Complete response of recurrent perihilar cholangiocarcinoma following sintilimab combined with lenvatinib plus S-1: a case report and review of literature.

Perihilar cholangiocarcinoma is a refractory malignancy with an unfavorable prognosis and a high probability of recurrence. Systemic chemotherapy is critical for palliative treatment, but effective therapeutic strategies for perihilar cholangiocarcinoma after first-line chemotherapy failure are scarce. Here, we introduced a sustained benefit following sintilimab combined with lenvatinib plus S-1 in a patient with recurrent perihilar cholangiocarcinoma. A 52-year-old female patient was admitted to our hospital due to yellow skin and sclera, and further radiological examination revealed perihilar cholangiocarcinoma. The patient underwent surgery and histopathological results confirmed moderately differentiated adenocarcinoma with metastatic lymph nodes. Postoperative adjuvant chemotherapy with gemcitabine and S-1 was given. One year after surgery, the patient experienced hepatic recurrence. Then, she received radiofrequency ablation combined with gemcitabine and cisplatin. Unfortunately, radiological assessment revealed progressive disease with multiple liver metastases after treatment. Subsequently, she received sintilimab combined with lenvatinib plus S-1 and the lesions were completely regressed following 14 cycles of combination therapy. The patient recovered well without disease recurrence at the last follow-up. Sintilimab combined with lenvatinib plus S-1 may be an alternative therapeutic option for chemotherapy-refractory perihilar cholangiocarcinoma, and further evaluation in a larger number of patients is needed.

Real-world experience of postoperative adjuvant chemoimmunotherapy in patients with perihilar cholangiocarcinoma at high risk of recurrence.

BACKGROUND: This study aimed to assess whether postoperative adjuvant chemoimmunotherapy could lead to better clinical outcomes for high-risk patients with perihilar cholangiocarcinoma (pCCA). METHODS: In the cohort study, we retrospectively reviewed patients who received surgical resection for pCCA with curative intent from January 2018 to December 2021 at the Sun Yat-sen Memorial Hospital. The patients at high risk for relapse were further analyzed. Among them, 20 patients received adjuvant chemoimmunotherapy, 28 patients received adjuvant chemotherapy, and 33 patients received surgery alone. The oncological outcomes and drug-associated adverse events were evaluated. RESULTS: The 2-year overall survival (OS) rates in patients treated with adjuvant chemoimmunotherapy, adjuvant chemotherapy, and surgery alone were 80.0%, 49.4% and 22.6%, respectively. Univariable and multivariable Cox analyses showed that the treatment regimen and TNM stage were associated with adverse OS. Adjuvant chemoimmunotherapy led to an increase in OS compared with adjuvant chemotherapy [hazard ratio (HR) = 3.253; 95% confidence interval (CI) 1.072-9.870; P = 0.037] or surgery alone (HR = 7.560; 95% CI 2.508-22.785; P < 0.001). The median recurrence-free survival was 22.0 months for the adjuvant chemoimmunotherapy group, 17.0 months for the adjuvant chemotherapy group, and 13.2 months for the surgery alone group (P = 0.177); these differences were not significant. The chemoimmunotherapy group was associated with more frequent hematological side effects than the chemotherapy group, but the difference was not statistically significant. CONCLUSION: Postoperative adjuvant chemoimmunotherapy for resected pCCA patients showed improved OS compared with adjuvant chemotherapy or surgery alone, and further prospectively randomized controlled trials are necessary to validate these results.

Clinical and pathological analysis of hepatic artery aneurysm in a patient with systemic lupus erythematosus: report of a case.

A hepatic artery aneurysm is an unusual but life-threatening hepatobiliary complication occurring in patients with systemic lupus erythematosus (SLE), and early diagnosis and treatment of this complication are essential. A 31-year-old man with SLE presented with recurring epigastric pain and jaundice for 2 months; he was diagnosed with choledocholithiasis and underwent surgery. Hemobilia was found intraoperatively, and two hepatic artery aneurysms were identified in the left lateral lobe during postoperative arteriography. Major hemobilia occurred 6 days after the operation, and the patient was immediately treated with selective embolization of the hepatic artery. However, the major hemobilia recurred 2 days later, and he was treated with a left lateral lobectomy and ligation of the proximal hepatic artery. The patient recovered uneventfully and is in good condition. A histological analysis revealed small- and medium-sized arteritis as well as hepatic artery aneurysm. Systemic lupus erythematosus complicated by a hepatic artery aneurysm should be considered in the differential diagnosis of patients showing symptoms of abdominal pain, jaundice, or gastrointestinal bleeding.

Hepatitis C virus core protein induces malignant transformation of biliary epithelial cells by activating nuclear factor-kappaB pathway.

UNLABELLED: In an earlier study, we found that hepatitis C virus core protein, HCV-C, participated in the malignant transformation of HCV-C transfected normal human biliary epithelial (hBE) cells by activating telomerase. Here we further investigated the signaling of the malignant transformation. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunoprecipitation were used to analyze the expression of HCV-C, human telomerase reverse transcriptase (hTERT), nuclear factor-kappaB (NF-kappaB) and NF-kappaB inhibitor alpha (IkappaBalpha) genes and the phosphorylation level of IkappaBalpha protein. Electrophoretic mobility shift assays (EMSA) and NF-kappaB-linked luciferase reporter assays were carried out to measure NF-kappaB activity. RESULTS: The expression of HCV-C and hTERT was detected only in HCV-C-transfected hBE (hBE-HCV-C) cells but not in vector-transfected or parental hBE cells. More NF-kappaB protein accumulated in nuclear extracts of hBE-HCV-C cells rather than in those of control cells, though total NF-kappaB protein level showed no difference among these cells. DNA binding activity of NF-kappaB and the NF-kappaB-linked luciferase activity were much higher in HCV-C-transfected hBE cells than those in vector- or non-transfected hBE cells. In addition, the IkappaBalpha phosphorylation level, but not the IkappaBalpha mRNA or protein levels, was increased after HCV-C transfection. CONCLUSIONS: Hepatitis C virus core protein activates NF-kappaB pathway in hBE cells by increasing the phosphorylation of IkappaBalpha. The pathway may be responsible for HCV-C-induced malignant transformation of hBE cells.

[Nanoparticle-mediated endostatin gene therapy targeting hepatocellular carcinoma utilizing heat-inducible promoter].

OBJECTIVE: To investigate the inhibitory effect of nanoparticle-mediated endostatin gene therapy on hepatocellular carcinoma xenografts combined with local hyperthermia utilizing heat-inducible promoter. METHODS: Heat-inducible HSP70B promoter and fusion gene of Endo/EGFP were cloned into pcDNA3.1 (+) plasmid, thus obtaining recombinant plasmid of pcDNA3.1 (+)/HSP70-Endo/EGFP using restriction endonucleases BglII/HindIII and EcoRI/SalI. The nanoparticles polylactide-grafted dextran copolymer (DEX-g-PLA) encapsulating the recombinant plasmid DNA were prepared by the method of emulsification and evaporation of organic solvent, and the surface shape of nanoparticles was observed by transmission electron microscope. Human hepatocellular cells of the lines HepG2 and ECV304 were cultured and transfected with the recombinant plasmid utilizing the nanoparticles. Following thermal induction at 37 degrees C, 39 degrees C, 41 degrees C, 43 degrees C, and 45 degrees C for 30 min, the expression of enhanced green fluorescent protein (EGFP) was detected by fluorescence microscope and flow cytometry. The concentration of endostatin protein in the supernatant was tested by ELISA, and the growth inhibition on the HepG2 and ECV304 cells was tested by MTT method. Balb/c nude mice were inoculated with HeG2 cells and then randomly divided into 2 groups to undergo intra-tumor injection of nanoparticles (heated or not heated), Lipofectamine 2000. Mice were used as controls without intra-tumor injection. Four weeks the mice were killed to observe the tumor inhibition rate. RESULTS: The nanoparticles encapsulating recombinant plasmid were of round or elliptical shape 90 approximately 120 nm in diameter. The efficiency of gene transfection mediated by nanoparticles was about 30.65%. The expression of Endo/EGFP gene in the HepG2 cells was up-regulated along with the increase of temperature, peaked at 43 degrees C (with the EGFP expression level 3.3 times as that at 37 degrees C). The concentration of endostatin protein in the supernatant of the 43 degrees C group was (177 +/- 28) microg/L, significantly higher than that of the 37 degrees C group [(41 +/- 10) microg/L]. MTT results indicated that endostatin inhibited the growth of ECV304 cells with a inhibition rate of 96.3% at the time point of 72 h in the 43 degrees C group, however, it did not show influence on HepG2 cells no matter what was the temperature The tumor inhibition rate in the mice of endostatin with thermal induction group was 58.5%, significantly higher than that of the 37 degrees C group (34.9%, P < 0.05). CONCLUSION: Low temperature thermal induction enhances the expression and secretion of endostatin in hepatocellular cells transfected by nanoparticles, and inhibits the growth of hepatocellular carcinoma xenografts.

Malignant transformation of the cultured human normal biliary tract epithelial cells induced by hepatitis C virus core protein.

High level expression of hepatitis C virus core protein (HCV-C) was detected in hilar cholangiocarcinoma tissues in our previous studies. This protein played an important role in the process of cancer cell inversion and proliferation, by some direct and indirect effects on certain genes. Based on this observation, we investigated the effect of HCV-C on human normal biliary epithelial (hBE) cell transformation and tumor development. Plasmid pHCV-C encoding the gene of HCV core protein was constructed and transfected into hBE cells. The expression and the biological effect of HCV-C in HCV-C gene-modified hBE cells were determined in vitro and in vivo. The clone formation rates of hBE cells transfected with pHCV-C, pcdna3.1 and mock-transfected cells were 36, 2.5 and 1.5%, respectively. Tumor developed in 7 of 7 nude mice after incubated with pHCV-C transfected hBE cells, while no tumor appeared in mice injected with pcdna3.1- and mock-transfected hBE cells. To investigate the possible mechanism of malignant transformation, we further studied the telomerase activity and human telomerase reverse transcriptase (hTERT) expression in pHCV-C transfected hBE cells. The elevated expression of hTERT was confirmed by RT-PCR, immunocytochemistry (ICC) and Western blot analysis, which in turn elevated the telomerase activity, confirmed by TRAP-ELISA. These results indicated that HCV-C protein could participate in malignant transformation of human normal biliary epithelial cells and induce cholangiocarcinoma tumorigenesis, and the activation of telomerase was one of the possible mechanisms.

[Human mucin 1 promoter drives human sodium/iodide symporter gene targeting expression in pancreatic carcinoma cells].

OBJECTIVE: To clone human mucin 1 (MUC1) gene promoter and apply to drive human sodium/iodide symporter (hNIS) gene targeting expression in pancreatic carcinoma cells. METHODS: Human Mucin1 (MUC1) promoter was cloned from the 5' flanking region of the MUC1 gene by two-step nest PCR from human pancreatic carcinoma cells of the line CAPAN-I, II and then linked to pDC316 plasmid (pDC316-MUC1). Subsequently, a recombinant plasmid containing MUC1 and hNIS was constructed (pDC316-MUC1/hNIS). The recombinant plasmid pDC316-MUC1/hNIS, pD316-mCMV/NIS plasmid, and pDC316-mCMV/hNIS plasmid were transfected into the CAPAN-II cells, human pancreatic carcinoma cells of the line PANC-1, and human cervical carcinoma cells of the line HeLa respectively as experimental group, positive control group, and negative control group. 48 h after the transfection RT-PCR and immunofluorescence were used to confirm the expression of hNIS mRNA and hNIS protein. Then the cells were cultured in solution with 125I. The 125I uptake in the cells was measured by gamma-counting. RESULTS: The sequence data of regulatory element in MUC1 promoter genes was corresponded to those of reference report. The hNIS protein expression level was high in the MUC1 positive cells, as CAPAN-II cells and PANC-1 cells, but very low in the MUC1 negative cells, such as the HeLa cells. Two days after the transfection, the CAPAN-II cells and PANC-1 cells showed a high level of 125I uptake after transfection with pDC316-MUC1/hNIS, and the CAPAN-II cells, PANC-1 cells, and HeLa cells showed a high level of 125I uptake after transfection with pDC316-MCMV/hNIS. A7-12-fold increase in 125I uptake was observed in the pDC316-MUC1/hNIS transfected cells compared with the pDC316-MUC1 transfected cells. CONCLUSION: MUC1 promoter cloned from CAPAN-2 cells can be used to drive NIS genes expression in MUC1 positive pancreatic carcinoma cells. Therefore, this strategy can be used as a novel and potent gene-targeting therapy in the MUC1 positive pancreatic carcinoma in vivo.

[Preparation of 5-fluorouracil encapsulated in amphiphilic polysaccharide nano-micelles and its killing effect on hepatocarcinoma cell line HepG2].

BACKGROUND & OBJECTIVE: Biodegradable colloidal nano-micelles is a novel targeting drug delivery and controlled release system, which could prolong the biological half-life and lighten the toxicity of chemotherapeutant, meanwhile, present fine biocompatibility. This study was to prepare the biodegradable 5-fluorouracil (5-FU)/DEX-g-PLA nano-micelles, and investigate their killing effect on hepatocarcinoma cell line HepG2 in vitro and in vivo. METHODS: 5-FU/DEX-g-PLA nano-micelles were prepared by 'self-assembly'. Its morphology was observed by transmission electron microscopy. The encapsulating efficiency of 5-FU was determined by ultraviolet spectrophotometry. The in vivo releasing of 5-FU from nano-micelles was investigated by high-performance liquid chromatography (HPLC). The inhibitory effect of 5-FU/DEX-g-PLA on HepG2 cells in vitro was measured by MTT assay. RESULTS: 5-FU/DEX-g-PLA nano-micelles were round or elliptical; the diameter was about 50 nm. The encapsulating efficiency was about 9.3%. The concentration of 5-FU released from 5-FU/DEX-g-PLA nano-micelles was sustained for longer time than that of the naked drug. The in vitro inhibition rate of cell growth was similar in 5-FU/DEX-g-PLA group and naked 5-FU group (58.8% vs. 58.0%, P>0.05); the in vivo inhibition rate of tumor growth was significantly higher in 5-FU/DEX-g-PLA group than in naked 5-FU group (73.1% vs. 57.5%, P<0.05). CONCLUSION: 5-FU/DEX-g-PLA nano-micelles can effectively inhibit the growth of HepG2 cells.

[Study of immunological effect of dendritic cell transfected with survivin gene on the specific anti-alimentary tract tumor].

OBJECTIVE: To investigate the effects of dendritic cells (DCs) transfected with survivin gene, and to observe the effective and specific anti-tumor immunological effect induced by modified DC in vitro. METHODS: Survivin gene was transfected to DCs with liposomes. Survivin expression could be detected both in DCs cells and in cell culture with method of Western blot. Cytokines as well as cellular surface molecule such as IL-12, TNF-alpha, CD1 alpha, CD83, MHCII, CD80 and CD86 were detected. The competence of inducing human specific cytotoxic T lymphocyte (CTLs) was also detected with MTT. RESULTS: Survivin expression could be detected both in DCs which were transfected with survivin cDNA and in cell culture superior. The IL-12 and TNF-alpha level was (265.2 +/- 32.7), (437.1 +/- 83.5) pg/ml, and much higher in transgened DC cells than blank DC cells (P < 0.05). CD1 alpha, CD83, MHCII, CD80 and CD86 was high expressed in survivin-DC cells, however, it was low expressed in blank DC cells. The lyse rate to gastric cancer cell, colon cancer cell and bile duct cancer cell was 65%, 77%, and 85% respectively, and these were much higher than those of blank DC cells. CONCLUSIONS: DCs transfected with survivin gene could induce specific cytotoxic T lymphocytes and strikingly raised DC cell's antigen present function, and have specific CTL killing activity.

Inactivation of RASSF1A, the tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma.

AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma. METHODS: RT-PCR was used to investigate the transcriptional expressing and re-expression of RASSF1A. RASSF1A mutation was analyzed with SSCP and selective sequencing. PCR was performed to detect the loss of heterozygosity (LOH) at the region of chromosome 3p21.3. Genomic DNA were modificated bisulfite and the frequency of methylation of CpG islands in RASSF1A promoter were evaluated by methylation specific PCR (MS-PCR). RESULTS: In all 48 samples and one cell lines of extrahepatic cholangiocarcinoma, the RASSF1A mutation is rare (6.12%, 3/49), 33 samples (68.75%) and QBC-939 cell lines (chi2 = 14.270, P = 0.001 > 0.01) showed RASSF1A express inactivation with LOH at microsatellite loci D3S4604. Among these 33 samples and QBC-939, 28 of 33 (84.85%) tumor samples and 1 cell lines were methylated for majority of 16 CpGs, the average frequency is 73.42%. CONCLUSION: The data we present suggest that RASSF1A which we have been searching for at 3p21.3 may be one of the key tumor suppressor gene and play an important role in the pathogenesis of extrahepatic cholangiocarcinoma, and the promoter methylation and allelic loss are the major mechanism for inactivation of RASSF1A.

Inhibitory effect of methylation inhibitor 5-aza-2-deoxycytidine on bile duct cancer cell line in vivo and in vitro.

BACKGROUND: Since the resection rate is low for bile duct cancer and the drugs used for chemotherapy are less effective, we studied the inhibitory effects of 5-aza-2-deoxycytidine (ZdCyd) on bile duct cancer cell line QBC939 in vivo and in vitro and its possibility in clinical treatment. METHODS: The survival and apoptosis rates of QBC939 after treatment with different dose of ZdCyd were detected by methyl thiazoy tetrazolium (MTT) and flow cytometry. The cooperative effect of ZdCyd with other chemotherapeutic drugs was also studied with MTT. The cancer cells were transplanted into nude mice, which were pre-treated with ZdCyd after tumor occurrence. RESULTS: ZdCyd decreased the cell survival rate, blocked the cell cycle at G1 phase, and increased the apoptosis rate. These effects were dose and time-dependent. ZdCyd also increased the anti-tumor effects of other chemotherapeutic drugs when used in combination. The tumor occurrence rate was lower in the ZdCyd pre-treated cells than in the untreated cells in nude mice, and ZdCyd was found to inhibit tumor growth. CONCLUSION: ZdCyd can inhibit the growth of QBC939 in vivo and in vitro through induction of cell apoptosis and has the cooperative effect on bile duct cancer cell when it is used with other chemotherapeutic drugs.