Clinicopathological characteristics and prognostic factors of gastrointestinal stromal tumors in Chinese patients.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms in the gastrointestinal tract, exhibiting wide variability in their biological behavior. The aim of the present study was to investigate the clinicopathological characteristics and prognostic factors of GISTs in Chinese patients. All GIST cases (n=182) retrieved from the pathology database and the archived files in Shanghai Changzheng Hospital between January 2011 and December 2014 were reviewed. The clinical symptoms, preoperative investigations, treatments, pathological characteristics and follow-up data of these patients were reviewed, and univariate and multivariate survival analyses were performed. A total of 73.1% of the GISTs were located in the stomach, and the most common three symptoms included abdominal pain (30.2%), dyspepsia (23.1%) and gastrointestinal bleeding (21.4%). Univariate analysis revealed that larger tumor size (P<0.001), higher mitotic rate (P<0.001), aggressive behavior (P<0.001), negative smooth muscle actin expression (P=0.009) and palliative resection (P<0.001) contributed toward poor overall survival (OS). In addition, non-gastric disease location (P<0.001), larger tumor size (P<0.001), higher mitotic rate (P=0.004), aggressive behavior (P<0.001) and palliative resection (P<0.001) were associated with poor relapse-free survival (RFS). Multivariate analysis indicated that mitotic rate [hazard ratio (HR=3.761, P=0.015)] and aggressive behavior (HR=3.916, P=0.010) were independent risk factors for OS, while non-gastric location (HR=4.740, P=0.002) and aggressive behavior (HR=4.009, P=0.004) were independent risk factors for RFS. The present study provided information on the clinicopathological characteristics and epidemiology of GISTs in the Chinese population. Non-gastric disease location, higher mitotic rate and tumor metastasis or local invasion prior to treatment were identified as predictors of a poor prognosis.

Potentials of the elevated circulating miR-185 level as a biomarker for early diagnosis of HBV-related liver fibrosis.

Early diagnosis of liver fibrosis is critical for early intervention and prognosis of various chronic liver diseases. Conventional repeated histological assessment is impractical due to the associated invasiveness. In the current study, we evaluated circulating miR-185 as a potential biomarker to predict initiation and progression of liver fibrosis. We found that miR-185 was significantly up-regulated in blood specimens from patients with HBV-liver fibrosis and rats with liver fibrosis, the miR-185 levels were correlated with liver fibrosis progression, but not with the different viral loads in HBV-infected patients. miR-185 was observed in collagen deposition regions during advanced liver fibrosis. We found that differences in miR-185 levels facilitated the discrimination between early-staged or advanced-staged liver fibrosis and the healthy controls with high specificity, sensitivity, and likelihood ratio using receiver-operator characteristic analysis. miR-185 targeted SREBF1, and increased expression of COL1A1 and a-SMA genes that are hallmarks of liver fibrosis. Our data supported that circulating miR-185 levels could be used as potential biomarkers for the early diagnosis of liver fibrosis.

MiR-9a-5p regulates proliferation and migration of hepatic stellate cells under pressure through inhibition of Sirt1.

AIM: To reveal the functions of microRNAs (miRNAs) with respect to hepatic stellate cells (HSCs) in response to portal hypertension. METHODS: Primary rat HSCs were exposed to static water pressure (10 mmHg, 1 h) and the pressure-induced miRNA expression profile was detected by next-generation sequencing. Quantitative real-time polymerase chain reaction was used to verify the expression of miRNAs. A potential target of MiR-9a-5p was measured by a luciferase reporter assay and Western blot. CCK-8 assay and Transwell assay were used to detect the proliferation and migration of HSCs under pressure. RESULTS: According to the profile, the expression of miR-9a-5p was further confirmed to be significantly increased after pressure overload in HSCs (3.70 ± 0.61 vs 0.97 ± 0.15, P = 0.0226), which resulted in the proliferation, migration and activation of HSCs. In vivo, the up-regulation of miR-9a-5p (2.09 ± 0.91 vs 4.27 ± 1.74, P = 0.0025) and the down-regulation of Sirt1 (2.41 ± 0.51 vs 1.13 ± 0.11, P = 0.0006) were observed in rat fibrotic liver with portal hypertension. Sirt1 was a potential target gene of miR-9a-5p. Through restoring the expression of Sirt1 in miR-9a-5p transfected HSCs on pressure overload, we found that overexpression of Sirt1 could partially abrogate the miR-9a-5p mediated suppression of the proliferation, migration and activation of HSCs. CONCLUSION: Our results suggest that during liver fibrosis, portal hypertension may induce the proliferation, migration and activation of HSCs through the up-regulation of miR-9a-5p, which targets Sirt1.

Overexpression of miR-126 inhibits the activation and migration of HSCs through targeting CRK.

BACKGROUND & AIMS: MicroRNAs (miRNAs) have been shown to play essential roles in HSCs activation which contributes to hepatic fibrosis. Our previous miRNA microarray results suggested that miR-126 might be decreased during HSCs activation as other studies. The aim of this study is to investigate the role of miR-126 during HSCs activation. METHODS: In this study, the expression of miR-126 during HSCs activation was measured and confirmed by qRT-PCR. Then, miR-126 expression was restored by transfection of lentivirus vector encoding miR-126. Futhermore, cell proliferation was assayed by the cell counting kit-8 (CCK-8), cell migration was assayed by transwell assay, and the markers of activation of HSCs, α-SMA and collagen type I, were assayed by qRT-PCR, Western Blotting, Immunostaining and ELISA. Luciferase reporter assay was used to find the target of miR-126, and Western Blotting and Immunostaining was used to validate the target of miR-126. Then, the expression and the role of the target of miR-126 during HSCs activation was further assessed. RESULTS: The expression of miR-126 was confirmed to be significantly decreased during HSCs activation. Overexpression of miR-126 significantly inhibited HSCs migration but did not affect HSCs proliferation. The expression of α-SMA and collagen type I were both obviously decreased by miR-126 restoration. CRK was found to be the target of miR-126 and overexpression of miR-126 significantly inhibited CRK expression. And it was found that overexpression of CRK also significantly decreased miR-126 expression and promoted HSCs activation. CONCLUSIONS: Our study showed that overexpression of miR-126 significantly inhibited the activation and migration of HSCs through targeting CRK which can also decrease miR-126 expression and promote HSCs activation.

Multiple carcinosarcomas of the esophagus and stomach.

Carcinosarcoma is an uncommon biphasic malignant neoplasm consisting of both carcinomatous and sarcomatous components. We report a case of an 84-year-old male with multiple carcinosarcomas occurring in the esophagus and stomach. Endoscopically, a bulky pedunculated polypoid lesion was observed in the middle of the esophagus and a huge discoid lesion in the lesser curvature. The patient received esophageal endoscopic mucosal resection, and the specimen measured 4×2.5×1.5 cm. Microscopically, the esophageal tumor consisted of several polymorphic spindle cells mixed with squamous cells, while the gastric biopsies revealed carcinomatous cells with evident abnormal karyokinesis and polymorphous spindle cells. Immunohistochemically, the resected tumor stained positively for the epithelial markers, epithelial membrane antigen (EMA) and cytokeratin 19 (CK 19), and the mesenchymal markers, smooth muscle actin (SMA) and vimentin. The gastric lesion stained positively for CK AE1/AE3, actin and vimentin, but was negative for EMA. Both lesions were positive for neuron specific enolase (NSE), demonstrating neuroendocrine differentiation. The patient succumbed seven months after being discharged from hospital. To our knowledge, this is the first case in the literature that describes multiple carcinosarcomas arising from the esophagus and stomach. A review of the available literature is also presented.

Value of baseline platelet count for prediction of complications in primary biliary cirrhosis patients treated with ursodeoxycholic acid.

BACKGROUND: Decreased platelet count has been observed in various liver diseases, but its significance in primary biliary cirrhosis (PBC) remains unknown. The present study aimed to evaluate the predictive value of the platelet count at diagnosis for PBC-related complications in patients newly diagnosed with PBC and treated with ursodeoxycholic acid (UDCA). METHODS: Ninety-six PBC patients without complications treated with UDCA immediately after diagnosis were retrospectively reviewed. All hematologic and chemical parameters, Mayo risk score and PBC-related complications including upper gastrointestinal hemorrhage, presence of ascites, serum bilirubin concentration > 102.6 µmol/L and onset of hepatic encephalopathy were extracted. The associations between these parameters at diagnosis and complications were determined and the prognostic value of the platelet count was evaluated by receiver operating characteristics (ROC) analysis, Kaplan-Meier method and Cox proportional hazard model with the hazard ratio (HR) and 95% confidence interval (CI) calculated. RESULTS: Patients with PBC-related complications had significantly decreased platelet count and serum bilirubin concentration, prolonged prothrombin time, and increased Mayo risk score compared to those without complications. A platelet count of ≤ 132.5 × 10(9)/L was associated with the occurrence of complications, with an area under the ROC curve of 0.74 (95% CI: 0.64-0.85). The association remained even after adjustment for Mayo risk score (HR: 2.85; 95% CI: 1.46-5.54; p < 0.01), as shown in the Cox proportional hazard model. CONCLUSIONS: Decreased platelet count is a predictive factor for PBC-related complications. A cut-off value of ≤ 132.5 × 10(9)/L is recommended for the baseline platelet count to predict complications in patients newly diagnosed with PBC and treated with UDCA.

[Clinical observation on the safety and efficacy of ursodeoxycholic acid and fuzheng huayu capsule in the treatment of primary biliary cirrhosis].

OBJECTIVE: To observe the clinical efficacy of ursodeoxycholic acid (UDCA) and Fuzheng Huayu Capsule (FHC) in the treatment of primary biliary cirrhosis (PBC). METHODS: Eighty PBC patients were randomly assigned to two groups, the treatment group and the control group, 40 in each group. Patients in the treatment group took UDCA and FHC, while those in the control group were treated with UDCA alone. The treatment course was 48 weeks for both groups. The clinical symptoms and signs, liver function indices (ALT, AST, ALP, GGT, ALB, TBIL, and TBA), hepatic fibrosis indices (HA, LN, IV-CL, and PIIIP), immunologic indices (IgG, IgM, and autoimmune antibodies), changes of portal hemodynamics, and adverse reactions were observed before treatment, as well as at week 4, 12, 24, and 48 after treatment. RESULTS: After treatment the skin itching and fatigue were significantly improved in the treatment group, showing statistical difference when compared with the control group (P < 0.05, P < 0.01). After treatment the levels of ALT, AST, ALP, GGT, TBIL, and TBA obviously decreased in the two groups. They were lower in the treatment group than in the control group at the same time point (P < 0.05). The decrement was the largest at week 4. Besides, at week 48 after treatment the ALB level was improved in the treatment group (P < 0.05). The levels of HA and PIIIP obviously decreased at week 4, 12, and 24, the levels of LN and IV-C obviously decreased at week 4 and 12, the decrement of the hepatic fibrosis indices at week 4 were more obvious in the treatment group. But the levels of HA and PIIIP were lower than the pre-treatment levels at week 12 in the control group. The immunologic indices such as IgM and IgG were improved in the two groups, with better results obtained in the treatment group (P < 0.05, P < 0.01). In the treatment group ANA turned negative in 1 patient and AMA turned negative in 2 patients. After 48 weeks of treatment, the spleen was retracted, the inner diameters of the portal vein (PV) and the splenic vein (SV) were significantly reduced, and the blood flow velocity in the PV and SV increased in the treatment group (P < 0.01). At week 24 and 48, 33 patients (82.5%) and 26 patients (90.0%) in the treatment group had complete relief, better than those of the control group [22 cases (55.0%) and 28 cases (70.0%)]. No obvious adverse reaction was found in the two groups during the treatment course. CONCLUSIONS: The combination therapy of UDCA and FHC was effective and safe in anti fibrosis and improving the liver functions of PBC patients. It was safe and better than the application of UDCA alone. It was advocated to be combined use for a long term. It might improve the long-term efficacy.

Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation.

Activation and migration of resident stellate cells (HSCs) within the hepatic space of Disse play an important role in hepatic fibrosis, which accounts for the increased numbers of activated HSCs in areas of inflammation during hepatic fibrosis. Currently, microRNAs have been found to play essential roles in HSC differentiation, proliferation, apoptosis, fat accumulation and collagen production. However, little is known about microRNA mediated HSC activation and migration. In this study, the miRNA expression profiles of quiescent HSCs, partially activated HSCs and fully activated HSCs were compared in pairs. Gene ontology (GO) and GO-Map network analysis indicated that the activation of HSCs was regulated by microRNAs. Among them miR-335 was confirmed to be significantly reduced during HSC activation by qRT-PCR, and restoring expression of miR-335 inhibited HSC migration and reduced α-SMA and collagen type I. Previous study revealed that tenascin-C (TNC), an extracellular matrix glycoprotein involved in cell migration, might be a target of miR-335. Therefore, we further studied the TNC expression in miR-335 over-expressed HSCs. Our data showed that exogenous TNC could enhance HSC migration in vitro and miR-335 restoration resulted in a significant inhibition of TNC expression. These results demonstrated that miR-335 restoration inhibited HSC migration, at least in part, via downregulating the TNC expression.

Analysis of HLA alleles polymorphism in Chinese patients with primary biliary cirrhosis.

BACKGROUND: A familial clustering of patients with primary biliary cirrhosis (PBC) and the presence of immunological abnormalities in family members suggest a genetic component involved in the pathogenesis of PBC. The aims of this study are to investigate the frequencies of human leukocyte antigen HLA-A, -B, and -DRB1 alleles in Chinese patients with PBC by polymerase chain reaction (PCR)-based techniques, and to assess the correlation of the above-mentioned HLA with some clinical and laboratory features. METHODS: Genotyping of HLA alleles were performed in 65 well-characterized PBC patients and 431 healthy controls with sequence-specific primers PCR amplification. RESULTS: HLA-DRB1*07 allele detected in 19 of the 65 (29.2%) PBC patients was subtyped as DRB1*0701, as well as in 13.9% of controls (PC<0.05, OR=2.55, 95%CI: 1.4-4.6). An increased frequency of DRB1*03 (18.4% vs. 7.2% in healthy controls) and a decreased frequency of DRB1*12 (16.9% vs. 28.8%) in PBC patients were statistically significant. There was no association with HLA-DRB1*08 reported. The frequencies for HLA-A, B and the other DRB1 alleles were similar between patients and healthy controls. CONCLUSIONS: The susceptibility to PBC in Chinese individuals is associated with DRB1*0701 allele. This association differs from that in North Americans, South Americans, North Europeans and even Japanese, but it is not restricted to any particular subgroup of patients.

Correlation of Chlamydia pneumoniae infection with primary biliary cirrhosis.

AIM: To evaluate the association between Chlamydia pneumoniae (Cpn) infection and primary biliary cirrhosis (PBC). METHODS: Cpn IgG and IgM were determined by enzyme-linked immunosorbent assay (ELISA) in 41 well-established PBC patients and two race-matched control groups (post-hepatitis cirrhosis, n = 70; healthy controls, n = 57). RESULTS: The mean level and seroprevalence of Cpn IgG in PBC group and post-hepatitis cirrhosis (PHC) group were significantly higher than those in healthy controls (46.8+/-43.4 RU/mL, 49.5+/-45.2 RU/mL vs 28.3+/-32.7 RU/mL; 68.3%, 71.4%, 42.1%, respectively; P<0.05). There was a remarkably elevated seroprevalence of Cpn IgM in patients with PBC (22.0%) compared to the PHC and healthy control (HC) groups. For the PBC patients versus the HCs, the odds ratios (ORs) of the presence of Cpn IgG and IgM were 2.7 (95% CI 0.9-6.1) and 5.1 (95% CI 1.4-18.5), respectively. Though there was no correlation in the level of Cpn IgG with total IgG in sera of patients with PBC (r = -0.857, P = 0.344>0.05), Cpn IgM was related with the abnormally high concentrations of total IgM in PBC group. CONCLUSION: The results of this study do not support the hypothesis that infection with Chlamydia pneumoniae may be a triggering agent or even a causative agent in PBC, but suggest that Chlamydia pneumoniae infection probably contributes to the high level of IgM present in most patients with PBC.

[Analysis of HLA alleles polymorphism in Chinese patients with primary biliary cirrhosis].

OBJECTIVE: To investigate the frequencies of human leuckocyte antigens (HLA) -A, B and DRB1 alleles in Chinese patients with primary biliary cirrhosis (PBC) using polymerase chain reaction-based techniques, and to assess the correlation of HLA molecules with other clinical and laboratory profiles. METHODS: Genotyping of HLA-A, B, and DRB1 were performed in 65 well-characterized patients with primary biliary cirrhosis and 431 healthy controls with PCR amplification with sequence-specific primers (PCR-SSP). RESULTS: The frequency of DRB1*0701 was increased to 29.2% compared with 13.9% in the controls (PC < 0.05, OR = 2.55, 95% CI: 1.4 approximately 4.6). No association was found with HLA-DRB1*08 which had been constantly reported. The A*2 allele (53.8%) was more frequent in the PBC patient group but without a significant statistical difference. The frequencies for the other A, B and DRB1 alleles were similar between patients and healthy controls. There was no difference between patients with or without DRB1*0701 in some clinical and laboratory profiles. CONCLUSION: Susceptibility to primary biliary cirrhosis in Chinese is associated with DRB1*0701 allele and differs from people in North America, South America, North Europe and even in Japan, but the association is not restricted to any particular subgroup of patients. Valine at position 78 of HLA DRbeta1 may play an important role in the pathogenesis of primary biliary cirrhosis.

[The value of antimitochondrial antibody and its subtypes in the diagnosis of primary biliary cirrhosis].

OBJECTIVE: A study on the value of antimitochondrial antibody (AMA) and its subtypes anti-M2, anti-M4, and anti-M9 in diagnosing primary biliary cirrhosis (PBC). METHODS: Antimitochondrial antibody was detected by indirect immunofluorescence and anti-M2, anti-M4 and anti-M9 by Western blotting. AMA and anti-M2 of 78 PBC patients, of 35 non-PBC hepato-biliary disease patients and 20 healthy controls were studied and anti-M2, anti-M4 and anti-M9 were studied in 30 of the 78 PBC patients. RESULTS: 96.2% (75/78) of PBC patients were AMA positive and 94.9% (74/78) of PBC patients were anti-M2 positive. Only three among the 35 non-PBC patients were positive for AMA (one with very low titre). None of the 35 non-PBC patients was anti-M2 positive. AMA and anti-M2 were negative in all the healthy controls. Among the 30 anti-M2 positive patients, 16 patients were anti-M4 positive (16/30, 53.3%) and 4 patients were anti-M9 positive (4/30, 13.3%). CONCLUSION: AMA and its subtypes (special anti-M2) are important sero-immunological markers for the diagnosis of PBC.

[Prediction and identification of autoepitopes of PDC-E2 specific CD8+ CTL in primary biliary cirrhosis patients].

OBJECTIVE: To identify autoepitopes of E2 subunit of pyruvate dehydrogenase complex (PDC-E2) specific CD8+ CTL in primary biliary cirrhosis (PBC) patients. METHODS: An online database SYFPEITHI was applied to predict HLA-A*0201 restricted epitopes which located in PDC-E2 30-50 aa and 150-190 aa where B-cell epitopes clustered with CD4+ T-cell epitopes. T2 cell line reconstitution and stabilization assay, induction of specific CTL lines from peripheral blood mononuclear cells (PBMCs) of patients with PBC and cytotoxicity of peptides-induced CTL were performed to screen the epitopes from those candidates. RESULTS: Five potential epitopes were predicted by database. Of the 5 candidates, two peptides 159-167 aa and 165-174 aa, with highly binding activity to HLA-A*0201 molecules, could stimulate PBMCs from most HLA-A*0201 positive PBC patients to proliferate and peptide-induced CTL lines showed specific cytotoxicity. CONCLUSION: Peptides of KLSEGDLLA (159-167 aa) and LLAEIETDKA (165-174 aa) in the inner lipoyl domain of PDC-E2 are HLA-A*0201 restricted CD8+ CTL immunodominant epitopes in PBC.