Early Human Hepatocellular Carcinoma Research Articles

HMBS is the most suitable reference gene for RT-qPCR in human HCC tissues and blood samples.

Reverse transcription-quantitative (RT-q) PCR is the most feasible and useful technique for identifying and evaluating cancer biomarkers; however, the method requires suitable reference genes for gene expression analysis. The aim of the present study was to identify the most suitable reference gene for the normalization of relative gene expression in human hepatocellular carcinoma (HCC) tissue and blood samples. First, 14 candidate reference genes were selected through a systematic literature search. The expression levels of these genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, PGK1, PPIA, RPLP0, RPL13A, SDHA, TBP, TFRC and YWHAZ) were evaluated using human multistage HCC transcriptome data (dataset GSE114564), which included normal liver (n=15), chronic hepatitis (n=20), liver cirrhosis (n=10), and early (n=18) and advanced HCC (n=45). From the 14 selected genes, five genes, whose expression levels were stable in all liver disease statuses (ACTB, GAPDH, HMBS, PPIA and RPLP0), were further assessed using RT-qPCR in 40 tissues (20 paired healthy tissues and 20 tissues from patients with HCC) and 40 blood samples (20 healthy controls and 20 samples from patients with HCC). BestKeeper statistical algorithms were used to identify the most stable reference genes, of which HMBS was found to be the most stable in both HCC tissues and blood samples. Therefore, the results of the present study suggest HMBS as a promising reference gene for the normalization of relative RT-qPCR techniques in HCC-related studies.

Serum level of cytokeratin 19 as a diagnostic and prognostic marker in patients with HCV-related hepatocellular carcinoma

BackgroundThe heterogeneous nature of human hepatocellular carcinoma (HCC) impedes both treatment strategies and prognostic predictions. Several markers have been proposed for the diagnosis of HCC. Cytoskeleton-associated proteins have been known as cellular integrators in neoplasm formation. Hepatic progenitor cells are thought to express alpha-fetoprotein (AFP) and hematopoietic as well as biliary markers such as cytokeratin 19 (CK 19) and cytokeratin 7. The aim of this study was to verify the role of serum CK 19 alone or in combination with AFP as a diagnostic marker of HCC and to assess the changes in its levels after ablation of HCV-related HCC to evaluate its role as a predictor marker for recurrence of HCC after ablation. The study was conducted on 102 HCV-related cirrhotic patients categorized into three different groups according to the clinical, laboratory, and radiological evaluation: group I—62 patients with early or intermediate HCC who underwent locoregional intervention, group II—20 patients with advanced HCC not fit for any intervention apart from best supportive treatment, and group III—20 cirrhotic patients with no evidence of HCC as proved by two imaging techniques.ResultsThe mean serum levels of CK 19 were 6.5 ± 5.7, 10.5 ± 12.5, and 6.8 ± 2.8 ng/ml in groups I, II, and III, respectively, with no significant difference between groups. Sensitivity, specificity, positive, and negative predictive values of combined AFP and human CK 19 at cutoff levels of 25.5 ng/ml and 6.25 ng/ml were 93.9%, 45%, 87.5%, and 64.3%, respectively. In group I patients, CK 19 levels were comparable in patients with ablated focal lesion and those who did not at baseline; then, it was significantly higher in ablated patients than in patients with residual tumor 1 and 6 months after the intervention.ConclusionsCombination of both AFP and CK 19 levels could increase the diagnostic accuracy of suspected HCCs. CK 19 levels are good predictors of ablation/recurrence in patients who underwent interventional procedures minimizing the need for follow-up imaging modalities.

Abstract 5183: Using transposon elements to elucidate the genetic mechanisms of HCC-associated lung metastases

Abstract Metastases account for most cancer-related deaths as a result of primary cancer spreading to distant sites. Amongst human hepatocellular carcinoma (HCC), there is a drastic decrease in the survival rate observed in patients with early stage localized HCC to late stage metastasis-associated HCC. Importantly, the genetic mechanisms associated with metastasis-associated HCC remains elusive. Therefore, a forward genetic screen in mice using Sleeping Beauty (SB) insertional mutagenesis system was developed to identify genes associated with metastasis-associated HCC. Truncated version of the epidermal growth factor receptor (Egfr) gene was frequently detected in both mouse primary liver tumors and lung metastases, which was confirmed by DNA sequencing and RNA sequencing. This indicates the importance of Egfr mutations for liver tumorigenesis and the metastasis process. More importantly, additional candidate genes involved in the metastasis process were also identified by our forward genetic screen and showed to be relevant in human HCC patients at the M1 cancer metastasis stage. Taken together, our study shows that the SB screen yields a high fraction of relevant events in human liver cancer and can provide valuable information on the evolution of metastasis-associated HCC. The authors declare that they have no competing interests. Citation Format: Lilian H. Lo, Amy P. Chiu, Xiao-Xiao Li, Barbara R. Tschida, Dewi K. Rowlands, David A. Largaespada, Vincent W. Keng. Using transposon elements to elucidate the genetic mechanisms of HCC-associated lung metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5183.

An HNF4α–microRNA-194/192 signaling axis maintains hepatic cell function

Hepatocyte nuclear factor 4α (HNF4α) controls the expression of liver-specific protein-coding genes. However, some microRNAs are also modulated by HNF4α, and it is not known whether they are direct targets of HNF4α and whether they influence hepatic function. In this study, we found that HNF4α regulates microRNAs, indicated by marked down-regulation of miR-194 and miR-192 (miR-194/192) in liver-specific Hnf4a-null (Hnf4aΔH) mice. Transactivation of the shared miR-194/192 promoter was dependent on HNF4α expression, indicating that miR-194/192 is a target gene of HNF4α. Screening of potential mRNAs targeted by miR-194/192 revealed that expression of genes involved in glucose metabolism (glycogenin 1 (Gyg1)), cell adhesion and migration (activated leukocyte cell adhesion molecule (Alcam)), tumorigenesis and tumor progression (Rap2b and epiregulin (Ereg)), protein SUMOylation (Sumo2), epigenetic regulation (Setd5 and Cullin 4B (Cln4b)), and the epithelial-mesenchymal transition (moesin (Msn)) was up-regulated in Hnf4aΔH mice. Moreover, we also found that miR-194/192 binds the 3'-UTR of these mRNAs. siRNA knockdown of HNF4α suppressed miR-194/192 expression in human hepatocellular carcinoma (HCC) cells and resulted in up-regulation of their mRNA targets. Inhibition and overexpression experiments with miR-194/192 revealed that Gyg1, Setd5, Sumo2, Cln4b, and Rap2b are miR-194 targets, whereas Ereg, Alcam, and Msn are miR-192 targets. These findings reveal a novel HNF4α network controlled by miR-194/192 that may play a critical role in maintaining the hepatocyte-differentiated state by inhibiting expression of genes involved in dedifferentiation and tumorigenesis. These insights may contribute to the development of diagnostic markers for early HCC detection, and targeting of the miR-194/192 pathway could be useful for managing HCC.

The miR-200b-ZEB1 circuit regulates diverse stemness of human hepatocellular carcinoma.

Accumulating evidence suggests that human hepatocellular carcinoma (HCC) can be derived from cancer stem cells (CSCs), which contribute to tumor initiation, metastasis, chemoresistance, and recurrence. A great variety of HCC CSCs resulting in diverse clinical manifestations have been reported. However, how CSC diversity is regulated and generated remains unclear. Here we report that the miR-200b-ZEB1 circuit is closely involved with the induction and maintenance of a diverse group of CSCs. We found that miR-200b downregulation occurred in early HCC and associated with poor prognosis. The downregulation was attributable to genome deletion and promoter methylation of the miR-200a/b/429 gene. Ectopic expression of miR-200b or silencing of ZEB1 led to a decrease in CD13+ and CD24+ HCC CSCs and an increase in EpCAM+ HCC CSCs. Mechanistically, miR-200b directly suppressed BMI1 and ZEB1 expressions. ZEB1 recognized promoters of CD13, CD24, and EpCAM genes resulting in CD13 and CD24 upregulation and EpCAM downregulation. Neither miR-200b nor ZEB1 had obvious effects on CD133 or CD90 expression. Silencing CD13 or CD24 expression suppressed tumorigenicity of HCC cells. Ectopic expression of CD24 reversed the suppression of tumorigenicity by ectopic expression of miR-200b. Clinically, miR-200b downregulation was coupled with ZEB1 upregulation in approximately two-thirds of HCC patients. ZEB1 expression was positively correlated with CD13 and CD24 expressions in HCCs, while miR-200b expression was positively correlated with EpCAM. Our findings suggest that the miR-200b-ZEB1 circuit is a master regulator of diverse stemness of HCC, which differentiates HCCs into those containing CD13+ /CD24+ CSCs from those containing EpCAM+ CSCs.

Abstract 1387: The importance of FGF pathway in hepatocellular carcinoma and its role in acquired resistance to anti-angiogenic therapies

Abstract INTRODUCTION: Fibroblast Growth Factor (FGF) signaling has an important role in different biological functions and human cancers, being some of its members described as driver oncogenes and components of evasive resistance to anti-angiogenic therapies. AIMS: 1) To identify molecular alterations of FGF signaling in human hepatocellular carcinoma (HCC), 2) to study the mechanism of acquired resistance to anti-angiogenic therapy in HCC to identify novel targets, and 3) to evaluate the role of brivanib in overcoming acquired resistance in animal models of HCC. METHODS: FGF pathway was analyzed in 104 early HCCs by whole-genome expression, genome-wide methylation status, microRNA expression and chromosomal aberrations. Subcutaneous tumors in nude mice (n=26) were treated with sorafenib (30 mg/kg/day) until development of acquired resistance, afterwards mice were randomized into either brivanib (BMS, 100 mg/kg/day) or sorafenib treatment. Anti-tumoral effect and survival were assessed. The mechanisms involved in acquired resistance were studied by gene expression profiling and protein evaluation. RESULTS: 31% of the HCC samples presented overexpression of oncogenic ligands (i.e. FGF19, FGF 21 and FGF2), and 19% overexpression of FGFR3 and FGFR4 -feature clustered in the proliferation HCC subclass (Chiang, Cancer Res, 2008). Promoter hypermethylation of FGFR2 (FDR=0.015) and regulation of FGFR3 expression by miR-483-5p (6 fold-change, FDR<0.05) were also observed, as well as high level amplification in FGF19 locus (8% of samples), and allelic losses of FGFR2 and Spry2 (p<0.05). FGF2 overexpression was associated with WNT-beta-catenin HCC subclass and correlated with expression of WNT-liver specific targets (i.e. GLUL, LGR5, TBX3, SPARCL1, p<0.05) suggesting a crosstalk among FGF and WNT pathways in HCC. Regarding the in vivo model, 65% mice acquired resistance after sorafenib therapy [median: 42 days; tumor size: 855 mm3 resistant group vs 316 mm3 sorafenib-responder group (p=0.002)]. This acquired resistance phenotype has been evaluated by gene profiling and GSEA revealing an enrichment of gene signatures of poor prognosis and resistance to cancer therapies (FDR<0.05), and identifying IGF-1R and FGFR1 overexpression as the potential drivers of the development of this resistance. Brivanib delayed tumor growth (p=0.03), increased survival after development of acquired resistance (median survival: 32 days vs 15 days on sorafenib, p=0.07) and inhibited FGF downstream signaling, reverting the resistant phenotype. CONCLUSIONS: FGF pathway is activated in a subclass of early HCC due to deregulation of ligands and receptors by different mechanisms. Acquired resistance to sorafenib in experimental models seems to be driven by FGF and IGF signaling pathways. Brivanib significantly delayed tumor growth and increased survival in the sorafenib-resistant in vivo model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1387. doi:1538-7445.AM2012-1387

Upregulation of Rac GTPase-Activating Protein 1 Is Significantly Associated with the Early Recurrence of Human Hepatocellular Carcinoma

To assess the significance of Rac GTPase-activating protein 1 (RACGAP1) expression in identifying HBV-positive human hepatocellular carcinoma (HCC) patients who are at high risk for recurrent disease. The prognostic significance of RACGAP1 was compared with clinicopathologic parameters available at diagnosis using multivariate and log-rank test. RACGAP1 expression and outcome in recurrence was compared between 35 patients with recurrence and 41 patients without recurrence using Kaplan-Meier analysis. RACGAP1-targeted molecules and pathways were identified and characterized by inhibition with siRNA duplexes. Kaplan-Meier analysis showed that the level of RACGAP1 expression is sufficient to predict the early recurrence of HCC: high RACGAP1 expression correlates with high risk of postresection recurrent HCC (P < 0.0005). Silencing of RACGAP1 in Hep3B and MHCC97-H HCC cells with high endogenous RACGAP1 expression inhibited cell migration and invasion. Using Ingenuity Pathway Analysis, the target molecules silenced in the RACGAP1 interactome were mostly genes related to the mitotic roles of the polo-like kinases. These included PRC1, AURKB, CDC2, ECT2, KIF23, PAK1, and PPP2R5E. In providing clinical corroboration of these results, when expression of these transcripts was analyzed in an expression database that we have established previously for HBV-positive HCC patients, these genes was mostly upregulated in patients who exhibited early recurrent disease and hence provided important corroboration of these results. siRNA-silencing RACGAP1 mainly targeted genes in an interactome clinically relevant to early HCC recurrence. Besides being an independent informative prognostic biomarker, RACGAP1 could also be a potential molecular target for designing therapeutic strategies for HCC.

Abstract 2173: Cytokeratin-19, a predictive marker and an early determinant of progenitor-derived hepatocellular carcinoma

Abstract Background: Human hepatocellular carcinoma (HCC) is a heterogeneous disease of distinct clinical subgroups. A principal source of tumor heterogeneity may be cell type of origin which in liver includes hepatocyte and/or adult stem/progenitor cells. Objective: To address this issue, we investigated the molecular mechanisms underlying the fate of the enzyme-altered preneoplastic lesions in the resistant hepatocyte (RH) model. Methods: Sixty samples classified as focal lesions, adenoma, early and advanced HCCs were micro-dissected after morphological and immunohistochemical evaluation and subjected to global gene expression profiling. Results: The analysis of progression of the persistent GSTP+ focal lesions to fully developed HCC showed that about 50% of persistent nodules and all HCCs expressed CK19 whereas 14% of remodeling nodules were CK19+. Unsupervised hierarchical clustering of the expression profiles also grouped the samples according to CK19 expression. Further, supervised analysis using the differentially expressed genes in each cluster combined with the gene connectivity tools identified 1308 unique genes and a predominance of the AP-1/JUN network in the CK19+ lesions. In contrast, the CK19-negative cluster exhibited only limited molecular changes (156 differentially expressed genes vs. normal liver) consistent with remodeling towards differentiated phenotype. Finally, comparative functional genomics revealed a stringent clustering of CK19+ early lesions and advanced HCCs with human HCCs characterized by poor prognosis. Furthermore, the CK19 associated gene expression signature accurately predicted the patient survival (P&amp;lt;0.009) and tumor recurrence (P&amp;lt;0.006). Conclusion: Our data establish CK19 as a prognostic marker of early neoplastic lesions and strongly suggest the progenitor derivation of HCC in the rat RH model. The capacity of the CK19 associated gene signature to stratify HCC patients according to clinical prognosis indicates the usefulness of the RH model for studies of stem/progenitor-derived HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2173.

Progenitor-derived hepatocellular carcinoma model in the rat

Human hepatocellular carcinoma (HCC) is a heterogeneous disease of distinct clinical subgroups. A principal source of tumor heterogeneity may be cell type of origin, which in liver includes hepatocyte or adult stem/progenitor cells. To address this issue, we investigated the molecular mechanisms underlying the fate of the enzyme-altered preneoplastic lesions in the resistant hepatocyte (RH) model. Sixty samples classified as focal lesions, adenoma, and early and advanced HCCs were microdissected after morphological and immunohistochemical evaluation and subjected to global gene expression profiling. The analysis of progression of the persistent glutathione S-transferase (GSTP)(+) focal lesions to fully developed HCC showed that approximately 50% of persistent nodules and all HCCs expressed cytokeratin 19 (CK19), whereas 14% of remodeling nodules were CK19(+). Unsupervised hierarchical clustering of the expression profiles also grouped the samples according to CK19 expression. Furthermore, supervised analysis using the differentially expressed genes in each cluster combined with gene connectivity tools identified 1308 unique genes and a predominance of the AP-1/JUN network in the CK19(+) lesions. In contrast, the CK19-negative cluster exhibited only limited molecular changes (156 differentially expressed genes versus normal liver) consistent with remodeling toward differentiated phenotype. Finally, comparative functional genomics showed a stringent clustering of CK19(+) early lesions and advanced HCCs with human HCCs characterized by poor prognosis. Furthermore, the CK19-associated gene expression signature accurately predicted patient survival (P < 0.009) and tumor recurrence (P < 0.006). Our data establish CK19 as a prognostic marker of early neoplastic lesions and strongly suggest the progenitor derivation of HCC in the rat RH model. The capacity of CK19-associated gene signatures to stratify HCC patients according to clinical prognosis indicates the usefulness of the RH model for studies of stem/progenitor-derived HCC.

Epigenetic Inhibition of Nuclear Receptor Small Heterodimer Partner Is Associated With and Regulates Hepatocellular Carcinoma Growth

Aberrant hypermethylation of promoter regions in cytosine-guanine dinucleotides (CpG) islands has been shown to be associated with transcriptional silencing of tumor-suppressor genes in many cancers. This study evaluated the methylation profile and the tumor-suppressive function of the small heterodimer partner (SHP, NR0B2) in the development of human hepatocellular carcinoma (HCC). Human HCC pathologic specimens and cell lines were used as model systems in this study. The expression of SHP is diminished in HCC pathologic specimens and cell lines by epigenetic silencing owing to SHP promoter hypermethylation. In vitro methylation decreased SHP promoter transactivation and nuclear receptor LRH-1 binding, an event that was reversed by demethylation. Overexpression of SHP inhibited HCC foci formation, arrested HCC tumor growth in xenografted nude mice, and increased the sensitivity of HCC cells to apoptotic stimuli. Further analysis of a total of 19 normal liver and 57 HCC specimens showed that down-regulation of SHP gene expression may be a common denominator of HCC. We propose that SHP functions as a novel tumor suppressor in the development of HCC. These findings provide new insight into the molecular mechanisms leading to this common cancer and may have both diagnostic and therapeutic applications.

Genome-scale profiling of gene expression in hepatocellular carcinoma: Classification, survival prediction, and identification of therapeutic targets

The heterogeneous nature of human hepatocellular carcinoma (HCC) has hampered both treatment and prognostic predictions. Gene expression profiles of human HCC were analyzed to define the molecular characteristics of the tumors and to test the prognostic value of the expression profiles. By applying global gene expression analyses, including unsupervised and supervised methods, 2 distinctive subclasses of HCC that were highly homogeneous for both the underlying biology and the clinical outcome were discovered. Tumors from the low survival subclass had strong cell proliferation and antiapoptosis gene expression signatures. In addition, the low survival subclass displayed higher expression of genes involved in ubiquitination and sumoylation, suggesting an etiologic involvement of these processes in accelerating the progression of HCC. Genes most strongly associated with survival were identified by using the Cox proportional hazards survival analysis. This approach identified a limited number of genes that accurately predicted the length of survival and provided new molecular insights into the pathogenesis of HCC. Future studies will evaluate potential diagnostic markers and therapeutic targets identified during the global gene expression studies. Furthermore, cross-species similarity of gene expression patterns will also allow prioritization of a long list of genes obtained from human gene expression profiling studies and focus on genes whose expression is altered during tumorigenesis in both species.

Orthotopic implantation of human hepatocellular carcinoma in mice: analysis of tumor progression and establishment of the BCLC-9 cell line.

To allow the longitudinal investigation of molecular events associated with the progression of human hepatocellular carcinoma (HCC), we sought to develop a murine model by orthotopic implantation of tumor fragments obtained from patients diagnosed at early stage. Tumor pieces (2 x 2 mm) were implanted on the liver surface of nu/nu mice. After xenograft growing, subsequent passages were performed to achieve long-term implant viability. Isolation of tumoral hepatocytes was done to establish new cell lines. HCC characteristics, proliferation rate, apoptotic index (terminal deoxynucleotidyl transferase-mediated nick end labeling), and expression of cell-cycle regulators (cyclins E and A, p21(Cip1), p27(Kip1), p16(INK4a), pRb, and p53) were assessed by Western Blot and immunohistochemistry, to correlate them with tumor progression. Five (50%) of the 10 primary HCCs resulted in small slow-growing liver implants. Three of them are viable after 48 months, whereas the remaining two survived for 15 and 13 months. Xenografts throughout passages exhibited a more aggressive phenotype with a poorer degree of differentiation, intense proliferation, moderate apoptosis, cell-cycle deregulation, p53 alterations, microvascular invasion, and dissemination. In one single passage, we observed critical growth delay, which was associated with significant p27(kip1) overexpression. We established the anchor-free growing BCLC-9 cell line from one xenograft. This has gains of chromosomes 7, 5p, 6q, and 9q, is hepatitis B virus-DNA positive, does not secrete alpha-fetoprotein, and has TP53 missense mutations in codons 192 and 242. The orthotopic implantation of early HCC fragments in nude mice provides a useful model to investigate the mechanisms of human HCC evolution and to establish new cell lines.

Expression of the retinoblastoma gene product in human hepatocellular carcinoma

Using the mouse anti—human retinoblastoma gene product (pRB) monoclonal antibody, PMG3-245, pRB was detected immunohistocytochemically in human hepatocellular carcinoma (HCC) tissues and a human HCC cell line, designated OCUH-16, recently established in our laboratory. This antibody reacted with human pRB and yielded a single band of approximately 110 kd from cultured OCUH16 cells. The granules that stained for pRB were found mostly in the HCC cell nuclei, with a few granules observed in the rough endoplasmic reticulum by electron microscopy. Most of the stained granules were located in the euchromatin-rich areas. The percentage of OCUH-16 cells that expressed pRB or DNA polymerase alpha (DNA-PA) decreased over time as the number of OCUH-16 cells increased. The number of HCC cells that stained for pRB in the biopsy specimens from 11 patients varied and pRB expression was well maintained in early and advanced HCC. The level of pRB expression did not correlate with the differentiation of HCC cells or the clinical prognosis. The expression of pRB statistically correlated with that of DNA-PA ( P < .01; r = .92). Some sinusoidal cells also stained for pRB. These findings imply that large deletions in the pRB gene are rare in the initiation or promotion of HCC. The correlation between pRB and DNA-PA may suggest that stained pRB participates in the proliferation of both HCC and non-HCC cells.

Phenotype Changes in Tumor Vessels Associated with the Progression of Hepatocellular Carcinoma

Human hepatocellular carcinoma (HCC) is characterized by hypervascularity and tumor staining in angiograms, and tumor angiogenesis is considered indispensable for tumor growth. HCC is also characterized by an obvious multistage process of tumor progression. To find out in which stage of human hepatocarcinogenesis angiogenesis occurs, we have carried out a pathological study, of the phenotypic changes in tumor vessels taken from surgically resected liver tumors showing each step of the progression. Eleven early advanced HCCs (advanced HCC component in early HCC nodule, eAd HCC), seven early HCCs (eHCC) and six adenomatous hyperplasias (AH), the non-tumorous liver surrounding each and five normal livers were studied by lectin histochemistry and immunohistochemistry. The sinusoidal endothelial cells from the non-tumorous liver were shown to be negative for UEA-I (Ulex europaeus I), but the endothelial cells from the sinusoidal tumor vessels in advanced HCC components were strongly positive. In AH and eHCC, half the tumors were negative and the other half focally positive. In the early HCC lesion of eAdHCC, the rate of positivity for UEA-I was a little higher than in AH and eHCC but lower than in advanced HCC lesions in eAdHCC. Immunohistochemically, laminin was not detected in the sinusoids in the non-tumorous liver or in the sinusoidal tumor vessels in AH and eHCC. In eAdHCC, however, two early HCC lesions and four advanced HCC lesions were positive. An immunohistochemical examination for muscle actin revealed an increase in arterial tumor vessels in six advanced HCC lesions and one early HCC lesion of eAdHCC. The results indicate the emergence of UEA-I-positive sinusoidal tumor vessels to be most pronounced during the progression from eHCC to advanced HCC, and laminin-positive sinusoidal tumor vessels or actin-positive arterial tumor vessels to emerge mainly in advanced HCC.

Serum Copper and Ceruloplasmin in Early and in Advanced Hepatocellular Carcinoma: Diagnostic and Prognostic Relevance

To evaluate the prognostic value of serum copper (S-Cu) and ceruloplasmin and their pathophysiologic significance in human hepatocellular carcinoma (HCC), we studied 49 patients with HCC (20 of which were submitted to partial hepatectomy) compared with 110 patients with liver cirrhosis. In HCC both S-Cu and ceruloplasmin were higher than in cirrhosis; moreover, S-Cu was correlated with the extension of HCC, evaluated by instrumental data and by surgical inspection. In cirrhotic patients, mean S-Cu was 122.9 micrograms/dl (SD, 29.3), in early HCC, 153.0 micrograms/dl (SD, 34.5), and in advanced HCC, 193.1 micrograms/dl (SD, 37.7). Variance analysis gave F = 59.4. In HCC patients S-Cu was positively correlated with ceruloplasmin and with fibrinogen. Survival, evaluated by Mantel's test stratified for surgical therapy, was longer in patients with S-Cu levels lower than 175 micrograms/dl and in those at an earlier stage. We therefore conclude that S-Cu has a relevant diagnostic value in detecting HCC also in early stage and allows prognostic evaluation as regards survival.