION: Intermittent Pringle manoeuvre induced relatively higher expression of heat shock genes, which are related to intracellular homeostasis, and is consistent with the well maintenance of liver ultrastructure.