Identification of latent biomarkers in hepatocellular carcinoma by ultra-deep whole-transcriptome sequencing.

There is an urgent need to identify biomarkers for hepatocellular carcinoma due to limited treatment options and the poor prognosis of this common lethal disease. Whole-transcriptome shotgun sequencing (RNA-Seq) provides new possibilities for biomarker identification. We sequenced ∼250 million pair-end reads from a pair of adjacent normal and tumor liver samples. With the aid of bioinformatics tools, we determined the transcriptome landscape and sought novel biomarkers by further empirical validations in 55 pairs of adjacent normal and tumor liver samples with various viral statuses such as HBV(+), HCV(+) and HBV(-)HCV(-). We identified a novel gene with coding regions, termed DUNQU1, which has a tissue-specific expression pattern in tumor liver samples of HCV(+) and HBV(-)HCV(-) hepatocellular carcinomas. Overexpression of DUNQU1 in Huh7 cell lines enhances the ability to form colonies in soft agar. Also, we identified three novel differentially-expressed protein-coding genes (ALG1L, SERPINA11 and TMEM82) that lack documented expression profiles in liver cancer and showed that the level of SREPINA11 is correlated with pathology stages. Moreover, we showed that the alternative splicing event of FGFR2 is associated with virus infection, tumor size, cirrhosis and tumor recurrence. The findings indicate that these new markers of hepatocellular carcinoma may be of value in improving prognosis and could have potential as new targets for developing new treatment options.

Identification of SOX4 target genes using phylogenetic footprinting-based prediction from expression microarrays suggests that overexpression of SOX4 potentiates metastasis in hepatocellular carcinoma.

A comprehensive microarray analysis of hepatocellular carcinoma (HCC) revealed distinct synexpression patterns during intrahepatic metastasis. Recent evidence has demonstrated that synexpression group member genes are likely to be regulated by master control gene(s). Here we investigate the functions and gene regulation of the transcription factor SOX4 in intrahepatic metastatic HCC. SOX4 is important in tumor metastasis as RNAi knockdown reduces tumor cell migration, invasion, in vivo tumorigenesis and metastasis. A multifaceted approach integrating gene profiling, binding site computation and empirical verification by chromatin immunoprecipitation and gene ablation refined the consensus SOX4 binding motif and identified 32 binding loci in 31 genes with high confidence. RNAi knockdown of two SOX4 target genes, neuropilin 1 and semaphorin 3C, drastically reduced cell migration activity in HCC cell lines suggesting that SOX4 exerts some of its action via regulation of these two downstream targets. The discovery of 31 previously unidentified targets expands our knowledge of how SOX4 modulates HCC progression and implies a range of novel SOX4 functions. This integrated approach sets a paradigm whereby a subset of member genes from a synexpression group can be regulated by one master control gene and this is exemplified by SOX4 and advanced HCC.

Significance of serum hepatocyte growth factor levels in patients with hepatocellular carcinoma undergoing hepatic resection.

BACKGROUND: Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen and may stimulate the proliferation and invasiveness of human hepatocellular carcinoma (HCC) cells through the c-met receptor. This study evaluates the significance of serum HGF levels in patients undergoing HCC resection. STUDY DESIGN: The peripheral and portal sera and HCC and non-tumorous tissues of 40 HCC patients, with tumor TNM stage I (n=12), II (n=17), and III (n=11) diseases, who underwent hepatic resection were prospectively collected. Serum HGF levels were determined by enzyme-linked immunosorbent assay. The c-met protein expressions were examined by immunohistochemistry. Median follow-up time was 69 months. RESULTS: The prehepatectomy portal HGF levels (median, 622pg/mL) were significantly higher than peripheral HGF levels (564pg/mL) (P=0.026). The posthepatectomy portal HGF levels (699pg/mL) were significantly higher than prehepatectomy portal HGF levels (P<0.001). C-met expression was detected in 87.5% HCC and in 85.0% non-tumorous liver tissues. By Cox multivariate analysis, posthepatectomy portal HGF level >699pg/mL (P<0.001), multiple tumors (P=0.042), and TNM stages II (P=0.019) and III (P=0.009) were independent factors related with survival. Patients with a posthepatectomy portal HCG level >699pg/mL and with a positive c-met expression in HCC tissue have the worst survival. CONCLUSIONS: In HCC patients, high peripheral and portal HGF serum levels related with poor prognosis after hepatic resection. Hepatocyte growth factor and c-met receptor can be targets of future HCC postoperative treatment.

Clinicopathological significance of survivin expression in patients with hepatocellular carcinoma.

AIMS: Survivin, a newly discovered member of the inhibitor of apoptosis protein family, is suggested to be involved in liver carcinogenesis. The aim was to investigate the clinical significance of survivin expression in resected hepatocellular carcinoma (HCC) and paired adjacent non-tumour tissue. METHODS AND RESULTS: Immunohistochemistry, reverse transcriptase-polymerase chain reaction and Western blots were used to examine survivin mRNA and protein levels in 94 specimens of HCC tissues at different TNM stages and the data were correlated with the clinicopathological profiles. Patients were categorized into those with high tumour survivin protein levels (T-N >or= -1) and those with low levels (T-N < -1). Follow-up data were collected prospectively. mRNA levels of survivin and its splice variants in tumour tissue were significantly higher than in paired non-tumour tissue. However, survivin protein levels in paired non-tumour tissue were significantly higher than in tumour tissue from all three TNM stages. Additionally, high tumour survivin protein levels (T-N >or= -1) correlated with a better prognosis and low levels (T-N < -1) with a worse survival rate. CONCLUSIONS: High cytoplasmic survivin protein levels in HCC tissues seem to be an indicator of better prognosis in HCC patients after resection.

DDX3, a DEAD box RNA helicase, is deregulated in hepatitis virus-associated hepatocellular carcinoma and is involved in cell growth control.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide and is highly correlated with hepatitis virus infection. Our previous report shows that a DEAD box RNA helicase, DDX3, is targeted and regulated by hepatitis C virus (HCV) core protein, which implicates the involvement of DDX3 in HCV-related HCC development. In this study, the potential role of DDX3 in hepatocarcinogenesis is investigated by examining its expression in surgically excised human HCC specimens. Here we report the differential deregulation of DDX3 expression in hepatitis virus-associated HCC. A significant downregulation of DDX3 expression is found in HCCs from hepatitis B virus (HBV)-positive patients, but not from HCV-positive ones, compared to the corresponding nontumor tissues. The expression of DDX3 is differentially regulated by the gender and, moreover, there is a tendency that the downregulation of DDX3 expression in HCCs is more frequent in males than in females. Genetic knockdown of DDX3 with small interfering RNAs (siRNA) in a nontransformed mouse fibroblast cell line, NIH-3T3, results in a premature entry to S phase and an enhancement of cell growth. This enhanced cell cycle progression is linked to the upregulation of cyclin D1 and the downregulation of p21(WAF1) in the DDX3 knockdown cells. In addition, constitutive reduction of DDX3 expression increases the resistance of NIH-3T3 cells to serum depletion-induced apoptosis and enhances the ras-induced anchorage-independent growth, indicating the involvement of DDX3 in cell growth control. These findings together with the previous study suggest that the deregulation of DDX3, a DEAD box RNA helicase with cell growth-regulatory functions, is involved in HBV- and HCV-associated pathogenesis.

Alteration of the copy number and deletion of mitochondrial DNA in human hepatocellular carcinoma.

Somatic mutations in mitochondrial DNA (mtDNA) have been detected in hepatocellular carcinoma (HCC). However, it remains unclear whether mtDNA copy number and mitochondrial biogenesis are altered in HCC. In this study, we found that mtDNA copy number and the content of mitochondrial respiratory proteins were reduced in HCCs as compared with the corresponding non-tumorous livers. MtDNA copy number was significantly reduced in female HCC but not in male HCC. Expression of the peroxisome proliferator-activated receptor gamma coactivator-1 was significantly repressed in HCCs (P<0.005), while the expression of the mitochondrial single-strand DNA-binding protein was upregulated, indicating that the regulation of mitochondria biogenesis is disturbed in HCC. Moreover, 22% of HCCs carried a somatic mutation in the mtDNA D-loop region. The non-tumorous liver of the HCC patients with a long-term alcohol-drinking history contained reduced mtDNA copy number (P<0.05) and higher level of the 4977 bp-deleted mtDNA (P<0.05) as compared with non-alcohol patients. Our results suggest that reduced mtDNA copy number, impaired mitochondrial biogenesis and somatic mutations in mtDNA are important events during carcinogenesis of HCC, and the differential alterations in mtDNA of male and female HCC may contribute to the differences in the clinical manifestation between female and male HCC patients.

Recurrent hepatocellular carcinoma after hepatic resection: prognostic factors and long-term outcome.

AIM: The prognosis of patients with recurrent hepatocellular carcinoma (HCC) after hepatic resection varies widely. This study analyzed long-term survival and prognostic factors of patients with recurrent HCC after hepatectomy. METHODS: From July 1991 to December 2000, 623 patients underwent hepatic resection for HCC. Of those, 347 (56.5%) patients had tumour recurrence, and 286 patients with follow-up time more than 24 months after recurrence were enrolled. Twenty-seven clinicopathologic factors underwent both univariate and multivariate analysis. RESULTS: Of these 286 patients, survival times after tumour recurrence were mean 672+/-619 days; median 468 days; and, range 10-3753 days. The overall 1-, 3-, 5-, and 10-year post-recurrence survival rates were 61.5, 33.4, 18.2, and 9.0%, respectively. Seventy (24.5%) patients were alive at the time of study, and 10 of the 34 patients who underwent re-resection were disease-free. By Cox regression analysis, multiple initial tumours (relative risk (RR) 1.428), recurrent multiple (RR 1.372), extrahepatic recurrence (RR 2.434), recurrent tumour size >2 cm (RR 1.926), post-hepatectomy period until recurrence <1 year (RR 1.769), and non-resectional treatment of recurrent tumours (RR 3.527) were independent prognostic factors for post-recurrent survival rates. CONCLUSIONS: In patients with recurrent HCC after hepatectomy, both initial and recurrent tumour factors influenced their prognosis. Early detection of recurrent tumours is important. Re-resection correlated with better post-recurrent survival rates.

Clinicopathologic and prognostic differences between patients with hepatitis B- and C-related resectable hepatocellular carcinoma.

BACKGROUND AND PURPOSE: Hepatitis B and C viral infections are important factors in the development of hepatocellular carcinoma (HCC). This study examined the clinicopathologic and prognostic differences in patients with hepatitis B- and C-related resectable HCC. METHODS: A total of 270 HCC patients who underwent hepatic resection were enrolled. Among these patients, 211 were positive for hepatitis B surface antigen (HBsAg) and 59 were positive for anti-hepatitis C virus antibody (anti-HCV). The clinical manifestations, pathologic features, and treatment outcomes were compared between the HBsAg-positive and anti-HCV-positive groups. RESULTS: Compared to anti-HCV-positive patients, HBsAg-positive patients were significantly younger, had a higher familial incidence of HCC, larger tumor size, and a higher incidence of multiple tumors. HCC patients who were anti-HCV positive had worse liver function and a higher incidence of history of blood transfusion. DNA flow cytometric analysis revealed significantly more proliferative activity in the non-tumor part of the liver in HBsAg-positive HCC patients. The 1-, 3-, and 5-year overall survival rates of HBsAg-positive patients were 79%, 57%, and 48%, respectively, and for anti-HCV-positive patients were 91%, 75%, and 62%, respectively. HBsAg-positive patients had a significantly lower overall survival rate than anti-HCV-positive patients (p = 0.018). CONCLUSIONS: HBsAg-positive patients with resectable HCC had a less favorable survival rate after tumor resection than anti-HCV-positive HCC patients. This survival difference might have been related to the relatively advanced stage of disease and the higher proliferative activity of the non-tumor part of the liver in HBsAg-positive HCC patients.

Potent antitumor effects of intra-arterial injection of fibroblasts genetically engineered to express IL-12 in liver metastasis model of rat: no additional benefit of using retroviral producer cell.

Interleukin-12 (IL-12) mediates significant antitumor effects in animal models but associated with dose-dependent toxicity in human. To achieve local expression of IL-12 at the tumor site without systemic toxicity, we performed intra-arterial administration of fibroblasts genetically engineered to produce IL-12 protein with or without retrovirus (CRIP- IL-12 or 3T3-IL-12) in liver metastasis model. Rat breast cancer cells ( MADB - 106) were injected into the portal vein of syngeneic Fisher rats on day 0, and fibroblasts were injected into the hepatic artery on day 7. On day 21, liver weight and number of liver tumors were examined. As controls, CRIP cells expressing retrovirus carrying lacZ marker gene (CRIP-lacZ) or saline (Hanks balanced salt solution, HBSS) were injected. Administration of CRIP-IL-12 significantly reduced tumor metastasis in liver measured by number of foci (CRIP- IL-12: 45.2 +/- 36.7, CRIP-lacZ: >250, HBSS: >250, P<.05) and by liver weight (CRIP-IL-12: 13.0+/-2.5 g, CRIP-lacZ: 30.4+/-8.5 g, HBSS: 26.0+/-7.6 g, P<.05). 3T3-IL-12, which produced only IL-12 protein but not IL-12 retrovirus, also had significant antitumor effects equivalent to CRIP-IL-12. Intra-arterial injection of IL-12--producing fibroblasts into the liver may be an effective therapy for liver tumors reducing systemic toxicity, and could be developed for clinical application.

Male predominance in hepatocellular carcinoma: new insight and a possible therapeutic alternative.

Hepatocellular carcinoma is one of the most common cancers in the world. The male to female ratio is 3-6 to 1 in patients with hepatocellular carcinoma. Although steroid hormones and receptors have been examined extensively for their role in the growth regulation of hepatocellular carcinoma, the direct stimulation of hepatocellular carcinoma by steroid hormones still awaits elucidation. On the other hand, clinical trials using antagonists for steroid hormones to treat hepatocellular carcinoma were found to be mostly ineffective. Recently it has been found that 2-methoxyestradiol - an estrogen metabolite - is effective in growth inhibition of various tumor cells as well as in angiogenesis inhibition. Since estrogen is metabolized in the liver, it is conceivable that females with menstruation cycles have more estrogen metabolized in their liver, consequently more 2-methoxyestradiol produced which could inhibit tumor growth in situ. We propose that the low incidence and mortality of hepatocellular carcinoma found in females may have resulted from the high levels of 2-methoxyestradiol produced in the liver during their reproductive years. Consequently, the growth of hepatocellular carcinoma in females is delayed significantly as compared to males. The potential of using 2-methoxyestradiol for treatment of patients with hepatocellular carcinoma after resection of tumor should be explored.

Comparison of 2-methoxyestradiol-induced, docetaxel-induced, and paclitaxel-induced apoptosis in hepatoma cells and its correlation with reactive oxygen species.

BACKGROUND: Previously, the authors observed that paclitaxel treatment of hepatoma cells resulted in differential cytotoxicity. Whether other antimicrotubule agents (docetaxel and 2-methoxyestradiol) are more effective than paclitaxel is not clear. Moreover, whether the modulation of reactive oxygen species (ROS) is involved in the drug-induced growth inhibition of hepatoma cells is not known. METHODS: The authors examined the effects of 2-methoxyestradiol, paclitaxel, and docetaxel on HepG2, Hep3B, HA22T/VGH, and Hepa1-6 hepatoma cell lines. The parameters examined included cell viability, cell membrane permeability, cell cycle distribution, DNA fragmentation, and ROS generation. RESULTS: Docetaxel and paclitaxel inhibited the growth of hepatoma cells at submicromolar concentrations, whereas that of 2-methoxyestradiol was within a micromolar range. This drug-induced growth inhibition was cell cycle dependent. 2-Methoxyestradiol-treated (10-50 microM) cells resulted in G2/M block prior to apoptosis. High dose (0.1 microM) docetaxel- and paclitaxel-treated cells resulted in a G2/M arrest followed by generation of polyploidy or apoptosis; however, low dose (0.01 microM) treatment induced apoptosis without G2/M arrest. The low dose effect was more significant in docetaxel-treated cells than in paclitaxel-treated cells. Although these antimicrotubule agents increased the formation of ROS, antioxidant treatment did not block drug-induced cell cycle and growth inhibition effects. CONCLUSIONS: The current results suggest that the growth inhibition of hepatoma cells induced by 2-methoxyestradiol, paclitaxel, and docetaxel was mediated through G2/M-phase arrest, caspase activation, and DNA fragmentation. The drug-induced apoptosis was independent of ROS formation. Docetaxel was more effective than paclitaxel in killing hepatoma cells. The potential of using 2-methoxyestradiol and docetaxel for the treatment of patients with hepatoma is worthy of further study.

Life-threatening haemorrhage from a sternal metastatic hepatocellular carcinoma.

Rupture of the tumour is a catastrophic complication of hepatocellular carcinoma. The prognosis in patients with a ruptured hepatocellular carcinoma is usually unfavourable. We describe a 46-year-old man who suffered from visible massive tumour haemorrhage due to a hepatitis B-related hepatocellular carcinoma that metastasized to the sternal bone. The prominent tumour mass was bulging over the anterior chest wall on the sternum of the patient, and bled spontaneously. This episode of life-threatening haemorrhage was stopped by surgical ligation of the bleeding site. Palliative radiotherapy shrank the tumour mass size and prevented further possible bleeding. This is likely to be the first reported case with a visible spontaneous tumour bleeding from a sternal metastatic hepatocellular carcinoma.

Prognostic significance of heat shock protein-27 expression in hepatocellular carcinoma and its relation to histologic grading and survival.

BACKGROUND: The expression of heat shock protein-27 (HSP-27) has been detected in some human tumors. In this study the authors investigated HSP-27 expression in patients with hepatocellular carcinoma (HCC) and examined its prognostic significance. METHODS: Expression of HSP-27 was studied in 58 HCC and adjacent noncancerous liver tissues by immunohistochemical stain. The relation between its expression and eight known prognostic factors was evaluated. RESULTS: Of the 58 HCC tissues studied, the presence of HSP-27 was demonstrated in 45 tissues (77.6%); low expression ( 25%) was demonstrated in 28 tissues. A significantly higher distribution of HSP-27 expression in HCC tissues compared with adjacent noncancerous liver tissues was obtained (P < 0.0001). Patients with high HSP-27 expression had a significantly higher histologic tumor grade than those with low HSP-27 expression (P = 0.001). The 5-year disease free survival rate of patients with high HSP-27 expression was 21.4% versus 59.3% for patients with low HSP-27 expression (P < 0.001). A similar relation was observed with overall survival (33.3% vs. 64. 8%; P = 0.009). HSP-27 expression was also identified to be a significant and powerful prognostic indicator for disease free survival (odds ratio = 2.25; P = 0.034) and for overall survival (odds ratio = 2.72; P = 0.015). CONCLUSIONS: The current study data suggest that HSP-27 expression is a powerful prognostic indicator and is related to histologic grade and survival of patients with HCC.

Clinical significance of microscopic tumor venous invasion in patients with resectable hepatocellular carcinoma.

BACKGROUND: Tumor venous invasion in patients with resectable hepatocellular carcinoma (HCC) is frequent and can be macroscopic and microscopic or microscopic alone. Although macroscopic invasion is a well-established prognostic indicator, the clinical significance of microscopic invasion remains unclear. METHODS: There were 322 patients enrolled who had undergone curative resection for HCC. The clinicopathologic factors and prognostic significance associated with macroscopic and microscopic venous invasion were analyzed. RESULTS: Macroscopic invasion was observed in 50 patients (15.5%) and microscopic invasion in 190 (59.0%). The larger the tumor, the more the incidence of venous invasion. There were 140 patients with microscopic invasion only (Group 1). Patients with macroscopic invasion (Group 2, n = 50) also had microscopic invasion. Compared with patients without venous invasion (Group 3, n = 132), Group 1 had a higher alpha-fetoprotein level, a larger tumor size, and more tumors without encapsulation. For group 1, the 1-, 3-, and 5-year disease-free survival rates were 65.6%, 41.6%, and 30.8%, respectively. The 1-, 3-, and 5-year overall survival rates were 87. 8%, 60.0%, and 52.7%, respectively. The survival rates of group 1 were lower than those of group 3 and higher than those of group 2 (P <.05). Multivariate analysis indicated that microscopic and macroscopic venous invasion, surgical margin, indocyanine-green retention, and tumor size and number were significant predictors of postresectional survival. CONCLUSIONS: In HCC patients, microscopic venous invasion is frequent and related independently to postresectional outcome.

Better post-resectional survival in female cirrhotic patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma is notably more prevalent in male. The purpose of this study was to assess the surgical results in male and female cirrhotic patients. METHODOLOGY: The surgical outcomes of 129 hepatocellular carcinoma patients with cirrhosis, including 109 males and 20 females, who had undergone hepatic resection were studied. The clinical, histologic features, DNA ploidy and proliferative phase fraction of tumor and cirrhotic liver were compared between male and female patients. RESULTS: Female patients had significantly lower incidences of history of smoking (5.6% vs. 52.9%, P < 0.001), alcohol intake (5.6% vs. 42.3%, P = 0.003) and hepatitis B surface antigen positivity (47.1% vs. 73.5%, P = 0.028) than male. Cell-cycle analysis of tumor part revealed female had a significant lower G2M phase fraction (3.4%) than male (5.7%) (P = 0.027). The 1-, 3-, and 5-year disease-free survival rates in male and female patients were 65.5% and 88.2%, 36% and 64.4%, and 29.7% and 64.4%, respectively. Female patients had a significantly better disease-free survival than male (P = 0.034, log-rank test). CONCLUSIONS: Female hepatocellular carcinoma with cirrhosis had lower incidences of hepatitis B surface antigenemia, alcohol abuse and lower DNA postsynthetic phase fraction in tumor tissue than male. Consequently, female hepatocellular carcinoma with cirrhosis had better survival than male.

Perioperative safety and prognosis in hepatocellular carcinoma patients with impaired liver function.

BACKGROUND: The benefits of liver resection for hepatocellular carcinoma (HCC) patients with concomitant impaired liver function were often considered questionable because of poor postoperative prognosis. This study will clarify whether an acceptable operative risk exists and whether limited resection will compromise the outcomes of these patients. STUDY DESIGN: Between July 1991 and December 1996, a total of 168 patients with HCC who underwent hepatectomies were enrolled and divided into normal (group A) and impaired (group B) liver function groups according to the value of indocyanine green retention rate at 15 minutes. Clinical features, surgical related features, pathologic features, and disease-free and overall survivals were compared between the groups. RESULTS: Operative morbidity and mortality in group A were 27.3% and 1.6%, and in group B were 40.0% and 2.5%, respectively (p = 0.129 and 0.506). Disease-free survival and overall survival at 5 years in group A were 43.2% and 59.6%, respectively, and in group B they were 30.6% and 56.8%, respectively (p = 0.607 and 0.378). CONCLUSIONS: Limited liver resection is safe and provides favorable prognosis in HCC patients with concomitant impaired liver function.

Preferentially deleted chromosome region 9p21 in large hepatocellular carcinomas.

The role of somatic deletions in chromosome 9 and chromosome 22 loci in hepatocellular carcinomas (HCC) was studied. Twenty-one paired HCC and adjacent tumor-free liver tissue samples were examined for loss of heterozygosity at six chromosome 9 and ten chromosome 22 loci. Among informative cases, the highest LOH rates were observed at 9p21 (40% or 4/10 at IFNA) and 9q23 (23% or 3/13 at D9S318). Our observed LOH rate at 9p21 was significantly higher than the background level previously reported for the same tumor type. Clinical data indicate that chromosome 9p21 deletions occurred preferentially in larger tumors (>5 cm diameter). However, a sequence analysis of the MTS1 gene coding region in cases of 9p21 LOH did not reveal any change, suggesting another tumor suppressor gene as the LOH target.