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We developed an efficient microRNA (miRNA) model that could predict the risk of lymph node metastasis (LNM) in hepatocellular carcinoma (HCC). We first evaluated a training cohort of 192 HCC patients after hepatectomy and found five LNM associated predictive factors: vascular invasion, Barcelona Clinic Liver Cancer stage, miR-145, miR-31, and miR-92a. The five statistically independent factors were used to develop a predictive model. The predictive value of the miRNA-based model was confirmed in a validation cohort of 209 consecutive HCC patients. The prediction model was scored for LNM risk from 0 to 8. The cutoff value 4 was used to distinguish high-risk and low-risk groups. The model sensitivity and specificity was 69.6 and 80.2 %, respectively, during 5 years in the validation cohort. And the area under the curve (AUC) for the miRNA-based prognostic model was 0.860. The 5-year positive and negative predictive values of the model in the validation cohort were 30.3 and 95.5 %, respectively. Cox regression analysis revealed that the LNM hazard ratio of the high-risk versus low-risk groups was 11.751 (95 % CI, 5.110–27.021; P < 0.001) in the validation cohort. In conclusion, the miRNA-based model is reliable and accurate for the early prediction of LNM in patients with HCC.

Background High frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma (HCC). microRNA (miR)-182-5p emerged as a high-priority miRNA in HCC and was found to be related to HCC metastasis. Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown. Methods Real-time PCR (RT-PCR) and in situ hybridization (ISH) were conducted to assess the expression of miR-182-5p in HCC cells and tissues. Cell Counting Kit-8 (CCK-8), transwell assays were performed to detected cells proliferation and migration ability. Flow cytometry assays were used to detect cell apoptosis rate, and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis. The target of miR-182-5p was validated with a dual-luciferase reporter assay and western blotting. Immunohistochemistry, immumoblotting, and immunoprecipitation were performed to test relative protein expression. Results We showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery. miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo. miR-182-5p directly targets 3′-UTR of FOXO3a and repressed FOXO3a expression, activating AKT/FOXO3a pathway to promote HCC proliferation. Notably, miR-182-5p activated Wnt/β-catenin signaling by inhibiting the degradation of β-catenin and enhancing the interaction between β-catenin and TCF4 which was mediated by repressed FOXO3a. Conclusions Consistently, miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery, and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.

Background Hepatectomy generally offers the best chance of long-term survival for patients with hepatocellular carcinoma (HCC). Many studies have shown that hepatectomy accelerates tumor metastasis, but the mechanism remains unclear. Methods An orthotopic nude mice model with palliative HCC hepatectomy was performed in this study. Metastasis-related genes in tumor following resection were screened; HCC invasion, metastasis, and some molecular alterations were examined in vivo and in vitro. Clinical significance of key gene mRNA expression was also analyzed. Results Metastasis suppressor 1 (MTSS1) located in the central position of gene function net of residual HCC. MTSS1 was up-regulated in residual tumor after palliative resection. In hepatitis B-related HCC patients undergone palliative hepatectomy, those with higher MTSS1 mRNA expression accompanied by activation of matrix metalloproteinase 2 (MMP2) in residual HCC, had earlier residual HCC detection after hepatectomy and poorer survival when compared to those with lower MTSS1. In different cell lines, the levels of MTSS1 mRNA increased in parallel with metastatic potential. MTSS1 down regulation via siRNA decreased MMP2 activity, reduced invasive potentials of HCC by 28.9 % in vitro, and averted the deteriorated lung metastatic extent in vivo. Conclusions The poor prognosis of hepatitis B-related HCC patients following palliative hepatectomy associates with elevated MTSS1 mRNA expression; therefore, MTSS1 may provide a new research field for HCC diagnosis and treatment. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0361-8) contains supplementary material, which is available to authorized users.

Purpose: Hepatocellular carcinoma (HCC) most often develops in patients infected with hepatitis B or hepatitis C virus. Differential gene expression profiling is useful for investigating genes associated with lymph node metastasis (LNM). We screened genes to identify potential biomarkers for LNM in HCC. Experimental Design: RNA was extracted from formalin-fixed specimens of paired intratumoral and peritumoral tissues of patients with lymph node–positive (n = 36) or negative (n = 36) HCC. A cDNA-mediated annealing, selection, extension, and ligation assay was done with an array of 502 known cancer-related genes to identify differentially expressed genes in 20 pairs of patients with or without LNM. Candidate biomarkers were evaluated by using immunohistochemistry and tissue microarrays in an independent cohort of 309 HCC patients who had undergone hepatectomy. Of the 309 patients, 235 (76.1%) patients were infected with hepatitis B. Results: Compared with lymph node–negative patients, lymph node–positive patients had 17 overexpressed genes and 19 underexpressed genes in intratumoral tissues, and 25 overexpressed genes and 22 underexpressed genes in peritumoral tissues. Hypoxia-inducible factor (HIF)-1α, VEGF, and matrix metalloproteinase (MMP)-2 were selected for analysis in the cohort of 309 HCC patients. We found that intratumoral protein levels of HIF-1α, VEGF, and MMP-2 were independent risk factors for developing LNM. Conclusion: We identified 83 cancer genes that were differentially expressed in lymph node–positive and lymph node–negative HCC. Our findings show that the combination of intratumoral HIF-1α, VEGF, and MMP-2 may be useful as a molecular prediction model for LNM. Clin Cancer Res; 17(16); 5463–72. ©2011 AACR.

An immune function assay has been proposed as a new strategy to monitor immunosuppression after organ transplantation. However, there are limited data regarding its role in liver transplant recipients with hepatocellular carcinoma (HCC). In this study, we sought to determine the utility of this functional assay in assessing the risk of infection, rejection, and tumor recurrence in liver transplant recipients.Immune function was determined by ImmuKnow assay that measures the amount of adenosine triphosphate (ATP) produced by CD4 (+) T cells to monitor the global immune status in 342 whole blood samples from 105 liver transplant recipients. The association between ATP value and post-transplant tumor recurrence was evaluated in 60 HCC patients. The ATP value in predicting tumor recurrence in other independent cohort of 92 recipients with HCC was analyzed prospectively.The mean ATP values of liver transplant recipients with infection (145.2 ± 87.0 ng/ml) or acute rejection (418.9 ± 169.5 ng/ml) were different from those with stable state (286.6 ± 143.9 ng/ml, P 0.05). In recipients with HCC who developed recurrent tumors, the values were significantly lower than those without recurrence (137.8 ± 66.4 vs. 289 ± 133.9 ng/ml, P 0.01); the optimal threshold value to predict post-transplant tumor recurrence was 175 ng/ml. Comparing with the patients in lower immune group (ATP ≤ 175 ng/ml), patients in the higher immune group (ATP 175 ng/ml) experienced significantly better disease-free survival (P 0.01). Multivariate Cox regression analysis showed the ATP value was an independent predictor of HCC recurrence.The immune function assay has the potential to assess the risk of infection and rejection in liver transplantation and to predict post-transplant tumor recurrence in recipients with HCC.

Primary clear cell carcinoma of the liver (PCCCL) is an uncommon variant of hepatocellular carcinoma (HCC). The prognostic factors influencing its recurrence and survival are not clarified. This study is to evaluate the predictive factors, the therapy, and prognosis of intrahepatic recurrences after resection of PCCCL. A total of 214 PCCCL patients treated by curative resection from January 1996 to March 2006 were retrospectively analyzed. Intrahepatic recurrences were classified into early (≤1 year) and late (>1 year) recurrences. The 1-, 3-, and 5-year overall survival (OS) rates for PCCCL patients were significantly better than those of HCC patients (P = .001). Serum a-fetoprotein (AFP) level, tumor size, liver cirrhosis, and vascular invasion were independent risk factors for both OS and disease-free survival (DFS) of PCCCL. Early and late intrahepatic recurrences developed in 28 patients and 71 patients, respectively. In multivariate analysis, ALT level and vascular invasion were independent risk factors for early recurrence, whereas age was the only significant risk factor for late recurrence. OS of late-recurrence group was significantly better than that of early-recurrence group (P = .001), and re-resection rate was higher in late than in early-recurrence group (P = .04). The 1-, 3-, and 5-year OS of patients with recurrence who received curative treatment was comparable to those who never had tumor recur (P = .71). PCCCL has a better prognosis and tends to recur later than HCC. Early and late recurrences of PCCCL are linked to different predictive factors. The time to recurrence and feasibility of curative treatment are the best determinants for the prognosis.

Metastasis-related recurrence often occurs in hepatocellular carcinoma (HCC) patients who receive curative therapies. At present, it is challenging to identify patients with high risk of recurrence, which would warrant additional therapies. In this study, we sought to analyze a recently developed metastasis-related gene signature for its utility in predicting HCC survival, using 2 independent cohorts consisting of a total of 386 patients who received radical resection. Cohort 1 contained 247 predominantly HBV-positive cases analyzed with an Affymetrix platform, whereas cohort 2 contained 139 cases with mixed etiology analyzed with the NCI Oligo Set microarray platform. We employed a survival risk prediction algorithm with training, test, and independent cross-validation strategies and found that the gene signature is predictive of overall and disease-free survival. Importantly, risk was significantly predicted independently of clinical characteristics and microarray platform. In addition, survival prediction was successful in patients with early disease, such as small (

Th1/Th2-like cytokine mRNA levels in non-cancerous hepatic tissues from patients with hepatocellular carcinoma (HCC) are associated with metastases and recurrence. This study evaluated the prognostic values of intratumoral and peritumoral Th1/Th2 cytokine protein levels in patients with HCC after curative resection.Two independent cohorts (A and B) of 453 patients with HCC were enrolled. Twelve Th1/Th2 cytokines in tumour and peritumoral hepatic tissues from cohort A (n=192) were quantified with enzyme-linked immunosorbent assays. This cohort was split into training and test sets which were used to identify and verify the prognostic cytokines. The prognostic values of identified cytokines were further validated in cohort B (n=261) using tissue microarray and immunohistochemical staining.In the training set, higher interleukin (IL)-2 and IL-15 levels in peritumoral liver tissues, but not in tumour tissues, were significantly associated with a decreased incidence of recurrence of intrahepatic tumour and a prolonged overall survival. This association was verified in the testing set and further validated in patients in cohort B. Importantly, this correlation remained significant in patients with early HCC. Univariate and multivariate analyses indicated that the prognostic performance of peritumoral IL-2 (HR for recurrence=0.4, 95% CI 0.3 to 0.6, p0.0001; HR for death=0.6, 95% CI 0.4 to 0.8, p=0.005) and IL-15 (HR for recurrence=0.7, 95% CI 0.5 to 0.95, p=0.025) was independent of other clinicopathological factors.Peritumoral IL-2 and IL-15 levels are useful for stratifying patients, even those with early-stage HCC, into subgroups with different prognoses after curative resection.

Background. Macrophage colony-stimulating factor 1 receptor (CSF-1R) expression in hepatocellular carcinoma (HCC) and its prognostic values are unclear. This study evaluated the prognostic values of the intratumoral and peritumoral expression of CSF-1R in HCC patients after curative resection. Methods. Tissue microarrays containing material from cohort 1 (105 patients) and cohort 2 (32 patients) were constructed. Immunohistochemistry was performed and prognostic values of these and other clinicopathological data were evaluated. The CSF-1R mRNA level was assessed by quantitative real-time polymerase chain reaction in cohort 3 (52 patients). Results. Both the CSF-1R density and its mRNA level were significantly higher in peritumoral liver tissue than in the corresponding tumor tissue. CSF-1R was distributed in a gradient in the long-distance peritumoral tissue microarray, with its density decreasing as the distance from the tumor margin increased. High peritumoral CSF-1R was significantly associated with more intrahepatic metastases and poorer survival. Peritumoral CSF-1R was an independent prognostic factor for both overall survival and time to recurrence and affected the incidence of early recurrence. However, intratumoral CSF-1R did not correlate with any clinicopathological feature. Peritumoral CSF-1R was also associated with both overall survival and time to recurrence in a subgroup with small HCCs (≤5 cm). Conclusions. Peritumoral CSF-1R is associated with intrahepatic metastasis, tumor recurrence, and patient survival after hepatectomy, highlighting the critical role of the peritumoral liver milieu in HCC progression. CSF-1R may become a potential therapeutic target for postoperative adjuvant treatment.

Metabolic syndrome and insulin resistance have been linked to increased risk of occurrence and mortality of hepatocellular carcinoma (HCC). Recently, retinol-binding protein 4 (RBP4) was clarified as a specific serological marker of insulin resistance. The aim of this study was to determine whether serum RBP4 could be used as a potential marker for predicting prognosis in patients with HCC after curative resection.Western immunoblotting and Enzyme-linked immunosorbent assay were used to measure the RBP4 expression in cell lines, supernatant, and serum. Serum RBP4 levels were compared with clinicopathological features and outcomes of patients with HCC. Furthermore, we investigated the impact of serum RBP4 level, serum C-peptide level, and HOMA-IR on overall survival (OS) and disease-free survival (DFS) of patients with HCC in the training cohort (156 patients with HCC), and then were validated in the validation cohort (105 patients with HCC).RBP4 protein overexpressed in HCC cell lines compared with normal liver cell line (P0.001) and correlated with metastatic potential. Serum RBP4 levels were associated with OS [hazard ratio (HR) = 2.208, P0.001] and DFS (HR = 1.878, P = 0.029) of patients with HCC. By multivariate analysis, the serum RBP4 level was identified as an independent factor for OS (HR = 2.170, P = 0.004) and DFS (HR = 1.769, P = 0.037) of patients with HCC. The prognostic value of serum RBP4 level was confirmed in the validation cohort.The serum RBP4 level is potential to be a useful prognostic factor for HCC after curative resection.

BACKGROUND: Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men. We examined microRNA expression patterns, survival, and response to interferon alfa in both men and women with the disease. METHODS: We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003. MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women. In addition, to validate our findings, we used quantitative reverse-transcriptase-polymerase- chain-reaction assays to measure microRNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent, prospective, randomized, controlled trials of adjuvant interferon therapy. RESULTS: In patients with hepatocellular carcinoma, the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men. Tumors had reduced levels of miR-26 expression, as compared with paired noncancerous tissues, which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma. Moreover, tumors with reduced miR-26 expression had a distinct transcriptomic pattern, and analyses of gene networks revealed that activation of signaling pathways between nuclear factor κB and interleukin-6 might play a role in tumor development. Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA. CONCLUSIONS: The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma. The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa. Copyright © 2009 Massachusetts Medical Society. All rights reserved., published_or_final_version

The peritumoral inflammatory environment is critical for the progression of intrahepatic recurrence of hepatocellular carcinoma (HCC) after curative resections. Here, we investigated the relevance of peritumoral mast cells (MCs) to HCC outcomes. Peritumoral tryptase+ MCs in addition to Foxp3+ T-regulatory cells (Tregs) were evaluated using immunohistochemistry enumeration in tissue microarrays containing 207 randomly selected HCC patients. Clinicopathological factors and postoperative outcomes were compared between high and low subgroups of MCs or Tregs. Compared to low denstiy, higher peritumoral MCs were associated with poorer clinical outcomes, and independently related to elevated 5-year recurrence incidence (54.1%vs 39.2%, P = 0.026). High-dense MCs were especially related to increased probability of early recurrence (within 2 years) (P = 0.004). We also found that peritumoral Tregs were positively correlated with MCs in density (r = 0.353, P

The ratio of serum gamma-glutamyl transferase (GGT) to alanine aminotransferase (ALT) is a parameter for assessing responses to antiviral therapies. The relationship between the prognosis of hepatitis B-associated hepatocellular carcinoma (HCC) and preoperative GGT/ALT is studied in hepatectomized Child-Pugh A patients. Two hundred and nineteen patients with hepatitis B virus-related HCC were included. Serum ALT and GGT levels were examined preoperatively and the GGT/ALT ratios were calculated. Their prognostic capabilities were evaluated by Cox-regression model. As dichotomized variables, GGT and ALT were distributed unequally (P

The inflammatory components of the liver remnant after hepatocellular carcinoma (HCC) resection are of prognostic importance. We evaluated prognostic potential of peritumoral activated hepatic stellate cells (HSCs) in 130 HCC cases. The messenger RNA (mRNA) levels of the functional genes in HSCs (ie, seprase, osteonectin, and tenascin-C), quantitated by real-time quantitative polymerase chain reaction, and the density of peritumoral Foxp3+ T-regulatory cells (Tregs) and CD68+ macrophages (MΦ), assessed immunohistochemically in tissue microarray sections, were positively correlated with the density of peritumoral activated HSCs. The density (P = .007 for recurrence-free survival [RFS] and P =.021 for overall survival [OS]) and functional genes (seprase, P = .001 for RFS; osteonectin, P = .007 for RFS and P =.021 for OS) of peritumoral activated HSCs independently contributed to high recurrence or death rates, as did peritumoral Tregs or MΦ. Moreover, peritumoral HSCs were related to more early recurrences. It is important to note that the density of peritumoral activated HSCs, in combination with seprase and osteonectin mRNA or density of Tregs and MΦ, might predict prognoses more effectively.

To examine expression profile and prognostic significance of vascular endothelial growth factor (VEGF) and its receptors in hepatocellular carcinoma (HCC) and peritumoral tissue.Expression of VEGF-A, VEGF-C, and VEGF receptor 1(VEGFR-1), VEGFR-2, and VEGFR-3 in tumor and peritumoral liver tissue was studied by immunohistochemistry in a tissue microarray from 107 patients with HCC. Unsupervised hierarchical cluster analyses were conducted to identify relevant clusters.Staining of VEGF-A, VEGF-C, VEGFR-2, and VEGFR-3 was mostly found on the tumor cells and peritumoral hepatocytes, but VEGFR-1 was mostly expressed in stromal cells. In most of the cases, the expression of VEGF-A, VEGFR-1, VEGFR-2, and VEGFR-3 in was higher in peritumoral liver tissue, while VEGF-C expression was higher in tumor. Unsupervised hierarchical clustering analysis identified four prognostically different clusters, of which cluster A was classified into the "poor prognosis group," and the other three clusters were classified into the "good prognosis group" (P = 0.047). Further analysis with a set of seven markers reproduced the same four cluster groups with significantly different recurrence free probability (RFP) (P = 0.018), and the low RFP group was associated with more intrahepatic satellite lesions. Multivariate analysis showed that classification defined by seven biomarkers was of prognostic significance (P = 0.000).Expression of VEGF and its receptors was higher in peritumoral tissue than in tumor in HCC. Seven biomarkers predicted patients' RFP, which consisted of tumoral expression of VEGF-A, VEGFR-1, and VEGF-C as well as peritumoral expression of VEGF-A, VEGFR-1, VEGFR-2, and VEGFR-3.

Prognostic factors in patients with hepatocellular carcinoma (HCC) with tumor thrombosis are not well established, especially for those given external-beam radiation therapy (EBRT). Patients (n = 136) with HCC who had portal vein (PV) or inferior vena cava (IVC) tumor thrombus received EBRT between January 1998 and October 2007. Demographic variables, laboratory values, tumor characteristics, and treatment modalities were determined at diagnosis and before EBRT. The total radiation dose ranged from 30 to 60 Gy (median, 50 Gy) and was focused on the tumor thrombi. Predictors of survival were identified using the univariate and multivariate analysis. Of the 136 patients, the tumor thrombus completely disappeared in 41 patients (30.1%), 36 patients (26.5%) had a partial response, 49 patients (36%) had stable disease, and 10 patients (7.4%) had progressive disease. On multivariate analysis, pretreatment unfavorable predictors were associated with lower albumin, higher gamma-glutamyltransferase and alpha-fetoprotein levels, poorer Child-Pugh classification, intrahepatic multifocality, lymph node metastases, poorer response to EBRT, and 2-dimension EBRT technique. Survival rates at 1, 2, and 3 years were 31.8%, 17.5%, and 8.8% for patients with PV tumor thrombi; 66.3%, 21.1%, and 15.8% for IVC tumor thrombi; and 25%, 8.3%, and 0% for PV plus IVC tumor thrombi, respectively. Overall median survival was 9.7 months. This study provides detailed information about the survival outcomes and prognostic factors of HCC with tumor thrombi in a relatively large cohort of patients treated with radiation, and the results will help in understanding the potential factors that influence survival for patients with HCC after EBRT.

Purpose: Cytokeratin 10 (CK10) was found to be expressed differently in human hepatocellular carcinoma (HCC) cell lines with different metastatic potentials in our previous research. The aim of this study was to assess the value of CK10 alone or in combination with cytokeratin 19 (CK19) in predicting tumor recurrence after curative resection in HCC patients. Experimental Design: CK10 expression in stepwise metastatic HCC cell lines and tumor tissues from 50 HCC patients was investigated using immunofluorescence assay, quantitative real-time reverse transcription-PCR, and Western blot analyses. Tumor tissue microarrays of 300 HCC patients who underwent curative resection between 1997 and 2000 were used to detect the expressions of CK10 and CK19. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Results: CK10 was overexpressed in the high metastatic HCC cell line and in tumor tissues of recurrent patients. Both univariate and multivariate analyses revealed that CK10 was a significant predictor for overall survival (OS) and disease-free survival, and that CK19 was a significant predictor for OS. CK10 expression was correlated with poor prognosis regardless of α-fetoprotein, tumor-node-metastasis stage, and vascular invasion. The 7-year OS and disease-free survival rates in CK10+ and/or CK19+ patients were 30.0% and 37.6%, respectively, which were significantly lower than that of CK10−/CK19− patients (56.1% and 60.0%, respectively; P < 0.001). Conclusion: CK10 is associated with HCC invasiveness. CK10 alone, or in combination with CK19, can be a novel predictor for poor prognosis of HCC patients after curative resection.

The heterogeneous nature of hepatocellular carcinoma (HCC) and the lack of appropriate biomarkers have hampered patient prognosis and treatment stratification. Recently, we have identified that a hepatic stem cell marker, epithelial cell adhesion molecule (EpCAM), may serve as an early biomarker of HCC because its expression is highly elevated in premalignant hepatic tissues and in a subset of HCC. In this study, we aimed to identify novel HCC subtypes that resemble certain stages of liver lineages by searching for EpCAM-coexpressed genes. A unique signature of EpCAM-positive HCCs was identified by cDNA microarray analysis of 40 HCC cases and validated by oligonucleotide microarray analysis of 238 independent HCC cases, which was further confirmed by immunohistochemical analysis of an additional 101 HCC cases. EpCAM-positive HCC displayed a distinct molecular signature with features of hepatic progenitor cells including the presence of known stem/progenitor markers such as cytokeratin 19, c-Kit, EpCAM, and activated Wnt-β-catenin signaling, whereas EpCAM-negative HCC displayed genes with features of mature hepatocytes. Moreover, EpCAM-positive and EpCAM-negative HCC could be further subclassified into four groups with prognostic implication by determining the level of α-fetoprotein (AFP). These four subtypes displayed distinct gene expression patterns with features resembling certain stages of hepatic lineages. Taken together, we proposed an easy classification system defined by EpCAM and AFP to reveal HCC subtypes similar to hepatic cell maturation lineages, which may enable prognostic stratification and assessment of HCC patients with adjuvant therapy and provide new insights into the potential cellular origin of HCC and its activated molecular pathways. [Cancer Res 2008;68(5):1451–61]

SummaryHepatocellular carcinoma (HCC) is an aggressive malignancy mainly due to metastases or postsurgical recurrence. We postulate that metastases are influenced by the liver microenvironment. Here, we show that a unique inflammation/immune response-related signature is associated with noncancerous hepatic tissues from metastatic HCC patients. This signature is principally different from that of the tumor. A global Th1/Th2-like cytokine shift in the venous metastasis-associated liver microenvironment coincides with elevated expression of macrophage colony-stimulating factor (CSF1). Moreover, a refined 17 gene signature was validated as a superior predictor of HCC venous metastases in an independent cohort, when compared to other clinical prognostic parameters. We suggest that a predominant humoral cytokine profile occurs in the metastatic liver milieu and that a shift toward anti-inflammatory/immune-suppressive responses may promote HCC metastases.

To evaluate the role of radiotherapy (RT) for hepatocellular carcinoma (HCC) patients with abdominal lymph node (LN) metastasis at our institution in the past 7 years.We identified 125 patients with HCC metastasis to regional LNs treated with or without external beam RT (EBRT) between 1998 and 2004. Clinical characteristics collected included alpha-fetoprotein status, gamma-glutamyltransferase, status of intrahepatic tumors (size and number), previous therapy for intrahepatic tumors, metastatic LN status (location, number, and size), tumor thrombi, and Child-Pugh classification. Of the 125 patients, 62 received local limited EBRT and were classified as the EBRT group. They received locoregional LN irradiation. The tumor dose ranged from 40 to 60 Gy in daily 2.0-Gy fractions, 5 times weekly. Another 63 patients, who did not receive EBRT, were selected from hospitalized patients in the same period and were classified as the non-EBRT group. The parameters studied included survival rates and tumor response to EBRT both as demonstrated by clinical symptoms and as seen on CT. The Kaplan-Meier method was used to evaluate the survival rates, and the Cox regression model was used to identify predictors of outcome.After EBRT, partial responses and complete responses were observed in 37.1% and 59.7% of patients, respectively. The median survival was 9.4 months (95% confidence interval 5.8-13.0) for the EBRT group and 3.3 months (95% confidence interval, 2.7-3.9) for the non-EBRT group (p0.001). Multivariate analysis showed that multiple intrahepatic primary tumors, occurrence of tumor thrombi, no therapy for intrahepatic tumors, and greater Child-Pugh classification were related to a poorer prognosis in all patients. In the EBRT group, the survival periods decreased as the distance of LN involvement from the liver increased (following the natural flow of lymph) and was also associated with the intrahepatic primary tumor size. The incidence of death resulting from LN-related complications was lower in the EBRT group.Lymph node metastasis from HCC is sensitive to EBRT. EBRT with 25 fractions of 2 Gy is an effective palliative treatment for patients with LN metastases from HCC presenting with good performance status and may prolong overall survival.

The model of Notch-driven carcinogenesis and development of hepatocellular carcinoma remains controversial and is based on observations of developmental stage- and dose-dependent Notch activation. In this study, the relevance of the spatial distribution of Notch cascade members to the promotion of hepatocellular carcinoma metastasis was evaluated. The spatial expression patterns of the members of the Jagged1 (JAG1)/Notch1 cascade in HCC were evaluated in a tissue microarray of 112 tumors and 46 peri-tumors. Regulation of JAG1/Notch1 on osteopontin (OPN) was evaluated by RNA interference. Tumor cells with JAG1 expressed on the membrane (JAG1(Mem)) were more likely to undergo extrahepatic metastasis [p

BACKGROUND: Peritumoral liver tissue could play a potential role in hepatocellular carcinoma (HCC) progression and patient survival via angiogenesis- and lymphangiogensis-related factors. The prognostic role of these factors in hepatocytes and stromal cells in HCC patients after curative resection remains to be explored. METHODS: Tumor tissue and surrounding peritumoral tissue were obtained from 145 resected HCC patients without lymph node metastasis (LNM) and 37 resected HCC patients with LNM. Tissue microarrays were constructed from duplicate cores of tumor tissue and surrounding peritumoral tissue from each resected specimen. Immunohistochemistry and real-time polymerase chain reaction were used to evaluate the expression of vascular endothelial growth factor-A (VEGF-A), VEGF-C, VEGF receptor-1(VEGFR-1), VEGFR-2, and VEGFR-3. Macrophage infiltration was determined by CD68 staining. Correlations between the expression of these factors and overall survival (OS) and time to recurrence (TTR) were studied. RESULTS: The peritumoral expression of VEGF-A, VEGF-C, VEGFR-1, VEGFR-2, and VEGFR-3 were significantly higher than expression of these factors in tumors. VEGFR-1 was mostly located in peritumoral macrophages, while VEGF-C and VEGFR-3 were mostly located in peritumoral hepatocytes. HCC with high peritumoral co-expression of VEGF-C, VEGFR-1, and VEGFR-3 was associated with higher peritumoral distribution of macrophages (0.87%±0.26% versus 0.45%±0.20%), LNM (32.4% versus 12.0%), shorter TTR (10.2 months versus 34.5 months), and poor prognosis (19.4 months versus 49.3 months). CONCLUSION: Expression of VEGF-C, VEGFR-1, and VEGFR-3 in peritumoral liver tissue is associated with a unique type of HCC that has a poorer outcome after hepatectomy.

To identify independent predictors of survival in patients with lymph node (LN) metastases from hepatocellular carcinoma (HCC) after external beam radiotherapy (EBRT). There were 191 patients with LN metastases from HCC received EBRT enrolled in the study cohort. EBRT was designed to focus on the LNs and a median dose of 50 Gy (range 40–60 Gy) was delivered. Treatment response was assessed by the WHO response criteria. Factors such as demographic data, tumor characteristics, and treatment modalities were determined before EBRT. Predictors of survival were identified by univariate and multivariate analysis. The median survival was 8.0 months for all patients. Factors including Child-Pugh status (p = 0.009), intrahepatic tumor control (p = 0.015), LN location (p = 0.015), and response to EBRT (p

Two decades have gone by since the earlier trials of alpha-fetoprotein (AFP) screening for hepatocellular carcinoma (HCC) were conducted in Africa and China. It is accepted that early detection, diagnosis and treatment of HCC remains an important target to be achieved before a breakthrough appears on the primary prevention of HCC. In the present study, screening investigations were performed in a high risk population of HCC, defined as persons who had hepatitis, blood transfusions, a family history of HCC, and were hepatitis B virus carriers. Ultrasonography combined with AFP serosurvey was accepted as an effective screening procedure to detect small HCC. Early diagnosis of HCC was not difficult if tumour markers and medical imaging were combined. Early resection has been proven to prolong survival of patients with small HCC. Repeated intralesional ethanol injection is an alternative treatment to surgery, while transcatheter arterial embolization is a less effective alternative. Re-resection of subclinical recurrence after curative resection has proven of merit in prolonging survival even further. Resection of small HCC remains an important approach in getting long-term HCC survival and to improving 5-year survival rates. It is more effective than treatment of large HCC. Studies on the secondary prevention of HCC have stimulated research into tumour markers, the natural history and cellular origin of HCC and oncogenes. However, the issue of 'cost-effectiveness' remains to be evaluated.

The poor prognosis of hepatocellular carcinoma (HCC) was partly a result of the majority of unresectable HCCs in clinical patients. Fortunately, with the progress of regional cancer therapies and multimodality treatment, some of the localized unresectable HCCs were converted to resectable ones. During the period 1960-1994, 72 of the 663 patients with surgically verified unresectable HCCs have been converted to resectable. Successful cytoreduction with median diameter reduced from 10 cm to 5 cm was mainly a result of the triple or double combination treatment with hepatic artery ligation, hepatic artery cannulation with infusion, radioimmunotherapy, and fractionated regional radiotherapy. The interval between the first operation and the sequential resection was 5 months. The operative mortality was 1.4% for sequential resection, and the 5-year survival was 62.1%. Analysis of factor influencing sequential resection rate revealed HCCs that were single nodule, well encapsulated, situated at right lobe or hepatic hilum, associated with micronodular cirrhosis, and treated with triple or double combination modalities had higher sequential resection rate as compared to their counterparts. Analysis of factors influencing survival after sequential resection revealed that HCCs with a solitary tumor confined in one lobe, without tumor embolus, and without residual cancer in specimen of sequential resection, had longer survival. It is suggested that localized unresectable, solitary, well encapsulated, right lobe or hilar HCC, associated with micronodular cirrhosis, will be good candidates for cytoreduction and sequential resection; and HCCs with unilateral involvement, without tumor embolus, and with complete necrosis of tumor after multimodality treatment favored better prognosis.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. The aim of this study was to elucidate the expression and mechanism of LRP1 in hepatocellular carcinoma (HCC). METHODS: LRP1 expression in 4 HCC cell lines and 40 HCC samples was detected. After interruption of LRP1 expression in a HCC cell line either with specific lentiviral-mediated shRNA LRP1 or in the presence of the LRP1-specific chaperone, receptor-associated protein (RAP), the role of LRP1 in the migration and invasion of HCC cells was assessed in vivo and in vitro, and the expression of matrix metalloproteinase (MMP) 9 in cells and the bioactivity of MMP9 in the supernatant were assayed. The expression and prognostic value of LRP1 were investigated in 327 HCC specimens. RESULTS: Low LRP1 expression was associated with poor HCC prognosis, with low expression independently related to shortened overall survival and increased tumor recurrence rate. Expression of LRP1 in non-recurrent HCC samples was significantly higher than that in early recurrent samples. LRP1 expression in HCC cell lines was inversely correlated with their metastatic potential. After inhibition of LRP1, low-metastatic SMCC-7721 cells showed enhanced migration and invasion and increased expression and bioactivity of MMP9. Correlation analysis showed a negative correlation between LRP1 and MMP9 expression in HCC patients. The prognostic value of LRP1 expression was validated in the independent data set. CONCLUSIONS: LRP1 modulated the level of MMP9 and low level of LRP1 expression was associated with aggressiveness and invasiveness in HCCs. LRP1 offered a possible strategy for tumor molecular therapy.

In the present study, we evaluated the prognostic value of intratumoral and peritumoral expression of connective tissue growth factor (CTGF), transforming growth factor-beta 1 (TGF-β1), and interleukin-11 (IL-11) in patients with hepatocellular carcinoma (HCC) after curative resection. Expression of CTGF, TGF-β1, and IL-11 was assessed by immunohistochemical staining of tissue microarrays containing paired tumor and peritumoral liver tissue from 290 patients who had undergone hepatectomy for histologically proven HCC. The prognostic value of these and other clinicopathologic factors were evaluated. The median follow-up time was 54.3 months (range, 4.3–118.3 months). High intratumoral CTGF expression was associated with vascular invasion (P = 0.015), intratumoral IL-11 expression correlated with higher tumor node metastasis (TNM) stage (P = 0.009), and peritumoral CTGF overexpression correlated with lack of tumor encapsulation (P = 0.031). Correlation analysis of these proteins revealed that intratumoral CTGF and IL-11 correlated with high intratumoral TGF-β1 expression (r = 0.325, P

Background. Hepatocellular carcinoma (HCC) most commonly develops in patients who have a viral infection, especially in the case of hepatitis B virus (HBV), and in patients with a chronic liver disease. HCC patients with bone metastasis (BM) suffer from pain and other symptoms that significantly reduce their quality of life. Identification of patients who are at high risk for BM after undergoing potentially curative treatment for HCC remains challenging. Here, we aimed to identify HCC BM-related genes and proteins to establish prediction biomarkers. Methods. RNA was extracted from 48 pairs of intratumoral and peritumoral formalin-fixed, paraffin-embedded tissue from HCC patients with and without BM. A cDNA-mediated annealing, selection, extension and ligation assay containing 502 cancer-related genes was used to identify novel BM-associated genes. An additional independent study with 350 HCC patients who had undergone hepatectomy was conducted to evaluate the expression of candidate genes at the protein level using immunohistochemistry on tissue microarrays (TMAs). Of the 350 patients, 273 (78.0%) were infected with HBV. Results. Seven intratumoral genes and 17 peritumoral genes were overexpressed in patients with BM, whereas 15 intratumoral genes and 28 peritumoral genes were underexpressed in patients with BM. We selected the following four genes for further analysis because they were differentially expressed in the cancer gene-specific microarray and were previously reported to be associated with BM: connective tissue growth factor (CTGF), matrix metalloproteinase-1 (MMP-1), transforming growth factor β1 (TGF-β1), and interleukin-11 (IL-11). We assessed the protein expression of these selected genes using immunohistochemistry on TMAs including 350 HCC patient specimens. We determined that expression of intratumoral CTGF, intratumoral IL-11, and peritumoral MMP-1 were independent prognostic factors for developing BM in HCC patients. Combining intratumoral CTGF and IL-11 expression was also an independent risk factor for BM development. Conclusions. Sixty-seven genes were differentially expressed in HCC patients with and without BM. High intratumoral CTGF, positive IL-11, and high peritumoral MMP-1 expression were associated with BM after hepatectomy. Intratumoral CTGF expression combined with IL-11 expression may serve as a useful predictive biomarker for HCC BM.

In the 1950s, hepatic lobectomy for huge hepatocellular carcinoma (HCC) has benefited 5-10% of HCC patients; in the 1970s, limited resection for small HCC and reresection for recurrence have benefited another 5-10% HCC patients. Cytoreduction and sequential resection for unresectable HCC might be of benefit to a further 5-10% HCC patients in the 1990s. Analysis of 1,642 patients with pathologically proven HCC in 1959-1991 demonstrated that the series 5-year survival has increased from 3.0% (n = 136) in the 1960s, to 12.2% (n = 440) in the 1970s, to 40.2% (n = 1,066) in the 1980s, which was correlated to the increasing number of limited resections for small HCC, reresections for subclinical recurrence, and cytoreductions and sequential resections for portions of unresectable HCC. With the advances in early detection, multimodality treatment, and changing concepts in surgical oncology, the role of surgery in the treatment of HCC has increased.

We performed our study to determine whether plasma macrophage migration inhibitory factor (MIF) levels have diagnostic and prognostic value in hepatocellular carcinoma (HCC) patients. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used to measure the expression of MIF in plasma and tissues, respectively. Plasma MIF levels were compared to HCC occurrence, clinicopathological features and outcomes. Cutpoints of plasma MIF levels for diagnosis and prognosis were, respectively, determined by receiver operating characteristic analysis and X-tile in corresponding training cohort, and then were confirmed in the validation cohort. The postoperative plasma MIF levels of HCC patients were detected in an independent cohort (80 HCC patients). As a result, MIF expression in situ was mainly observed in the cytoplasm of HCC cells. Intratumoral MIF expression was positively correlated with plasma MIF levels (r = 0.759, p < 0.001). Compared to serum α-fetoprotein (AFP), plasma MIF had a higher diagnostic value for discrimination of HCC from controls at 35.3 ng/ml. With determined cutpoints, plasma MIF levels demonstrated a significant association with overall survival (OS) and disease-free survival (DFS) of HCC patients even in patients with normal serum AFP levels and Tumor Node Metastasis (TNM) stage I. In addition, the plasma MIF levels were identified as an independent factor for OS [hazard ratio (HR) = 1.754; p = 0.012] and DFS (HR = 2.121; p < 0.001). Plasma MIF levels decreased markedly within 30 days after tumor resection (p < 0.001). Therefore, plasma MIF levels have potential as a diagnostic and prognostic factor for HCC.

Vascular endothelial growth factor-targeted therapy is a promising treatment for hepatocellular carcinoma (HCC), but its clinical benefit is often accompanied by acquired resistance. In animal studies, antiplacental growth factor therapy is effective with less resistance. The role of placental growth factor (PlGF) in the progression of HCC is not clear. In our study, we used immunohistochemistry in tissue microarrays to investigate PlGF expression in tumor and peritumoral liver tissues from 105 patients with HCC. Intratumoral and peritumoral PlGF mRNA expression was analyzed in another cohort of 37 patients. Peritumoral PlGF expression was significantly higher than intratumoral PlGF expression (p < 0.001). Intratumoral PlGF expression was not associated with patients' overall survival (OS) or time to recurrence (TTR). However, peritumoral PlGF expression, which was associated with tumor size, presence of intrahepatic metastasis, TNM stage and Barcelona Clinic Liver Cancer stage, was an independent risk factor for OS (p = 0.026) and TTR (p = 0.041). The prognostic value of peritumoral PlGF expression was further validated in a validation cohort (n = 394). We inferred that the elevation of PlGF in peritumoral liver might be induced by hypoxia. We found that peritumoral PlGF expression was associated with hypoxia-inducible factor-1α (p = 0.017). PlGF expression was elevated in L02, a hepatic cell line, under hypoxic conditions in vitro. These findings indicate that high peritumoral PlGF expression is associated with tumor recurrence and survival after resection of HCC. PlGF could be a target of adjuvant therapy and deserves further investigations.

Purpose Lymph node metastasis (LNM) is a chief cause of morbidity and mortality in patients with hepatocellular carcinoma (HCC) after hepatectomy. The aim of this study was to investigate the relationship between the expression of CXCR4 and vascular endothelial cell growth factor (VEGF)-C and the clinicopathological features of HCC with LNM. Methods Immunohistochemical staining for CXCR4 and VEGF-C was performed on tissue microarrays that were constructed using tumor specimens from patients with HCC with (N = 123) or without (N = 145) LNM. The relationship between the clinicopathological features of HCC and the expression of CXCR4 and VEGF-C was analyzed using the Pearson chi(2) test, logistical regression analysis, and receiver operating characteristic analysis. Results Nuclear CXCR4 expression and VEGF-C expression were positively correlated with LNM and poor outcome in HCC. Moreover, nuclear CXCR4 expression was positively correlated with VEGF-C expression (correlation coefficient 0.256). Receiver operating characteristic analysis revealed that both factors were predictive of HCC LNM {CXCR4: area under the curve, 0.695 [95% confidence interval (CI), 0.630-0.759; VEGF-C: area under the curve, 0.629 (95% CI, 0.562-0.695]}. Patients with tumors exhibiting high nuclear CXCR4 expression or high VEGF-C expression had significantly poorer overall survival than those with low tumor expression of the corresponding factors. Multivariate analysis showed that UICC T stage [odds ratio (OR), 1.615, 95% CI, 1.306-1.997], nuclear CXCR4 expression (OR, 3.998; 95% CI, 2.706-5.907), and VEGF-C expression (OR, 1.903; 95% CI, 1.203-3.011) were independent risk factors for developing HCC LNM. Discussion These findings suggest that nuclear CXCR4 expression and VEGF-C expression can be used to identify patients with HCC at high risk for developing LNM.

BACKGROUND: The current study was performed to identify clinical features and independent predictors of survival in patients with bone metastases from hepatocellular carcinoma (HCC). METHODS: Patients (n = 205) with bone metastases from HCC received external beam radiotherapy (EBRT) between 1997 and 2007. Demographic variables, laboratory values, tumor characteristics, and treatment modalities were determined before EBRT. The total radiation dose ranged from 32 to 66 grays (Gy) (median, 50 Gy) and was focused on the involved bone. RESULTS: In 80 of 205 (39.0%) patients with bone metastasis from HCC, tumors were characterized by osteolytic, expansile soft-tissue masses. Overall pain relief from EBRT occurred in 204 patients (99.5%). No consistent dose-response relation was found for palliation of bone metastases with doses between 32 and 66 Gy (P = .068), but the retreatment rate was higher in patients with expansile soft tissue. On univariate analysis, shorter survival was associated with poorer Karnofsky performance status (KPS), higher γ-glutamyltransferase and α-fetoprotein levels, tumor size >5 cm, uncontrolled intrahepatic tumors, multifocal bone lesions, involvement of spinal vertebrae, extraosseous metastases, and a shorter disease-free interval after an initial diagnosis of HCC. On multivariate analysis, pretreatment-unfavorable predictors were associated with lower KPS, higher tumor markers, and uncontrolled intrahepatic tumor when KPS was considered. The median survival was 7.4 months. CONCLUSIONS: The results of the current study provide detailed information regarding clinical features, survival outcomes, and prognostic factors for HCC with bone metastases in a relatively large cohort of patients treated with EBRT. These prognostic factors will help in determining which dose and fraction are appropriate. Cancer 2009. © 2009 American Cancer Society.

Background. Osteopontin (OPN) plays important roles in tumor progression and metastasis through binding to CD44 and integrin. The goal of this study was to elucidate the prognostic significance of OPN and CD44 in hepatocellular carcinoma patients. Methods. Tumor tissue microarray was used to detect the expression levels of OPN and CD44 in 302 hepatocellular carcinoma patients undergoing curative resection between 1997 and 2000 at our institute. Clinicopathologic data for these patients were investigated. The prognostic effects of OPN and CD44 were evaluated using the Kaplan–Meier method and compared using the log-rank test. The Spearman rank test and Fisher's exact test were applied to demonstrate correlations. Results. Both OPN and CD44 were independent predictors for overall survival and disease-free survival. When OPN and CD44 were taken into consideration together, the predictive range was extended and the sensitivity was improved, especially for those patients with normal serum α-fetoprotein levels. The 8-year overall survival and disease-free survival rates in OPN+ and/or CD44+ patients were 28.2% and 25.6%, respectively, which were significantly lower than those of OPN−CD44− patients (52.1% and 51.6%, respectively). Conclusions. OPN combined with CD44 is a promising independent predictor of tumor recurrence and survival in hepatocellular carcinoma patients.

Purpose To investigate prognostic values of the intratumoral and peritumoral expression of macrophage colony-stimulating factors (M-CSF) in hepatocellular carcinoma (HCC) patients after curative resection. Patients and Methods Expression of M-CSF and density of macrophages (MΦ) were assessed by immunohistochemistry in tissue microarrays containing paired tumor and peritumoral liver tissue from 105 patients who had undergone hepatectomy for histologically proven HCC. Prognostic value of these and other clinicopathologic factors was evaluated. Results Neither intratumoral M-CSF nor MΦ density was associated with overall survival (OS) or disease-free survival (DFS). High peritumoral M-CSF and MΦ density, which correlated with large tumor size, presence of intrahepatic metastasis, and high TNM stage, were independent prognostic factors for both OS (P = .001 and P < .001, respectively) and DFS (P = .001 and P = .003, respectively) and affected incidence of early recurrence. In a small HCC subset, peritumoral M-CSF was also correlated with both OS and DFS (P = .038 and P = .001, respectively). The combination of peritumoral M-CSF and MΦ had a better power to predict the patients' death and disease recurrence (P < .001 for both). Conclusion High peritumoral M-CSF and MΦ were associated with HCC progression, disease recurrence, and poor survival after hepatectomy, highlighting the importance of peritumoral tissue in the recurrence and metastasis of HCC. M-CSF and MΦ may be targets of postoperative adjuvant therapy.

BACKGROUND. Few studies have investigated the pathologic types and prognosis of hepatocellular carcinoma (HCC) with lymph node metastasis (LNM). The purpose was to explore pathologic types and pertinent therapy of HCC with LNM. METHODS. An immunohistochemical study for CK19 and OV-6 was performed on tissue microarrays of HCC with LNM (n = 47) and those without LNM (n = 125). The clinicopathologic factors and patient survival were analyzed. RESULTS. Immunopositivity of CK19 and OV-6 in HCC with LNM were higher than that in 125 HCC without LNM (27.7% vs 5.6%, P = .000; 29.8% vs 12.8%, P = .009); their expressions were significantly correlated in HCC with LNM (correlation coefficient: 0.637, P = .000). The CK19 expression and tumor (T) classification of American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) tumor staging system were 2 independent risk factors for developing LNM (odds ratio [OR], 5.170, 95% confidence interval [CI], 1.840-14.528, P = .002; OR, 1.879, 95% CI, 1.236–2.857, P = .003). The CK19(+) group had shorter median survival (7.7 months vs 21.7 months, P = .013); CK19 expression was the independent prognostic factor for overall survival in HCC with LNM and was correlated with proliferating cell nuclear antigen labeling index and matrix metalloproteinase-9 expression (correlation coefficient: 0.484, P = .001 and 0.459, P = .001, respectively). CONCLUSIONS. CK19 expression and AJCC/UICC T classification were 2 independent risk factors for developing LNM in HCC. CK19 expression was the independent prognostic factor for HCC with LNM. It is of clinical significance for treatment modalities to differentiate HCC with intrahepatic cholangiocarcinoma-like differentiation (CK19[+]) from one with a higher T classification (CK19[−]). Cancer 2008. © 2008 American Cancer Society.

MicroRNAs (miRNAs) have been used as cancer-related biomarkers. Hepatocellular carcinoma (HCC) is an aggressive cancer with a dismal outcome largely due to metastasis and postsurgical recurrence. We investigated whether the expression of certain miRNAs are associated with HCC metastasis. We examined the miRNA expression profiles of 482 cancerous and noncancerous specimens from radical resection of 241 patients with HCC. Using a supervised algorithm and a clinically well-defined cohort of 131 cases, we built a unique 20-miRNA metastasis signature that could significantly predict (P < 0.001) primary HCC tissues with venous metastases from metastasis-free solitary tumors with 10-fold cross-validation. However, significant miRNAs could not be identified from the corresponding noncancerous hepatic tissues. A survival risk prediction analysis revealed that a majority of the metastasis-related miRNAs were associated with survival. Furthermore, the 20-miRNA tumor signature was validated in 110 additional cases as a significant independent predictor of survival (P = 0.009) and was significantly associated with both survival and relapse in 89 cases of early stage HCC (P = 0.022 and 0.002, respectively). These 20 miRNAs may provide a simple profiling method to assist in identifying patients with HCC who are likely to develop metastases/recurrence. In addition, functional analysis of these miRNAs may enhance our biological understanding of HCC metastasis.

Purpose: Our previous studies have shown that chromosome 8p deletion correlates with metastasis of hepatocellular carcinoma (HCC). This study was to determine whether 8p deletion could be used in predicting the prognosis of patients with HCC, particularly in those with early stage of HCC. Experimental Design: A total of 131 patients with tumor-node-metastasis (TNM) stage I of HCC who underwent curative liver resection were enrolled. Loss of heterozygosity (LOH) was examined using 10 microsatellite markers at chromosome 8p, as well as 14 microsatellites at chromosome 1p, 17p, 4q, 13q, and 16q, and their association with 5-year overall survival (OS) and disease-free survival (DFS) of patients was analyzed. Results: In the entire cohort of patients, the mean LOH frequency at these 24 loci was 43.2%; LOH frequencies at D8S298 and D1S199 were 31.5% and 33.7%, respectively. LOH at D8S298 was associated with a worse 5-year OS (P = 0.008) and DFS (P = 0.038) in patients with TNM stage I of HCC. Likewise, the patients with LOH at D1S199 had a worse 5-year OS (P < 0.001) and DFS (P = 0.014) compared with those without LOH at D1S199. In multivariate analyses, LOH at D8S298 was an independent predictor of decreased DFS (hazard ratio, 0.372; 95% 95% confidence interval, 0.146-0.948; P = 0.038), whereas LOH at D1S199 was an independent predictor of decreased OS (hazard ratio, 0.281; 95% confidence interval, 0.123-0.643; P = 0.003). Conclusions: LOH at D8S298 and D1S199 is independently associated with a worse survival in patients with TNM stage I of HCC after curative resection and could serve as novel prognostic predictors for this subgroup of patients.

Background To study lymph node metastasis (LNM) and prognosis in patients with operable hepatocellular carcinoma (HCC) as well as the value of routine complete lymphadenectomy. Few studies have been reported on LNM in patients with operable HCC. Methods Lymph node enlargement of 968 patients with operable HCC was carefully explored and LNM was diagnosed by typical intraoperative findings or pathology. Results Forty-nine (5.1%) patients had LNM, which was associated with advanced tumor properties. The 1-, 3-, and 5-year overall survival in patients with LNM was poorer than those without LNM (62.0%, 31.0%, and 26.0% vs. 81.0%, 62.0%, and 47.0%, P = 0.000). The 1-, 3-, and 5-year overall survival in patients who received complete lymphadenectomy (n = 26) was poorer than those without LNM (68.0%, 31.0%, and 31.0% vs. 81.0%, 62.0%, and 47.0%, P = 0.017), and was not better than patients who received chemotherapy or radiotherapy (P = 0.944). Conclusion The incidence of LNM in operable HCC patients was low, and patients with LNM had a poorer prognosis. LNM status determined the disease-free survival but not the overall survival of HCC. The complete lymphadenectomy did not improve overall survival, as compared with chemotherapy or radiotherapy. J. Surg. Oncol. 2007;96:37–45. © 2007 Wiley-Liss, Inc.