Objective: To investigate the utility of albumin RNAscope in situ hybridization in the diagnosis and differential diagnosis of hepatocellular carcinoma and its mimics. Methods: One hundred and fifty-two cases of hepatocellular carcinoma and its mimics and 33 cases of normal tissue were selected from the pathology database of the Department of Pathology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from January 2013 to December 2019. Tissue microarrays were constructed and RNAscope in situ hybridization was performed to detect the expression of albumin mRNA. Results: No albumin mRNA expression was detected in normal tissues except for the liver. All hepatocellular carcinoma regardless of its degree of differentiation and primary or metastatic nature had detectable albumin mRNA, with strong and diffuse staining in 90.7% (49/54) of cases. While the positive rate of HepPar-1, Arg-1 or one of them by immunohistochemistry was 87.0% (47/54), 85.2% (46/54) and 92.6% (50/54) respectively. The positive rates of albumin mRNA in intrahepatic cholangiocarcinoma and biphenotypic hepatocellular carcinoma were 7/15 and 9/10, respectively. The former showed focal or heterogeneous staining, while the latter showed strong and diffuse staining. The positive rate of hepatoid adenocarcinoma was 8/19, and the albumin expression could be diffuse or focal. Sporadic cases of poorly differentiated gastric adenocarcinoma and metastatic colon adenocarcinoma showed focal staining of albumin mRNA. Conclusions: Detection of albumin mRNA by RNAscope in situ hybridization is of great value for the diagnosis and differential diagnosis of HCC, and the sensitivity may be improved by combining with HepPar-1 and Arg-1. It also offers different diagnostic clues according to different expression patterns.
Adenocarcinoma/diagnosis* ; Albumins/genetics* ; Bile Duct Neoplasms/pathology* ; Bile Ducts, Intrahepatic/pathology* ; Biomarkers, Tumor/genetics* ; Carcinoma, Hepatocellular/genetics* ; China ; Colonic Neoplasms ; Diagnosis, Differential ; Humans ; In Situ Hybridization ; Liver Neoplasms/pathology* ; RNA, Messenger

BACKGROUNDIn qualitative diagnosis of bile duct stenosis, single diagnostic measure is difficult to make a correct diagnosis, to combine several diagnostic techniques may be helpful to make an accurate diagnosis. The aim of this study was to evaluate the value of intraductal ultrasonography (IDUS), endoscopic brush cytology and K-ras, P53 gene mutation in the early diagnosis of malignant biliary stricture.

METHODSFrom February 2012 to February 2013, 84 patients with suspected malignant biliary stricture were performed IDUS firstly, then endoscopic brush cytology and finally K-ras, P53 gene mutation detection, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of all above ways were evaluated and compared.

RESULTSOf 84 patients, 52 cases were ultimately diagnosed malignant biliary stenosis; of which, 9 cases had no recurrence or metastasis to other organs after radical operation during the follow-up period. IDUS combined with brush cytology and K-ras + P53 gene mutation detection had obvious advantage in the sensitivity, accuracy and negative predictive value than any other joint detection and single detection (the advantage was more significant compared with IDUS + brush cytology or any single detection P < 0.01). There were obvious statistical significance in the sensitivity and accuracy between IDUS + brush cytology + P53 or IDUS + brush cytology + K-ras and IDUS + brush cytology or IDUS (P < 0.05). There was no statistical significance in the sensitivity, specificity, positive predictive value, negative predictive value and accuracy between IDUS + brush cytology + P53 and IDUS + brush cytology + K-ras (P > 0.05).

CONCLUSIONSIDUS combined with brush cytology and K-ras, P53 gene mutation detection is better than the separate detection and contribute to the early diagnosis of malignant biliary stricture. Its more widespread use is recommended.

Aged ; Aged, 80 and over ; Bile Duct Diseases ; diagnosis ; genetics ; Bile Duct Neoplasms ; diagnosis ; genetics ; Bile Ducts ; pathology ; Constriction, Pathologic ; diagnosis ; genetics ; Female ; Genes, p53 ; genetics ; Genes, ras ; genetics ; Humans ; Male ; Middle Aged ; Mutation

OBJECTIVETo investigate the expressions of different forms of ROS fusions in Chinese patients with cholangiocarcinoma (CCA).

METHODSRT-PCR was employed to examine formalin-fixed and paraffin-embedded CCA samples from stage I-IV patients for detection of ROS fusions involving Fused in Glioblastoma (FIG), solute carrier protein (SLC34A2) and major histocompatibility complex class II invariant chain (CD74). Serpin peptidase inhibitor clade A member 1 (SERPINA1) was detected as the reference gene.

RESULTSIn all the 56 CCA samples, 80.4% (45/56) were positive for SERPINA1 expression as evaluable samples. Of these evaluable samples, none expressed the ROS fusions.

CONCLUSIONROS fusions are not common in Chinese CCA patients.

Antigens, Differentiation, B-Lymphocyte ; genetics ; metabolism ; Bile Duct Neoplasms ; metabolism ; pathology ; Carrier Proteins ; genetics ; metabolism ; Cholangiocarcinoma ; metabolism ; pathology ; Female ; Gene Expression ; Histocompatibility Antigens Class II ; genetics ; metabolism ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Paraffin Embedding ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Sodium-Phosphate Cotransporter Proteins, Type IIb ; genetics ; metabolism

BACKGROUNDSmad4 is found mutated in many cancers. It acts as a tumor suppressor in the regulation of TGF-β signaling pathway. The objective of this work was to study the expression of Smad4 in intrahepatic cholangiocarcinoma (ICC) and its relationship with the biological behavior and prognosis of the disease.

METHODSForty-nine paraffin-embedded ICC specimens and nine normal liver tissues were analyzed by immunohistochemical methods using Smad4 monoclonal antibodies. The expression of Smad4 was compared with the clinical pathological characteristics of the patients.

RESULTSThe expression of Smad4 was 100% positive in normal liver tissues, which was higher than that in the ICC (44.9%). Negative labeling of the Smad4 protein was found in 26.1% (6/23) of well-differentiated ICCs and 61.5% (16/26) of poorly to moderately differentiated ICCs, and 34.3% (12/35) and 71.4% (10/14) showed negative Smad4 labeling (P = 0.018) of ICC at pathological Tumor Node Metastasis (pTNM) stage I-II and pTNM stage III-IV separately. Furthermore, 72% (8/11) of lymph node metastatic ICCs and 73.3% (11/15) of intrahepatic metastatic ICCs showed negative labeling of the Smad4 protein. The loss of Smad4 expression in those metastatic ICCs was significantly more severe compared with non-metastatic ICCs (P = 0.000).

CONCLUSIONSThe expression of Smad4 was associated with the histological grade, clinical stage, and metastasis of ICC (P < 0.05). The detection of Smad4 may be helpful in determining the degree of malignancy and prognosis of ICC.

Adult ; Aged ; Bile Duct Neoplasms ; Bile Ducts, Intrahepatic ; Cholangiocarcinoma ; chemistry ; pathology ; Female ; Humans ; Liver Neoplasms ; chemistry ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis ; Signal Transduction ; physiology ; Smad4 Protein ; analysis ; genetics ; physiology ; Transforming Growth Factor beta ; physiology

OBJECTIVETo construct a small interfering RNA (siRNA) vector targeting p63 and observe its effect on the proliferation and invasiveness of human cholangiocarcinoma cells in vitro.

METHODSReal-time PCR was used to examine the expression of p63 in human cholangiocarcinoma QBC939 cells. The recombinant lentivirus shRNA-p63 vector was constructed and transfected into QBC939 cells via Lipofectamine 2000 to establish a cholangiocarcinoma cell line with stable expression of siRNA-p63. The interfering efficiency of the siRNA targeting p63 was assessed using Western blotting. MTT and soft agar colony formation assays were used to evaluate the changes in the cell proliferation, and Boyden test was employed to observe the cell invasiveness after the transfection.

RESULTSQBC939 cells showed a high expression of p63. The recombinant lentivirus shRNA-p63 vector was successfully constructed as verified by sequencing. Transfection with the vector significantly suppressed the proliferation and invasiveness of QBC939 cells.

CONCLUSIONDown-regulation of p63 can inhibit the proliferation and invasiveness of human cholangiocarcinoma QBC939 cells in vitro.

Bile Duct Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cholangiocarcinoma ; genetics ; pathology ; Humans ; Membrane Proteins ; genetics ; metabolism ; Neoplasm Invasiveness ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo explore whether hepatitis C virus core protein (HCV C) regulates the expression of NFAT1 to participate in the progression and malignant biological behavior of intrahepatic cholangiocarcinoma cells.

METHODSThe recombinant plasmid pEGFP-N(3)-HCV C and the empty vector pEGFP-N(3) were cotransfected with enhanced green fluorescent protein (EGFP) into RBE cells using liposome. Real-time PCR and Western blotting were used to examine the expression of NFAT1 mRNA and protein in the transfected RBE cells. MTT assay was used to evaluate the changes in the cell proliferation, and the cell cycle changes were analyzed by flow cytometry.

RESULTSHCV C transfection significantly enhanced the expressions of NFAT1 mRNA and protein in RBE cells (P<0.05) and promoted the progression of cell cycle into G(2)/M phase to accelerate the cell proliferation.

CONCLUSIONTransfection with HCV C gene up-regulates NFAT1 expression and promotes the cell cycle progression and proliferation of intrahepatic cholangiocarcinoma cells, suggesting the involvement of HCV C in the progression of intrahepatic cholangiocarcinoma.

Bile Duct Neoplasms ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma ; pathology ; Gene Expression ; Humans ; NFATC Transcription Factors ; genetics ; Plasmids ; Transfection ; Viral Core Proteins ; genetics

OBJECTIVETo investigate the expression profile of microRNA-21 in human cholangiocarcinoma tissues and to validate its bona fide targets in human cholangiocarcinoma cells.

METHODSThe expression profile of microRNA-21 in human cholangiocarcinoma tissues and cholangiocarcinoma cell line, QBC939, was evaluated by using real-time PCR analysis. The bona fide targets of microRNA-21 were analyzed and confirmed by dual luciferase reporter gene assay and western blot, respectively. The expressional correlation of microRNA-21 and its targets was probed in human cholangiocarcinoma tissues by using real-time PCR, locked nucleic acid in situ hybridization (LNA-ISH), and immunohistochemistry analysis.

RESULTSReal-time PCR analysis revealed that microRNA-21 expression depicted a significant up-regulation in human cholangiocarcinoma tissues about 5.6-fold as compared to the matched normal bile duct tissues (P<0.05). The dual luciferase reporter gene assay revealed endogenous microRNA-21 in cholangiocarcinoma cell line, QBC939, inhibited the luciferase reporter activities of wild-type PTEN (P<0.01) and PDCD4 (P<0.05) and had no this effect on mutated PTEN and PDCD4. Moreover, loss of microRNA-21 function led to a significant increase of PTEN and PDCD4 protein levels in QBC939 cells. Elevated microRNA-21 levels were accompanied by marked reductions of PTEN and PDCD4 expression in the same cholangiocarcinoma tissue.

CONCLUSIONmicroRNA-21 expression is up-regulated in human cholangiocarcinoma and PTEN, PDCD4 are direct effectors of microRNA-21.

Apoptosis Regulatory Proteins ; genetics ; Bile Duct Neoplasms ; genetics ; pathology ; Bile Ducts, Intrahepatic ; metabolism ; pathology ; Cell Line, Tumor ; Cholangiocarcinoma ; genetics ; pathology ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Male ; MicroRNAs ; genetics ; physiology ; Middle Aged ; PTEN Phosphohydrolase ; genetics ; RNA-Binding Proteins ; genetics ; Transfection

OBJECTIVETo study the effects of sorafenib on lymphangiogenesis in transplanted human cholangiocarcinoma in nude mice.

METHODSThe model of transplanted human cholangiocarcinoma in nude mice was established by subcutaneous inoculation of cholangiocarcinoma cell line QBC 939 cells. Thirty-six nude mice were randomly divided into 3 groups after tumor formation: control group, sorafenib 30 mg × kg⁻¹ × d⁻¹ group and sorafenib 60 mg × kg⁻¹ × d⁻¹ group (n = 12 each), and then treated by gavage for 6 weeks. The tumor growth of the dose groups and control group was measured with calipers. Using immunohistochemical staining, the lymphatic microvessels at tumor edge were marked by LYVE-1 and counted. The expression of VEGFR-3 mRNA in paracancerous tissues was evaluated by RT-PCR.

RESULTSSorafenib significantly depressed the growth of cholangiocarcinoma. The inhibitory rate in the sorafenib 30 mg × kg⁻¹ × d⁻¹ group and 60 mg × kg⁻¹ × d⁻¹ group was 55.1% and 67.9%, respectively. The LMVDs of the control group, sorafenib 30 mg × kg⁻¹ × d⁻¹ group and 60 mg × kg⁻¹ × d⁻¹ group were 11.75 ± 3.19, 6.84 ± 2.18 and 5.03 ± 1.91, respectively. The LMVD of the control group was significantly higher than that in the dose groups (P < 0.01). The relative expressions of VEGFR-3 mRNA in the control group, sorafenib 30 mg × kg⁻¹ × d⁻¹ group and 60 mg × kg⁻¹ × d⁻¹ group were 2.158 ± 0.312, 1.027 ± 0.144 and 0.736 ± 0.149, respectively. The relative expression of VEGFR-3 mRNA in the control group was significantly higher than that in the dose groups (P < 0.05). No occurrence of lymph node metastasis was found in all groups.

CONCLUSIONSorafenib can significantly inhibit the growth of xenograft cholangiocarcinoma in nude mice. Sorafenib may reduce LMVD by down-regulation of the expression of VEGF-C/D and VEGFR-3 signaling axis.

Animals ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Benzenesulfonates ; administration & dosage ; pharmacology ; Bile Duct Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cholangiocarcinoma ; metabolism ; pathology ; Dose-Response Relationship, Drug ; Down-Regulation ; Humans ; Lymphangiogenesis ; drug effects ; Lymphatic Vessels ; drug effects ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Niacinamide ; analogs & derivatives ; Phenylurea Compounds ; Pyridines ; administration & dosage ; pharmacology ; RNA, Messenger ; metabolism ; Random Allocation ; Vascular Endothelial Growth Factor C ; metabolism ; Vascular Endothelial Growth Factor Receptor-3 ; genetics ; metabolism

Adult ; Aged ; Bile Duct Neoplasms ; blood ; virology ; Biomarkers ; blood ; DNA, Viral ; blood ; Female ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatitis, Viral, Human ; blood ; virology ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Liver ; metabolism ; pathology ; virology ; Male ; Middle Aged

OBJECTIVETo explore the possibility of using melanoma antigen (MAGE)-1 and MAGE-3 gene encoding proteins as an index of potential target for immunotherapy in intrahepatic cholangiocarcinoma (IHCC) patients.

METHODSThe expressions of MAGE-1 and MAGE-3 genes in tumor tissues and tumor adjacent non-IHCC liver tissues were examined by RT-PCR method. The relationship between positive expression rates of MAGE-1 and MAGE-3 genes and clinical data including sex, age, tumor diameters, tumor envelope, tumor nodules number, and hepatitis B virus surface antigen were determined.

RESULTSThe positive expression rates of MAGE-1 (35%) and MAGE-3 genes (45%) were significantly higher in the tumor tissues than in tumor adjacent tissues (0) (P<0.01). The positive expression rates of MAGE-1 and MAGE-3 genes had no relationship with the clinical data (P >0.05), except the morphology of tumor (P <0.05).

CONCLUSIONThe high expression rates of MAGE-1 and MAGE-3 genes in IHCC suggests the MAGE-1 and MAGE-3 gene may be a target for immunotherapy in IHCC patients.

Adult ; Aged ; Antigens, Neoplasm ; genetics ; Bile Duct Neoplasms ; genetics ; Bile Ducts, Intrahepatic ; pathology ; Cholangiocarcinoma ; genetics ; Female ; Humans ; In Vitro Techniques ; Liver Neoplasms ; genetics ; Male ; Melanoma-Specific Antigens ; Middle Aged ; Neoplasm Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction