Overall survival of patients with hepatocellular carcinoma treated with sintilimab and disease outcome after treatment discontinuation.

BACKGROUND: The use of Anti-PD-1 therapy has yielded promising outcomes in hepatocellular carcinoma (HCC). However, limited research has been conducted on the overall survival (OS) of patients with varying tumor responses and treatment duration. METHODS: This retrospective study analyzed HCC patients who received sintilimab between January 2019 and December 2020 at four centers in China. The evaluation of tumor progression was based on Response Evaluation Criteria in Solid Tumors version 1.1. The study investigated the correlation between tumor response and OS, and the impact of drug use on OS following progressive disease (PD). RESULTS: Out of 441 treated patients, 159 patients satisfied the inclusion criteria. Among them, 77 patients with disease control exhibited a significantly longer OS compared to the 82 patients with PD (median OS 26.0 vs. 11.3 months, P < 0.001). Additionally, the OS of patients with objective response (OR) was better than that of patients with stable disease (P = 0.002). Among the 47 patients with PD who continued taking sintilimab, the OS was better than the 35 patients who discontinued treatment (median OS 11.4 vs. 6.9 months, P = 0.042). CONCLUSIONS: In conclusion, the tumor response in HCC patients who received sintilimab affects OS, and patients with PD may benefit from continued use of sintilimab.

Hazard rate for postoperative recurrence in patients with hepatocellular carcinoma at Barcelona Clinic Liver Cancer stage 0 or A1: A multicenter observational study.

AIM: Surgical treatment is the first-line treatment for patients with Barcelona Clinic Liver Cancer (BCLC) stage 0 or A1 hepatocellular carcinoma (HCC), and postoperative monitoring improves long-term survival. We aimed to establish a reasonable short-interval follow-up duration for patients with HCC. METHODS: The cohort for this retrospective study included 1396 HCC patients with BCLC stage 0 or A1 disease who underwent curative resection from 2013 to 2016 at five centers in China. Hazard rates for recurrence were calculated using the hazard function. RESULTS: The recurrence rates in patients with BCLC stage 0 and A1 HCC were 46.4% and 58.0%, respectively. The hazard curve for stage 0 patients was relatively flat, and the hazard rate was consistently low (peak hazard rate 0.0163). The hazard rate curve for recurrence was initially high (peak hazard rate 0.0441) in patients with BCLC stage A1 disease and showed a rapid decreasing trend within 1 year, followed by a slow decreasing trend, reaching a low level (<0.0163) at approximately 36 months. The time to low risk was 47, 41, and 51 months in patients with cirrhosis, hepatitis B virus (HBV) infection, and satellite lesions, respectively. CONCLUSIONS: A short-interval follow-up of 1 year is sufficient for HCC patients with BCLC stage 0 disease, whereas a short-interval follow-up time of 3 years should be considered for patients with stage A1 disease. The follow-up period should be appropriately prolonged for patients with cirrhosis, HBV infection, and satellite lesions.

Transarterial chemoembolization plus a PD-1 inhibitor with or without lenvatinib for intermediate-stage hepatocellular carcinoma.

AIM: Transarterial chemoembolization (TACE) combined with a PD-1 inhibitor and TACE combined with a PD-1 inhibitor and lenvatinib have recently been reported as promising treatments to improve the prognosis of hepatocellular carcinoma (HCC) patients. This study aims to compare the efficacy of these two treatments. METHODS: A retrospective study was conducted, and patients were recruited from two centers in China. Progression-free survival (PFS) and overall survival (OS) were compared, and the objective response rate (ORR) and disease control rate (DCR) were evaluated according to the modified Response Evaluation Criteria in Solid Tumors (mRECIST). Treatment-related adverse events (AEs) were analyzed to assess safety. RESULTS: The median follow-up for the entire cohort was 11.4 months. Of the 103 patients included in this study, 56 received triple therapy, and 47 received doublet therapy. PFS was significantly higher in the triple therapy group than in the doublet therapy group (mPFS 22.5 vs. 14.0 months, P < 0.001). Similar results were obtained in terms of OS (P = 0.001). The ORR and DCR were also better in the triple therapy group (64.3% vs. 38.3%, P = 0.010; 85.7% vs. 57.4%, P = 0.002). The most common AEs in the triple therapy group were decreased albumin (55.3%), decreased platelet count (51.8%) and hypertension (44.6%). CONCLUSIONS: The combination of TACE with a PD-1 inhibitor and lenvatinib in patients with BCLC stage B HCC might result in significantly improved clinical outcomes with a manageable safety profile compared with TACE with a PD-1 inhibitor.

Effects of Stereotactic Body Radiation Therapy Plus PD-1 Inhibitors for Patients With Transarterial Chemoembolization Refractory.

Background and Aims: Patients with intermediate-stage hepatocellular carcinoma (HCC) who are refractory to transarterial chemoembolization (TACE) have a poor prognosis. This study aimed to explore whether stereotactic body radiation therapy (SBRT) combined with PD-1 inhibitors could improve the clinical outcomes of such patients. Methods: This retrospective cohort study included patients with intermediate-stage HCC who were diagnosed with TACE refractoriness between January 2019 and December 2020 in the Eastern Hepatobiliary Surgery Hospital and the First Affiliated Hospital of Wenzhou Medical University. The patients were divided into two groups: (1) those who switched from TACE to receive stereotactic body radiotherapy (SBRT) combined with PD-1 inhibitors; (2) those who continued TACE treatment and added PD-1 inhibitors. Progression-free survival (PFS), overall survival (OS), and tumour response were assessed in both groups after becoming refractory to TACE treatment. Results: Of the seventy-six patients included in this study, the median PFS was 19.6 months in the SBRT-IO group (n=31) and 10.1 months in the TACE-IO group (n=45, p<0.05). The SBRT-IO group also had a significantly higher OS than the TACE-IO group (p<0.05). The objective response rate (ORR) and disease control rate (DCR) were also better in the SBRT-IO group (ORR, 71.0% vs. 15.6%, OR=8.483, 95% CI 3.319-21.680, P < 0.001; DCR, 80.6% vs. 31.1%, OR=9.226, 95% CI 3.096-27.493, P < 0.001). Conclusions: SBRT combined with a PD-1 inhibitor improves PFS and OS in TACE-refractory patients with intermediate-stage HCC. Therefore, this therapy is a suitable option in cases of TACE treatment failure.

A novel classification in predicting prognosis and guiding postoperative management after R0 liver resection for patients with hepatocellular carcinoma and microvascular invasion.

BACKGROUND: Microvascular invasion (MVI) is a significant risk factor affecting survival outcomes of patients after R0 liver resection (LR) for hepatocellular carcinoma (HCC). The current classification of MVI is not refined enough to prognosticate long-term survival of these patients, and a new MVI classification is needed. METHODS: Patients with HCC who underwent R0 LR at the Eastern Hepatobiliary Surgery Hospital from January 2013 to December 2013 and with resected specimens showing MVI were included in this study with an aim to establish a novel MVI classification. The classification which was developed using multivariate cox regression analysis was externally validated. RESULTS: There were 180 patients in the derivation cohort and 131 patients in the external validation cohort. The following factors were used for scoring: α-fetoprotein level (AFP), liver cirrhosis, tumor number, tumor diameter, MVI number, and distance between MVI and HCC. Three classes of patients could be distinguished by using the total score: class A, ≤3 points; class B, 3.5-5 points and class C, >5 points with distinct long-term survival outcomes (median recurrence free survival (mRFS), 22.6, 10.2, and 1.9 months, P < 0.001). The predictive accuracy of this classification was more accurate than the other commonly used classifications for HCC patients with MVI. In addition, the mRFS of class C patients was significantly prolonged (1.9 months vs. 6.2 months, P < 0.001) after adjuvant transcatheter arterial chemoembolization (TACE). CONCLUSIONS: A novel MVI classification was established in predicting prognosis of HCC patients with MVI after R0 LR. Adjuvant TACE was useful for class C patients.

A positive feedback loop between Periostin and TGFß1 induces and maintains the stemness of hepatocellular carcinoma cells via AP-2α activation.

BACKGROUND: Liver cancer stem cells (LCSCs) play key roles in the metastasis, recurrence, and chemotherapeutic resistance of hepatocellular carcinoma (HCC). Our previous research showed that the POSTN gene is closely related to the malignant progression and poor prognosis of HCC. This study aimed to elucidate the role of POSTN in generating LCSCs and maintaining their stemness as well as the underlying mechanisms. METHODS: Human HCC tissues and matched adjacent normal tissues were obtained from 110 patients. Immunohistochemistry, western blotting (WB), and RT-PCR were performed to detect the expression of POSTN and stemness factors. The roles of transforming growth factor (TGF)-ß1 and AP-2α in the POSTN-induced stemness transformation of HCC cells were explored in vitro and in vivo using LCSCs obtained by CD133+ cell sorting. RESULTS: The high expression of POSTN was correlated with the expression of various stemness factors, particularly CD133, in our HCC patient cohort and in TCGA and ICGC datasets. Knockdown of POSTN expression decreased the abilities of HCC cell lines to form tumours in xenograft mouse models. Knockdown of POSTN expression also suppressed cell viability and clone formation, invasion, and sphere formation abilities in vitro. Knockdown of AP-2α attenuated the generation of CD133+ LCSCs and their malignant behaviours, indicating that AP-2α was a critical factor that mediated the POSTN-induced stemness transformation and maintenance of HCC cells. The role of AP-2α was verified by using a specific αvß3 antagonist, cilengitide, in vitro and in vivo. Activation of POSTN could release TGFß1 from the extracellular matrix and initiated POSTN/TGFß1 positive feedback signalling. Furthermore, we found that the combined use of cilengitide and lenvatinib suppressed the growth of HCC cells with high POSTN expression more effectively than the use of lenvatinib alone in the patient-derived xenograft (PDX) mouse model. CONCLUSIONS: The POSTN/TGFß1 positive feedback pathway regulates the expression of stemness factors and the malignant progression of HCC cells by regulating the transcriptional activation of AP-2α. This pathway may serve as a new target for targeted gene therapy in HCC.

Prognostic Value of Microvascular Invasion in Eight Existing Staging Systems for Hepatocellular Carcinoma: A Bi-Centeric Retrospective Cohort Study.

BACKGROUND: Microvascular invasion (MVI) is a significant risk factor affecting survival outcomes of patients after R0 liver resection (LR) for hepatocellular carcinoma (HCC). However, whether the existing staging systems of hepatocellular carcinoma can distinguish the prognosis of patients with MVI and the prognostic value of MVI in different subtypes of hepatocellular carcinoma remains to be clarified. METHODS: A dual-center retrospective data set of 1,198 HCC patients who underwent R0 LR was included in the study between 2014 and 2016. Baseline characteristics and staging information were collected. Homogeneity and modified Akaike information criterion (AICc) were compared between each system. And the prognostic significance of MVI for overall survival (OS) was studied in each subgroup. RESULTS: In the entire cohort, there were no significant survival differences between Cancer of the Liver Italian Program (CLIP) score 2 and 3 (p = 0.441), and between Taipei Integrated Scoring System (TIS) score 3 and 4 (p = 0.135). In the MVI cohort, there were no significant survival differences between Barcelona Clinic Liver Cancer stages B and C (p=0.161), CLIP scores 2 and 3 (p = 0.083), TIS scores 0 and 1 (p = 0.227), TIS scores 2 and 3 (p =0.794), Tokyo scores 3 and 4 (p=0.353), and American Joint Committee on Cancer Tumor-Node-Metastasis 7th stage I and II (p=0.151). Among the eight commonly used HCC staging systems, the Hong Kong Liver Cancer (HKLC) staging system showed the highest homogeneity and the lowest AICc value in both the entire cohort and MVI cohort. In each subgroup of the staging systems, MVI generally exhibited poor survival outcomes. CONCLUSIONS: The HKLC staging system was the most accurate model for discriminating the prognosis of MVI patients, among the eight staging systems. Meanwhile, our findings suggest that MVI may be needed to be incorporated into the current HCC staging systems as one of the grading criteria.

A blood-based 22-gene expression signature for hepatocellular carcinoma identification.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and lethal malignancies. Early detection of HCC could largely reduce mortalities. Ultrasonography (US) and serum Alpha Fetoprotein (AFP) test are the screening methods that are most frequently applied to high-risk populations. Due to the poor performance of AFP testing, and the highly operator-dependent nature of US, a biomarker for HCC early diagnosis is highly sought after. We developed a method for HCC screening using a 22-gene expression signature. METHODS: Peripheral whole blood of 98 patients were processed through microarrays for the first round of feature selection via two strategies, Minimal Redundancy Maximal Relevance and Least Absolute Shrinkage and Selection Operator combined with Support Vector Machine (SVM). Candidate genes were combined for further validation through qPCR in an enlarged population with 316 samples with 104 chronic hepatitis, 112 liver cirrhosis (LC), and 100 HCC. RESULTS: A 22-gene signature was established in classifying HCC and non-cancer samples with good performance. The area under curve reached 0.94 in all of the samples and 0.93 in the AFP -negative samples. CONCLUSIONS: We have established a blood mRNA signature with high performance for HCC screening. Our results show transcriptome of peripheral blood could be valuable source for biomarkers.

Diagnosis and surgical treatment of retroperitoneal paraganglioma: A single-institution experience of 34 cases.

The present study aimed at identifying the clinical, radiological and pathological characteristics of retroperitoneal paragangliomas, and determining the association between the tumor features and the prognosis of patients following surgery. A total of 34 patients with retroperitoneal paragangliomas, who underwent resection between November 1999 and December 2015, were included in the present retrospective study. The patients' demographics, clinical symptoms and signs, tumor functional status, surgical procedure, intraoperative results, tumor pathology, radiological results, and postoperative survival time were recorded and analyzed. Of the 34 patients, the most common type of presenting symptom was abdominal mass (46%), followed by hypertension (39%) and abdominal pain (32%). Functional tumors occurred in 20 patients (59%). Computed tomography (CT) and magnetic resonance imaging revealed soft-tissue masses, with marked enhancement in the arterial phase, indicative of retroperitoneal paragangliomas. The preoperative CT diagnostic accuracy rate between 2010 and 2015 was markedly improved, compared with that between 1999 and 2009. The tumors were primarily located close to the renal arteries and veins surrounding the abdominal aorta and inferior vena cava. With the exception of one malignant paraganglioma, the majority of paragangliomas were positive for chromogranin A, S-100 protein, vimentin and heat-shock protein 90, and exhibited decreased expression of Ki-67 antigen and insulin-like growth factor 2. All tumors were completely removed by surgery. Distant metastasis, but not tumor size, functional status and local invasion, was markedly associated with survival. The preoperative diagnostic accuracy rate of retroperitoneal paragangliomas may be improved by focusing on the predilection sites and CT characteristics. In addition, immunohistochemical markers were useful to determine tumor malignancy. Complete surgical resection was appropriate for all patients and postoperative survival time was identified to be associated with tumor metastasis.

Intra-thyroid thyroglossal duct cyst: a case report and review of literature.

Thyroglossal duct cyst is the most common congenital cyst in the head and neck, which is defined usually occurring in children. However, intra-thyroid thyroglossal duct cyst in an adult is unusually found. Here we describe a case of a 45-year-old woman who was found neck mass along the midline for 5 years. During the surgery we found a separated nodule in the left inferior pole of the thyroid. Surprisingly the diagnosis of the nodule was confirmed by pathology and histological examination demonstrating that it was the thyroglossal duct cyst. Intra-thyroid thyroglossal duct cyst in an adult is a rare finding, with few cases reported. For it is generally thought that any thyroid tissue found in the lateral aspect of the neck may indicate metastatic deposits from well-differentiated thyroid carcinoma. Although pathogenesis of an alone thyroglossal duct cyst in the left inferior pole of the thyroid remains unknown, our case could suggest thyroglossal duct cyst should not be excluded in the differential diagnosis of lateral neck masses especially when it simulates nodules in the thyroid.

GSK-3ß suppresses the proliferation of rat hepatic oval cells through modulating Wnt/ß-catenin signaling pathway.

AIM: Glycogen synthase kinase 3ß (GSK-3ß) plays a crucial role in hepatic biology, including liver development, regeneration, proliferation and carcinogenesis. In this study we investigated the role of GSK-3ß in regulation of growth of hepatic oval cells in vitro and in liver regeneration in partially hepatectomized rats. METHODS: WB-F344 cells, the rat hepatic stem-like epithelial cells, were used as representative of oval cells. Cell viability was examined using a WST-8 assay. The cells were transfected with a recombinant lentivirus expressing siRNA against GSK-3ß (GSK-3ßRNAiLV) or a lentivirus that overexpressed GSK-3ß (GC-GSK-3ßLV). Adult rats underwent partial (70%) hepatectomy, and liver weight and femur length were measured at d 7 after the surgery. The expression of GSK-3ß, phospho-Ser9-GSK-3ß, ß-catenin and cyclin D1 was examined with immunoblotting assays or immunohistochemistry. RESULTS: Treatment of WB-F344 cells with the GSK-3ß inhibitor SB216763 (5 and 10 µmol/L) dose-dependently increased the levels of phospho-Ser9-GSK-3ß, but not the levels of total GSK-3ß, and promoted the cell proliferation. Knockout of GSK-3ß with GSK-3ßRNAiLV increased the cell proliferation, whereas overexpression of GSK-3ß with GC-GSK-3ßLV decreased the proliferation. Both SB216763 and GSK-3ßRNAiLV significantly increased the levels of ß-catenin and cyclin D1 in the cells, whereas GSK-3ß overexpression decreased their levels. In rats with a partial hepatectomy, administration of SB216763 (2 mg/kg, ip) significantly increased the number of oval cells, the levels of phospho-Ser9-GSK-3ß, ß-catenin and cyclin D1 in liver, as well as the ratio of liver weight to femur length at d 7 after the surgery. CONCLUSION: GSK-3ß suppresses the proliferation of hepatic oval cells by modulating the Wnt/ß-catenin signaling pathway.

MicroRNA-155 promotes the proliferation and invasion abilities of colon cancer cells by targeting quaking.

The increasing expression of microRNA­155 (miR­155) and decreasing expression of RNA­binding protein quaking (QKI) in colon cells have been observed previously. In this study, we attempted to establish the correlation between miR­155 and QKI. In addition, we assessed whether the expression of miR­155 and QKI is linked to the proliferation and invasion capabilities of colon cells. Firstly, nineteen tumor samples, divided into two groups according to the presence or absence of lymphatic metastasis, were obtained from colon cancer patients at the First Affiliated Hospital of Wenzhou Medical University, China. The expression level of miR­155 and QKI was measured by quantitative polymerase chain reaction (qPCR). Secondly, the GES­1, SW480 and COLO205 cell lines were cultured and the expression level of QKI and miR­155 was also assessed by qPCR. Thirdly, a luciferase reporter gene assay was performed to detect the association between miR­155 and QKI, and qPCR and western blot analysis were performed to confirm the effects of miR­155 on the expression of QKI at the mRNA and protein level. Subsequently, the SW480 cells were used in the following experiments. Following treatment with miR­155 inhibitor and QKI overexpression vector, western blot analysis, propidium iodide (PI) staining and a cell scratch assay were carried out to assess the effects of miR­155 on the proliferation and invasion potential of colon cancer cells. qPCR findings revealed higher miR­155 expression and lower QKI expression in colon cancer tissues as well as the colon cancer cell lines SW480 and COLO205. The relative luciferase activity of the 3' untranslated region (3'UTR) was decreased by approximately 45% when SW480 cells stimulated by mimic­miR­155 were combined with the wild­type 3'UTR constructs. In addition, when the cells were treated with mimic­miR­155, QKI expression was significantly decreased at the mRNA and protein level. These outcomes revealed that miR­155 decreased the production of QKI by acting on the 3'UTR of the QKI gene. Furthermore, PI staining and the cell scratch assay revealed that miR­155 influenced the cell cycle and invasion abilities of colon cancer cells by directly targeting QKI and decreased the production of QKI by acting on the 3'UTR of the QKI gene. This study has demonstrated the correlation between miR­155 and QKI, in which miR­155 regulates the cell cycle and invasion ability of colon cancer cells via the modulation of QKI expression. Our study provides novel therapeutic strategies for colon cancer therapy.

Neutrophil-lymphocyte ratio: a controversial marker in predicting Crohn's disease severity.

Peripheral blood-derived inflammation-based scores such as the neutrophil-lymphocyte ratio (NLR) have recently been proposed as prognostic markers in ulcerative colitis. In some previous serological markers are commonly used to detect the severity of the Crohn's disease (CD), but their sensitivity and specificity are relatively low. So we want to use simple indicators which are easy to obtain to predict disease severity. Now, we investigated and compared the capacity of NLR and other inflammatory markers in detecting CD activity and differentiating CD patients from healthy controls. These CD patients had not received corticosteroid or immunosuppressive drugs within a defined period of time. Data from our hospital between 2010 and 2012 was used. Neutrophil-lymphocyte ratio (NLR), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), white blood cells (WBC), platelet count and albumin were measured in 44 patients with active CD, 66 patients with inactive CD, and 55 healthy blood donors. Disease activity was assessed by the Crohn's Disease Activity Index. In the active CD group, NLR values were found to be elevated compared to inactive CD patients and controls (6.00±7.38, 5.53±6.18 and 1.84±0.85, respectively), but statistical difference was not found between active and inactive CD groups. The overall accuracy of NLR (cutoff: 2.13 fl), CRP (cutoff: 10.5 mg/dl), ESR (cutoff: 19.5 mm/hour) and WBC (cutoff: 9.2 × 10(9)/l) in differentiating CD patients from healthy controls was 80.9%, 67.3%, 71% and 60% respectively. NLR values were found to be correlated with WBC and CRP levels. NLR increased in CD patients compared with healthy subjects. NLR had the best accuracy in determination of CD patients and healthy controls. NLR did not show a discriminative value in disease activity.

[Role of miR-21 in rat hepatic oval cell proliferation and activation].

OBJECTIVE: To investigate the possible mechanisms of miR-21-mediated regulation of proliferation and activation of hepatic oval cells. METHODS: The 2-acetamidofluorene/partial hepatectomy (2-AAF/PH) method was applied to generate hepatic oval cell activation model in male Sprague-Dawley rats; after the 7 days of 2-AAF/PH or PH alone (control), the rats were sacrificed at 0 h, 6 h, 12 h, 24 h, 72 h and 168 h. Expression of miR-21 was detected by real-time PCR and differences between groups were evaluated using the two-sample t-test. Differential transcription of miR-21 target genes was assessed bioinformatically, and with western blotting to detect changes in protein expression of the target gene. RESULTS: The rat hepatic oval cell activation model was successfully established.The 2-AAF/PH rats showed miR-21 expression beginning to increase at 12 h, peaking at 24 h, and decreasing thereafter until an increase at 168 h.For the control group, the miR-21 expression began to increase at 6 h, until 24 h when expression began steadily declining to reach the original level.Compared to the control group, the experimental group showed expression of miR-21 that was significantly less at 6 h (P=0.039, t =3.029) and significantly more at 24 h and 168 h (P=0.026, t =-3.433 and P=0.007, t =-5.105). Among the predicted target genes of miR-21 were WW domain containing E3 ubiquitin protein ligase 1 (WWD), Smad family member 7 (Smad7), and polybromo-1 (Pbrm1).Smad7 protein expression began to decrease at 6 h in the control group, until reaching its minimum at 24 h when it increased; in the experimental group, SMAD7 expression increased at 6 h, then began to decrease with the minimum detected at 168 hour.In the control group, the Smad7 mRNA expression decreased slightly at 6 h, then began to increase, reaching its peak at 24 h when the expression fell to the original level. In the experimental group, the Smad7 mRNA expression began to increase at 6 h and reached its peak at 24 h when it decreased; the expression was little more than its original level at 168 h.Smad7 protein expression was negatively correlated with miR-21, and Smad7 mRNA expression was positively correlated with miR-21 but negatively correlated with Smad7 protein expression. CONCLUSION: miR-21 may play a vital role in the activation and proliferation of hepatic oval cells.As a target gene of miR-21, Smad7 might be involved in the process.

Effect of NK4 transduction in bone marrow-derived mesenchymal stem cells on biological characteristics of pancreatic cancer cells.

Pancreatic cancer usually has a poor prognosis, and no gene therapy has yet been developed that is effective to treat it. Since a unique characteristic of bone marrow-derived mesenchymal stem cells (MSCs) is that they migrate to tumor tissues, we wanted to determine whether MSCs could serve as a vehicle of gene therapy for targeting pancreatic cancer. First, we successfully extracted MSCs from SD rats. Next, MSCs were efficiently transduced with NK4, an antagonist of hepatocyte growth factor (HGF) which comprising the N-terminal and the subsequent four kringle domains of HGF, by an adenoviral vector. Then, we confirmed that rat MSCs preferentially migrate to pancreatic cancer cells. Last, MSCs expressing NK4 (NK4-MSCs) strongly inhibited proliferation and migration of the pancreatic cancer cell line SW1990 after co-culture. These results indicate that MSCs can serve as a vehicle of gene therapy for targeting pancreatic cancer.

Correlation between tuberous sclerosis complex 2 and glycogen synthase kinase 3 beta levels, and outcomes of patients with hepatocellular carcinoma treated by hepatectomy.

AIM: Tuberous sclerosis complex 2 (TSC2), a tumor suppressor, may play an essential role in the regulation of cell growth and cell survival under energy stress conditions. In addition, TSC2 may act in concert with Wnt and energy signals by additional phosphorylation of glycogen synthase kinase 3ß (GSK3ß) to regulate cell growth. The expression levels and function of TSC2 and GSK3ß in hepatocellular carcinoma (HCC) remain unclear. METHODS: The protein levels of TSC2 and GSK3ß were measured by immunohistochemistry in normal liver (n = 20), HCC (n = 80) and pericancerous tissues (n = 80). The correlations between TSC2, and GSK3ß levels, clinicopathological features and patient survival were also analyzed. RESULTS: The protein levels of TSC2 and GSK3ß in HCC tissues were significantly lower than that in normal liver tissues and pericancerous tissues (P < 0.05). Decreased TSC2 and GSK3ß expression was found to be significantly correlated with advanced clinicopathological characteristics and poor prognosis. The results also showed that TSC2 protein levels were associated with GSK3ß expression in HCC specimens. CONCLUSION: This is the first demonstration that the decreases in TSC2 and GSK3ß levels may be associated with vascular invasion, histological grade and tumor-node-metastasis classification.

Experimental studies on treatment of pancreatic cancer with double-regulated duplicative adenovirus AdTPHre-hEndo carrying human endostatin gene.

BACKGROUND: Gene-virus targeted therapy is a promising new method of treating pancreatic cancer. To increase the efficacy and decrease the side-effect, we constructed a conditionally replicative adenovirus (CRAd) expressing human endostatin, with a human Telomoerase Reverse Transcriptase (hTERT) promoter for the regulation of the early stage of adenovirus expression of gene E1a and a Hypoxia Response Element (HRE) promoter to regulate the gene E1b. METHODS: A gene recombination technique was adopted to construct and generate the adenovirus AdTPHre-hEndo. Pancreatic cancer cells were studied both in vitro and in vivo. Western blotting was adopted to observe the expressions of protein E1A and E1B; duplication assay was applied to observe the selective duplication capability of recombinant cells. MTT assay was applied to measure the lethal effects of virus on pancreatic cancer cells, and ELISA was adopted to detect the human endostatin gene expression. A pancreatic cancer transplantation tumor model of nude mice was constructed to observe the antitumor effects of the virus. RESULTS: Double-regulated duplicative adenovirus AdTPHre-hEndo genes were successfully constructed. Duplication and lethal assays proved that AdTPHre-hEndo could replicate specifically in pancreatic cancer cells and kill them. The endostatin expression in a cultured supernatant from tumor cells was significantly higher than that obtained from non-duplicative adenovirus vectors carrying that gene. The animal experiment demonstrated that AdTPHre-hEndo has a high capability to limit pancreatic cancer growth. CONCLUSIONS: AdTPHre-hEndo has a special ability to duplicate and kill pancreatic cancer cells in in vitro and in vivo experiments, thus providing a new gene-virus-based treatment system for pancreatic cancer.

Decreased expression of LKB1 correlates with poor prognosis in hepatocellular carcinoma patients undergoing hepatectomy.

AIM: To study any correlation of LKB1 expression with prognosis in hepatocellular carcinoma (HCC) cases. METHODS: A total of 70 HCC patients and 20 primary intrahepatic stone patients in the first affiliated hospital of Wenzhou Medical College were enrolled in this study. LKB1 expression was detected by immunohistochemistry. Patients were followed-up and prognostic factors were evaluated. RESULT: LKB1 expression was decreased in the HCC samples. Loss of LKB1 expression in HCC was significantly related to histologic grade (P=0.010), vascular invasion (P=0.025) and TMN stage (P=0.011). Patients showing negative LKB1 expression had a significantly shorter disease-free and overall survival than those with positive expression (P = 0.001, P=0.000, respectively). Multivariate Cox regression analysis indicated that LKB1 expression level was an independent factor of survival (P = 0.033). CONCLUSION: HCC patients with decreased expression LKB1 have a poor prognosis. The loss of LKB1 expression is correlated with a lower survival rate.

[Effects of glycogen synthase kinase 3ß overexpression in rat and glycogen synthase kinase 3ß inhibitor SB-216763 on proliferation of hepatic oval cells].

OBJECTIVE: To research the effects of glycogen synthase kinase (GSK3ß) overexpression and GSK3ß inhibitor SB-216763 on the proliferation of hepatic oval cells in rats and its regulatory mechanisms by Wnt signaling pathway. METHODS: The hepatic oval cells WBF-344 were divided into the blank control group, GSK3ß over-expression group, DMSO control group and GSK3ß inhibitor groups, while the inhibitor groups set up three concentration gradients, that was 1, 5, 10 µmol/L. Using the GSK3ß over-expression lentivirus, which had been identified correctly, and SB-216763 dealt with the cells WBF-344. The cells morphology of each group was observed under the phase contrast inverted microscope, and the expression of fluorescence in the lentivirus-transfected group was observed under the fluorescent microscope. The proliferation of each group cells was tested by CCK8 kits. The cells' apoptosis was detected by AnnexinV-FITC/PI kits. The expression of GSK3ß, ß-catenin and cyclin D1 were detected by Western blot. RESULTS: The cells of GSK3ß over-expression group were fewer and obvious aging. However, in each inhibitor added group, the cells' division and proliferation was vigorous, and the condition was good. Moreover, the cells' proliferation was getting stronger with the concentration of SB-216763 increasing. A large number of green fluorescence was expressed in the lentivirus-transfected cells. The cells' proliferation in GSK3ß over-expression group restrained (t = 7.178, P < 0.01, as compared with control), while the cells' proliferation was vigorous in inhibitor groups (F = 45.030, P < 0.01, as compared with control). Flow Cytometry showed that the cells apoptosis was significant in GSK3ß over-expression group. Western blot showed that the expression of GSK3ß was increased, while the expression of ß-catenin and cyclin D1 was decreased in the over-expression group. The expression of GSK3ß had no significant difference among the control group and inhibitor groups. However, the expression of ß-catenin and cyclin D1 was significantly increased with the concentration of SB-216763 increasing. CONCLUSIONS: The overexpression of GSK3ß can inhibit the Wnt signaling pathway, thus restrain the cells' proliferation and promotes apoptosis. SB-216763 can activate the Wnt pathway, thus promotes cells' proliferation.

Angiogenesis and clinicopathologic characteristics in different hepatocellular carcinoma subtypes defined by EpCAM and α-fetoprotein expression status.

Recently, two hepatic lineage markers epithelial cell adhesion molecule (EpCAM) and α-fetoprotein (AFP) were used to classify hepatocellular carcinoma (HCC) into four subtypes with prognostic implication. In the present study, we further evaluated the clinicopathologic and angiogenic characteristics among these HCC subtypes. EpCAM expression was investigated by immunohistochemistry in 115 HCC primary tumors. Based on EpCAM immunostaining and serum AFP levels, 115 HCC cases were classified into four subtypes: EpCAM+AFP+ (26.1%), EpCAM-AFP+ (20.0%), EpCAM+AFP- (20.8%), and EpCAM-AFP- (33.1%). EpCAM+AFP+ and EpCAM-AFP+ HCC were associated with late TNM stages and high frequencies of venous invasion, whereas EpCAM+AFP- and EpCAM-AFP- subtypes were associated with early TNM stages and low frequencies of venous invasion. Furthermore, EpCAM+AFP+ HCC had a significantly higher microvessel density (MVD) and higher level of VEGF (Vascular epithelial growth factor) expression than the other three subtypes. In conclusion, our study indicated that subtype classification of HCC based on EpCAM and AFP status had clinicopathologic and biologic implications in aggressive phenotype and angiogenesis. We also suggest that the EpCAM+AFP+ HCC patients might be potential therapeutic candidates for anti-angiogenesis therapy.