Novel method for classification of prion diseases by detecting PrPres signal patterns from formalin-fixed paraffin-embedded samples.

Prion disease is an infectious and fatal neurodegenerative disease. Western blotting (WB)-based identification of proteinase K (PK)-resistant prion protein (PrPres) is considered a definitive diagnosis of prion diseases. In this study, we aimed to detect PrPres using formalin-fixed paraffin-embedded (FFPE) specimens from cases of sporadic Creutzfeldt-Jakob disease (sCJD), Gerstmann-Sträussler-Scheinker disease (GSS), glycosylphosphatidylinositol-anchorless prion disease (GPIALP), and V180I CJD. FFPE samples were prepared after formic acid treatment to inactivate infectivity. After deparaffinization, PK digestion was performed, and the protein was extracted. In sCJD, a pronounced PrPres signal was observed, with antibodies specific for type 1 and type 2 PrPres exhibited a strong or weak signals depending on the case. Histological examination of serial sections revealed that the histological changes were compatible with the biochemical characteristics. In GSS and GPIALP, prion protein core-specific antibodies presented as PrPres bands at 8-9 kDa and smear bands, respectively. However, an antibody specific for the C-terminus presented as smears in GSS, with no PrPres detected in GPIALP. It was difficult to detect PrPres in V180I CJD. Collectively, our findings demonstrate the possibility of detecting PrPres in FFPE and classifying the prion disease types. This approach facilitates histopathological and biochemical evaluation in the same sample and is safe owing to the inactivation of infectivity. Therefore, it may be valuable for the diagnosis and research of prion diseases.

Role of growth factor receptor bound protein 7 in hepatocellular carcinoma.

The human growth factor receptor-bound protein 7 (Grb7) is an adaptor molecule and is related to cell invasion. In this present study, we investigated the clinical and biological significance of Grb7 expression in human hepatocellular carcinoma (HCC). We reviewed 64 consecutive patients who had undergone liver resection for HCC, and we investigated the correlation between Grb7 expression and clinical outcome. To analyze the biological behavior of Grb7 in vitro and in vivo, we established Grb7 stable knockdown HCC cells using RNA interference technology. The positive staining of Grb7 protein was correlated with portal venous invasion (P < 0.01), hepatic venous invasion (P < 0.01), and intrahepatic metastasis (P < 0.05). Positive expression of Grb7 was significantly correlated with focal adhesion kinase (FAK) protein levels in HCC (P < 0.01). The Grb7- and FAK-positive group showed a significantly poorer prognosis as compared with the Grb7- and FAK-negative group (P < 0.05). Grb7 knockdown HCC cells exhibited significantly lower levels of invasion potential (P < 0.05) and motility (P < 0.05) than the control cells in vitro; moreover, Grb7 knockdown HCC cells showed delayed onset of the tumors compared with the control cells in vivo. Grb7 expression can modulate the invasive phenotype of HCC. Grb7 plays an important role in HCC progression and is strongly associated with expression of FAK. Grb7 could be a therapeutic target in HCC.

Prognosis of early hepatocellular carcinoma after hepatic resection.

BACKGROUND/AIMS: Clinicopathologic characteristics and postoperative prognosis of early hepatocellular carcinoma (eHCC) have not been clarified. METHODOLOGY: Early HCC was designated as tumor purely composed of well-differentiated HCC not containing moderately or poorly differentiated component. Twenty seven patients with early HCC among 515 patients underwent hepatic resection for HCC were analyzed. RESULTS: The survival rate at 5 years after hepatic resection of the patients with early HCC was 76%. No clinicopathologic factors correlated with the survival. The postoperative recurrence occurred in 19 (70%) patients, and nine patients survived more than five years free of recurrence. The levels of aspartate aminotransferase and alanine aminotransferase of the patients with recurrence within five years (n = 9) were much higher than those of the patients without recurrence (n = 18). The prognosis after recurrence of the patients with more than three tumors was significantly worse than that of the patients with one or two tumors (P < 0.01). CONCLUSIONS: The survival rate of early HCC after hepatic resection is favorable, however, recurrence is considerably frequent and it depends on the degree of inflammation of underling liver disease. Therefore, the control of inflammation of liver parenchyma and the preservation of liver function may be most important in patients with early HCC.

Expression profile of class I histone deacetylases in human cancer tissues.

Histone deacetylase (HDAC) activity is one of the widely used and well-established mechanisms for regulation of various genes in cancer. To identify which subtype of class I HDACs are overexpressed in cancers, we analyzed the expression of class I HDAC isotypes composed of HDAC1, 2, 3 and 8 in several cell lines and human cancer tissues, including cancer of the stomach, esophagus, colon, prostate, breast, ovary, lung, pancreas and thyroid. The results showed that >75% of human cancer tissues and their corresponding non-cancerous epithelium showed high expression of these class I HDACs. However, the immunoreactivity of HDAC8 in both prostatic cancer tissue and non-cancerous prostate glands was lower than that in other cancer tissues. Furthermore, 5-40% of cancer tissues overexpressed class I HDACs, when compared with normal epithelium. The results suggest the potential usefulness of HDAC inhibitors for the treatment of a wide variety of human cancers.

Reduced expression of focal adhesion kinase in intrahepatic cholangiocarcinoma is associated with poor tumor differentiation.

OBJECTIVE: Focal adhesion kinase (FAK) expression has been linked to tumor cell invasion and metastasis, but its role in intrahepatic cholangiocarcinoma (ICC) has not been addressed. The goal of this study was to investigate FAK expression in ICC and to assess whether its expression is correlated with clinicopathological factors or prognosis in patients with ICC. METHODS: FAK expression was examined using immunohistochemistry with sections from 56 resected ICC specimens. The correlations between FAK expression and clinical outcome were assessed. RESULTS: The patients were divided into two groups according to the degree of FAK expression: high FAK group (n = 16) and low FAK group (n = 40). A lower expression of FAK was correlated with tumor size, poor differentiation, lymph node metastasis, vascular invasion, and intrahepatic metastasis. In the low FAK expression group, multiple recurrence and distant metastases were more prevalent than in the high FAK expression group. The overall and disease-free survival analysis indicated worse outcomes of the low FAK expression group (p < 0.01). CONCLUSIONS: A low expression of FAK in ICC is associated with a poor outcome after a surgical resection.

Role of expression of focal adhesion kinase in progression of hepatocellular carcinoma.

PURPOSE: Although hepatocellular carcinoma (HCC) is the most common cancer of the human liver, the mechanisms that regulate HCC development and progression remain unclear. The aim of this study was to investigate whether focal adhesion kinase (FAK) is involved in the progression of human HCC. EXPERIMENTAL DESIGN: Western blot analysis for FAK was performed on three HCC cell lines. We reviewed 64 consecutive patients who had undergone initial liver resection for HCC without preoperative treatment. Immunohistochemistry analysis for FAK was performed on paraffin-embedded tissues. FAK expression was confirmed by Western blot analysis in several clinical samples. We investigated the correlation between FAK expression and clinical outcome. RESULTS: FAK proteins were detected in all HCC cell lines. Hepatocytes in the normal liver and chronic hepatitis with or without cirrhosis were negative for immunohistochemical staining for FAK expression. Cytoplasmic FAK expression was observed in 18 of 64 patients (28.1%), and this positive staining was correlated with gender (P < 0.05), a lower level of serum albumin (P < 0.05), and portal venous invasion (P < 0.01). Positive staining for FAK was associated with significantly poorer survival (P < 0.05). In multivariate analysis, FAK overexpression was an independent factor in determining the prognosis of patients. CONCLUSIONS: These data suggest that FAK plays an important role in promoting tumor progression, especially vascular invasion, in HCC. FAK could play an important role in HCC progression and would be a novel target for HCC therapeutics as well as a prognostic marker.

Transcriptional repressor snail and progression of human hepatocellular carcinoma.

PURPOSE: Snail protein is a suppressive transcriptional factor of E-cadherin that mediates cell-to-cell adhesion, tumor progression, and metastases. We explored the expression and function of Snail and its family member Slug in human hepatocellular carcinoma (HCC) to identify its role in tumor progression. EXPERIMENTAL DESIGN AND RESULTS: Transfection of Snail cDNA in Li-7, endogenous E-cadherin-positive human HCC cells, selectively induced the loss of E-cadherin protein expression. We then investigated the expression of Snail and Slug mRNA in 43 human tissue samples of HCC. Using in situ hybridization, Snail mRNA was determined to dominantly express in HCC cells, but not in bile duct cells, blood vessels or infiltrating leukocytes. The mRNA of Snail and Slug were quantified using real-time reverse transcriptase-PCR, and correlations with E-cadherin expression and clinicopathological factors were investigated. Snail mRNA was overexpressed in 7 cases (16%) of HCC compared with adjacent noncancerous liver tissue. E-Cadherin protein expression determined in the same 43 cases by immunohistochemistry was significantly down-regulated in those cases with Snail mRNA overexpression (P = 0.04). The tumor and nontumor ratio of Snail mRNA independently correlated with tumor invasiveness (P = 0.04). However, Slug mRNA correlated with neither E-cadherin expression nor tumor invasiveness. CONCLUSIONS: The data indicate that Snail both down-regulates E-cadherin expression and promotes the invasion in human HCC.

Intrahepatic cholangiocarcinoma: its mode of spreading and therapeutic modalities.

Intrahepatic cholangiocarcinoma (IHCC) is a primary adenocarcinoma of the liver, arising from the intrahepatic bile ducts. The prognosis is generally poor because locoregional extension is usually advanced at the time of diagnosis. Even after a resection, the outcome for patients with advanced IHCC is extremely poor, and the presence of lymph node metastasis has been reported in most previous studies to be the worst prognostic factor after a resection. There are no clear guidelines on lymph node dissection with IHCC. In this article, we review the mode of invasion and the therapeutic modalities: hepatic resection, lymph node dissection, liver transplantation, radiation, and chemotherapy for IHCC.

Clinicopathologic risk factors for recurrence after a curative hepatic resection for hepatocellular carcinoma.

BACKGROUND: The long-term prognosis after resection for patients with hepatocellular carcinoma is still unsatisfactory because of the high recurrence rate. The survival of patients with multiple intrahepatic or extrahepatic recurrence is especially poor. METHODS: Among the patients who underwent hepatic resection for hepatocellular carcinoma between 1981 and 2000, 216 patients with 3 or less than 3 intrahepatic recurrences (group B); 156 patients with more than 3 intrahepatic recurrences, extrahepatic recurrences, or both (group C); and 51 patients who survived more than 5 years without recurrence (group A) were clinicopathologically studied. RESULTS: The period to recurrence of group C was significantly earlier than that of group B and also showed a significantly poor prognosis after recurrence. Tumor factors, including size, portal venous invasion, intrahepatic metastasis, histologic grade, or the number of tumors at resection in group C was significantly worse than in groups A and B. Although no differences are recognized in the tumor factors between groups A and B, except for the alpha-fetoprotein level, liver function in group B was significantly worse than that in group A. In addition, the frequency of hepatitis B surface antigen in group B and that of hepatitis C virus in group B was significantly less and higher than that in group A, respectively. CONCLUSION: Similar to extrahepatic metastasis, multinodular recurrences are also mainly caused by metastatic recurrence from the main tumor by means of the portal system, and recurrences with up to 3 intrahepatic nodules are mainly caused by metachronous multicentric hepatocarcinogenesis. Because the mechanisms of recurrence differed, determining the patterns of recurrence on the basis of the clinicopathologic findings is important for selecting the optimal postoperative therapy for each individual patient.

C-met and hepatocyte growth factor expression in combined hepatocellular and cholangiocarcinoma.

The membranous tyrosine kinase receptor c-met and its natural ligand hepatocyte growth factor are prominent mitogens, motogens and morphogens for hepatocytes and many other cell types in vitro as well as in vivo. To clarify the significance of the c-met/hepatocyte growth factor system in the development and spread of combined hepatocellular and cholangiocarcinoma, surgical specimen from 30 patients, consisting of 4 double cancers, 20 combined types and 6 mixed types, were examined immunohistochemically. Immunoreactivity for HGF was significantly correlated with the differentiation degree of cholangiocellular components, being highest in well and moderately differentiated and lowest in poorly differentiated components (p=0.0119). No significant association was observed between expression of c-met or HGF and the presence of liver cirrhosis, vascular invasion, perineural invasion, lymphatic permeation, intrahepatic metastasis or lymph node metastasis. HGF may have an important impact on the differentiation of certain cHCC-CC. Other clinicopathologic factors related to tumor development and spread may not be influenced by c-met or HGF, at least not on the protein level.

Morphologic approach to hepatocellular carcinoma development in man: de novo or the so-called 'dysplastic nodule-carcinoma' sequence?

The so-called dysplastic nodule-carcinoma sequence in the liver is generally accepted because hepatocellular carcinoma is not an uncommon finding in precancerous lesions. In order to evaluate the existence and frequency of de novo hepatocarcinogenesis we studied 112 surgically resected early well-differentiated hepatocellular carcinomas showing replacing growth without less differentiated component in themselves. They were divided into two groups: carcinoma in dysplastic area (type A) and carcinoma without dysplastic area (type B) and were analyzed clinicopathologically. We encountered 77 cases of type A (68.8%) and 35 of type B (31.2%). The frequency of type A in cirrhotic group (74.7%) is statistically higher than that of non-cirrhotic group (54.5%) (p=0.0453). Using multivariate analysis, the occurrence of type A was related with higher age, the presence of cirrhosis and hepatitis B surface antigen positive. The tumor size and the presence of fatty change in the tumor tended to relate with type A. We propose two pathways morphologically in early hepatocarcinogenesis, one of which has a close relation to hepatitis B virus and/or cirrhosis.

Prognostic factors in node-negative intrahepatic cholangiocarcinoma with special reference to angiogenesis.

BACKGROUND: The aim of this study was to clarify prognostic factors and recurrence patterns in patients with node-negative intrahepatic cholangiocarcinoma (IHCC). METHODS: A retrospective study was performed to review prognostic factors and recurrence patterns (1) in 22 patients with node-negative IHCC after curative hepatic resection and (2) in 49 patients who underwent resection and lymph node dissection for IHCC. In addition to determining the clinicopathologic factors, the investigators also performed immunohistochemical examination of microvessel counts using antihuman CD-31 and antibody. RESULTS: The significant poor prognostic factors in node-negative IHCC were the presence of intrahepatic metastasis, portal vein invasion of cancer cells, and high microvessel counts. After multivariate analysis was conducted, the independent poor prognostic factors were the presence of intrahepatic metastases and high microvessel counts. Of 9 patients who had postoperative recurrence of their disease, intrahepatic recurrence was observed in 7 (78 %). CONCLUSIONS: The factors linked to poor prognosis in IHCC were tumor angiogenesis and the presence of intrahepatic metastasis. Because intrahepatic recurrence was common, regional and adjuvant chemotherapy to the liver may improve the outcome of patients with these risk factors and node-negative IHCC.

The role of thymidine phosphorylase and thrombospondin-1 in angiogenesis and progression of intrahepatic cholangiocarcinoma.

Thymidine phosphorylase (TP), an important regulator of angiogenesis, is correlated with progression, metastasis, and prognosis in various types of tumor. In contrast, both positive and negative effects of thrombospondin-1 (TSP-1) on angiogenesis have been reported. In the present study, we examined the expression of TP and TSP-1 in carcinoma cells in 67 primary intrahepatic cholangiocarcinomas (ICCs) immunohistochemically and its correlation with angiogenesis, clinicopathological features, and prognosis. Twenty-six (38.8%) cases were classified as exhibiting positive TP expression. TP expression showed a significant correlation with vascular invasion, lymphatic permeation, perineural invasion, and lymph node metastasis. Thirty-four (50.7%) cases were classified as exhibiting positive TSP-1 expression. TSP-1 expression was significantly correlated with only lymphatic permeation. The microvessel count in positive TP expression cases was significantly higher than that in negative cases. In contrast, the microvessel count in negative TSP-1 expression cases was significantly higher than that in positive cases. Survival in patients who were positive for both TP and TSP-1 expression was significantly poor. Our results suggest that the increased TP expression and decreased TSP-1 expression contribute to angiogenesis, but that the role of angiogenesis in ICC is not closely related to tumor aggressiveness. The TP and TSP-1 expression in ICC may enhance tumor aggressiveness.

Performance of radiological methods in diagnosing hepatocellular carcinoma preoperatively in a recipient of living related liver transplantation: comparison with step section histopathology.

PURPOSE: The aim of the present study was to evaluate the performance of multidetector-row CT (MDCT) and magnetic resonance imaging (MRI) in diagnosing hepatocellular carcinoma (HCC) preoperatively in living related liver transplantation (LRLT) recipients with liver cirrhosis and HCC. MATERIALS AND METHODS: A total of 25 LRLT recipients with 89 pathologically proved HCCs underwent dynamic 4-row MDCT (5 mm collimation) and MRI within 1 month before LRLT. The images were reviewed for the diagnosis of HCC on a tumor-by-tumor basis by three observers independently and randomly using explanted specimens as the gold standard. The diagnostic accuracy of these techniques in the detection of HCC was assessed with alternative free response receiver operating characteristic (ROC) analysis. The sensitivity and positive predictive values were evaluated. RESULTS: The average values of the area under the ROC curve (Az) of MRI images were higher than those obtained with MDCT; however, no significant difference was observed (P > 0.05). The overall sensitivity of HCC with MRI was higher than that with MDCT, especially in the case of HCCs <20 mm. CONCLUSION: A better diagnostic performance regarding HCCs in LRLT recipients was achieved with MRI than with MDCT, although no significant difference was observed.

Sclerosed hemangioma of the liver: possible diagnostic value of diffusion-weighted magnetic resonance imaging.

We present a case of a sclerosed hemangioma (SH) of the liver that showed a high apparent diffusion coefficient (ADC) value. The patient was undergoing preoperative evaluation for a metastatic breast cancer lesion when a liver mass with a diameter of 3 cm was found. It was described as a heterogeneously hyperechoic mass on ultrasonography and as a well-defined, lobulated mass with early peripheral enhancement and internal heterogeneous enhancement in the delayed phase on computed tomography. The fat-suppressed T2-weighted images demonstrated a heterogeneously hyperintense mass, which showed an ADC value of 2.01 x 10(-3) mm(2)/s. Liver metastasis and cholangiocellular carcinoma could not be excluded based on the imaging findings. After surgery, a definite diagnosis of SH was obtained. Microscopically, many hyalinized portions with poor cellular and fibrous components were observed in the tumor, and this hyalinization accompanied with liquiform degeneration, which may have been one of the causes of the high ADC value. We discuss the diagnostic value of diffusion-weighted imaging for SH of the liver.

The predictors of microvascular invasion in candidates for liver transplantation with hepatocellular carcinoma-with special reference to the serum levels of des-gamma-carboxy prothrombin.

The microvascular invasion of cancer cells (mvi) is a good prognostic factor after orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC). The aim of this study is to predict mvi in patients with HCC who were candidates for OLT. We studied 218 patients with HCC resections who had HCC without any extrahepatic metastases and vascular invasion detected during preoperative evaluation. We analyzed the clinico-pathological data of these patients to predict the mvi presence. The mvi prediction scoring system was made and the accuracy of this system was examined using independent clinico-pathologic factors. The size and histological grade of the tumor were significantly correlated with the mvi. The des-gamma-carboxy prothrombin (DCP) is a mvi predictor. The sensitivity of our mvi prediction system was 75% and the specificity was 85% in 32 patients who underwent living-donor liver transplantations for HCC. Our study shows that besides the tumor size and histological grade, a measurement of the serum DCP levels could be a good predictor for mvi. A tumor biopsy and a preoperative measurement of DCP could improve the selection of patients with HCC for OLT. Our scoring system for mvi provides us a precise prediction of the presence of mvi.

Delayed-phase dynamic CT enhancement as a prognostic factor for mass-forming intrahepatic cholangiocarcinoma.

PURPOSE: To retrospectively determine whether the degree of contrast material enhancement at delayed-phase dynamic computed tomography (CT) for intrahepatic cholangiocarcinoma (ICC) is related to the patient's prognosis after surgery. MATERIALS AND METHODS: Neither institutional review board approval nor informed consent was required for this retrospective evaluation. Thirty-two patients (22 men, 10 women; mean age, 60.8 years; range, 33-80 years) with mass-forming ICC underwent dynamic CT. Delayed CT images obtained 4-6 minutes after the injection of contrast material were evaluated by two radiologists. Patients were classified in consensus into one of two groups: Group 1 included those in whom more than two-thirds of the tumor showed enhancement on delayed-phase scans. Group 2 included those in whom less than two-thirds of the tumor showed enhancement on delayed-phase scans. The imaging findings were correlated with pathologic findings. Survival curves were drawn by using the Kaplan-Meier method, and the differences between the groups were compared with the log-rank test. Multivariate analysis was performed to clarify prognostic factors. RESULTS: There were 13 patients in group 1 and 19 in group 2. The degree of enhancement on the delayed-phase images showed statistically significant correlation with the amount of fibrous stroma (P < .001) and the frequency of perineural invasion (P < .01). The survival rate in group 1 was significantly lower than that in group 2 (P = .016). Multivariate analysis revealed that enhancement of more than two-thirds of the ICC was a significant and independent prognostic factor. CONCLUSION: The degree of enhancement on delayed-phase CT scans is a useful indicator for prediction of the prognosis of patients with mass-forming ICC.

Suppressed MKP-1 is an independent predictor of outcome in patients with hepatocellular carcinoma.

OBJECTIVE: An increase in the activity of mitogen-activated protein kinases (MAPKs) has been correlated with a more malignant phenotype in several tumor models in vivo. This study was designed to clarify the expression of MKP-1 in surgically resected hepatocellular carcinoma (HCC). METHODS: We reviewed the cases of 77 patients who had undergone initial liver resection for HCC without preoperative treatment. Immunohistochemical analysis of MKP-1 was performed on paraffin-embedded tissues. The correlation between MKP-1 expression and clinical outcome was investigated. RESULTS: Tumor cells were immunohistochemically stained for MKP-1 expression, and the same levels as in normal hepatocytes were detected in 66 (85%) of 77 HCC patients, being decreased in 11 (15%) HCCs. Decreased MKP-1 expression significantly correlated with serum alpha-fetoprotein levels and tumor size (p<0.05). The disease-free survival rates in MKP-1-negative and -positive patients were 0 and 31.0% at 5 years, respectively (p<0.01). The survival rates after a surgical resection in MKP-1-negative and -positive patients were 18.2 and 65.5% at 5 years, respectively (p<0.01). CONCLUSIONS: The MKP-1 expression in HCC was an independent prognostic factor for outcome in HCC patients. In the future, it will be useful to explore whether the phosphatase expression might account for the response to HCC treatments targeting at MAPK activation.

Detection of combined hepatocellular and cholangiocarcinomas on enhanced CT: comparison with histologic findings.

OBJECTIVE: The purpose of this study was to evaluate the performance of enhanced CT in making a diagnosis of combined hepatocellular and cholangiocarcinomas (HCC-CCs) by comparing CT findings with histologic findings. CONCLUSION: One third (nine of 27 cases) of the combined HCC-CCs were correctly diagnosed on enhanced CT by detailed analysis of the enhancing pattern around or within the mass. Various factors such as an atypical enhancing pattern, the size of each component, and the presence of a mass composed of intermediate tumor cells-that is, cells with intermediate characteristics between HCC and CC-were found to be the causes of misdiagnosis of combined HCC-CC on enhanced CT.

Tenascin expression at the invasive front is associated with poor prognosis in intrahepatic cholangiocarcinoma.

Tenascin and decorin are components of the extracellular matrix (ECM) that are implicated in cell proliferation in tumors. Here, we propose that abnormal expression of stromal ECM may play an important role in the progression of intrahepatic cholangiocarcinoma, which is characterized by desmoplastic reaction. To explore this hypothesis, we performed immunohistochemical analysis in order to examine the expression and distribution of tenascin and decorin in 75 cases of intrahepatic cholangiocarcinoma. In the intratumoral stroma, positive staining for tenascin was observed in 51 (68%) cases, and positive staining for decorin was observed in 61 (81%) cases. However, at the invasive front, positive staining for tenascin was found in 23 (31%) cases, and positive staining for decorin was found in 6 (8%) cases. Decorin staining was not correlated with aggressive behavior of intrahepatic cholangiocarcinoma, whereas intratumoral tenascin staining was correlated with lymphatic permeation and proliferative activity measured by Ki67. Tenascin staining at the invasive front was associated with tumor size, lymphatic permeation, lymph node metastasis, and proliferative activity and appeared to be a useful prognostic factor by univariate analysis, although it was not an independent prognostic factor. These results indicate that tenascin plays a role in tumor progression in cases of intrahepatic cholangiocarcinoma and that tenascin expression, especially at the invasive front, may be a useful marker in evaluating an unfavorable prognosis in patients with intrahepatic cholangiocarcinoma.