Fibroblast activation protein inhibitor (FAPi) positive tumour fraction on PET/CT correlates with Ki-67 in liver metastases of neuroendocrine tumours.

AIM: Gallium-68-labelled inhibitors of the fibroblast activation protein (FAPi) enable positron emission tomography/computed tomography (PET/CT) imaging of fibroblast activation. We evaluated if [68Ga]Ga-DATA5m.SA.FAPi PET/CT is related to Ki-67 as a marker of tumour aggressiveness in patients with liver metastases of NET. METHODS: Thirteen patients with liver metastases of a histologically confirmed NET who underwent PET/CT with [68Ga]Ga-DATA5m.SA.FAPi, [18F]FDG and [68Ga]Ga-DOTA-TOC were retrospectively analyzed. PET-positive liver tumour volumes were segmented for calculation of volume, SUVmax and PET-positive tumour fraction (TF). PET parameters were correlated with Ki-67. RESULTS: FDGSUVmax correlated positively (rho = 0.543, p < 0.05) and DOTATOCSUVmax correlated negatively (rho = -0.618, p < 0.05) with Ki-67, the correlation coefficients were in the moderate range. There was no significant correlation between FAPiSUVmax and Ki-67 (rho = 0.382, p > 0.05). FAPiTF correlated positively (rho = 0.770, p < 0.01) and DOTATOCTF correlated negatively (rho = -0.828, p < 0.01) with Ki-67, both significantly with high correlation coefficients. FDGTF also correlated significantly with Ki-67, with a moderate correlation coefficient (rho = 0.524, p < 0.05). The ratio FAPiVOL:DOTATOCVOL showed a significant and strong correlation with Ki-67 (rho = 0.808, p < 0.01). CONCLUSION: The ratio FAPiVOL:DOTATOCVOL might serve as a clinical parameter for the assessment of dedifferentiation and aggressiveness of liver metastases in patients with NET. [68Ga]Ga-DATA5m.SA.FAPi might hold potential for identification of high-risk patients. Further studies are warranted to evaluate its prognostic significance in comparison to [18F]FDG in patients with NET.

Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma.

Immune checkpoint inhibition suggests promising progress for the treatment of advanced hepatocellular carcinoma (HCC). However, the underlying cellular mechanisms remain unclear because liver cancer cells apparently do not upregulate inhibitory checkpoint molecules. Here, we analysed whether regulatory T cells (Tregs) can alternatively trigger checkpoint inhibition pathways in HCC. Using flow cytometry we analysed expression of checkpoint molecules (PD-1, PD-L1, CTLA-4, GITR, Tim-3) on peripheral CD4+CD25+Foxp3+ Tregs and their secretion of inhibitory mediators (IL-10, IL-35, TGF-beta, galectin-9) in 116 individuals (50 patients with HCC, 41 non-tumour bearing liver disease controls, 25 healthy controls). Functional activity of Tregs on T effector cells (IFN-gamma production, cytotoxicity) was characterized in vitro using a lectin-dependent cellular cytotoxicity (LDCC) assay against checkpoint inhibitor-negative P815 target cells. Unlike liver patients without malignancy and healthy controls, the frequency of checkpoint inhibitor-positive Tregs inversely correlated to age of patients with HCC (PD-L1, p = 0.0080; CTLA-4, p = 0.0029) and corresponded to enhanced numbers of Tregs producing IL-10 and IL-35 (p < 0.05 each). Tregs inhibited IFN-gamma secretion and cytotoxicity of CD8+ T cells when added to LDCC against P815 cells. Treg-induced inhibition of IFN-gamma secretion could be partially blocked by neutralizing PD-1 and PD-L1 antibodies specifically in HCC patients. In HCC peripheral Tregs upregulate checkpoint inhibitors and contribute to systemic immune dysfunction and antitumoural activity by several inhibitory pathways, presumably facilitating tumour development at young age. Blocking PD-L1/PD-1 interactions in vitro selectively interfered with inhibitory Treg -T effector cell interactions in the patients with HCC and resulted in improved antitumoural activity also against checkpoint inhibitor-negative tumour cells.

Combined photodynamic therapy with systemic chemotherapy for unresectable cholangiocarcinoma.

BACKGROUND: Chemotherapy with gemcitabine and cisplatin is the current standard for patients with unresectable cholangiocarcinoma. Local photodynamic therapy has also demonstrated benefit in patients with extrahepatic cholangiocarcinoma. AIM: To evaluate the benefit of photodynamic therapy in combination with systemic chemotherapy in advanced extrahepatic cholangiocarcinoma. METHODS: Three hundred and fifty-three patients diagnosed with cholangiocarcinoma between 2004 and 2016 were treated at the University Hospital of Bonn, Germany. Of these, 96 suffering from unresectable extrahepatic cholangiocarcinoma were included. Patients were stratified according to treatment: combination photodynamic therapy and chemotherapy (36 patients), photodynamic therapy alone (34 patients), and chemotherapy alone (26 patients). RESULTS: Combined photodynamic therapy with chemotherapy resulted in significantly longer overall survival than chemotherapy alone (P = 0.022). Median survival was 20 months in the combination group (95% CI: 16.38-23.62), 15 months in the photodynamic alone group (95% CI: 10.02-19.98) and 10 months in the chemotherapy alone group (95% CI: 8.45-11.55). In multivariate analysis, combination therapy and photodynamic therapy alone (HR: 0.41, 95% CI: 0.22-0.77, P = 0.006), metal stenting, and radiofrequency ablation were independent predictors of longer survival. CONCLUSIONS: Combination photodynamic therapy and chemotherapy was well tolerated and resulted in significantly longer survival than chemotherapy alone. Application of photodynamic therapy significantly correlated with longer survival, demonstrating benefit in advanced cholangiocarcinoma. Thus, photodynamic therapy should be considered during therapeutic decision making in advanced cholangiocarcinoma.

Toxic damage increases angiogenesis and metastasis in fibrotic livers via PECAM-1.

Excessive ethanol consumption is one of the main causes of liver fibrosis. However, direct effects of ethanol exposure on endothelial cells and their contribution to fibrogenesis and metastasis were not investigated. Therefore we analysed whether ethanol directly affects endothelial cells and if this plays a role during fibrogenesis and metastasis in the liver. Murine and human endothelial cells were exposed to ethanol for up to 72 hours. In vitro, effects on VEGF, HIF-1alpha, PECAM-1, and endothelial cell functions were analysed. In vivo, effects of continuous liver damage on blood vessel formation and metastasis were analysed by PECAM-1 immunohistochemistry. Ethanol increased HIF-1alpha and VEGF levels in murine and human endothelial cells. This resulted in enhanced intracellular signal transduction, and PECAM-1 expression as well as tube formation and wound healing. In vivo, toxic liver damage increased angiogenesis during fibrogenesis. Metastasis was also enhanced in fibrotic livers and located to PECAM-1 positive blood vessels compared to nonfibrotic mice. In conclusion, ethanol had strong effects on endothelial cells, which--at least in part--led to a profibrotic and prometastatic environment mediated by PECAM-1. Blockade of increased PECAM-1 expression could be a promising tool to inhibit fibrogenesis and metastasis in the liver.

Combination of hypoxia and RNA-interference targeting VEGF induces apoptosis in hepatoma cells via autocrine mechanisms.

Control of VEGF signaling is an intense objective of pre-clinical and clinical studies in HCC disease with steadily increasing clinical application. Despite its emerging role, several aspects of anti-VEGF based treatments are poorly investigated, like the impact on tumor cells themselves, such as the effect on intracellular signaling and apoptosis induction in hepatoma cells. Effects of siRNA-VEGF on VEGF, VEGF-receptor expression and VEGF-A signaling such as AKT and JNK phosphorylation were determined under normoxic or hypoxic conditions in murine hepatoma cells. Apoptosis induction was analyzed by SubG1-fraction, JC1-staining and caspase-8 activation. VEGF receptor expression was analysed by semiquantitative real time PCR. Independent of oxygen status, siRNA-VEGF reduced VEGF levels resulting in decreased AKT and increased JNK phosphorylation in Hepa129 cells. The VEGF-receptors neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2) were downregulated following siRNA-VEGF treatment or hypoxia induction respectively. Functionally, hypoxia significantly increased the apoptosis rate (as analyzed by SubG1-fraction, JC1-staining and JNKphosphorylation) which was further stimulated by siRNA-VEGF treatment. Our data indicate that antitumoral efficacy of an anti-VEGF based treatment with siRNA is partly based on negative autocrine feedback mechanisms which are even enhanced under hypoxic conditions. This observation helps to understand why antitumoral efficacy can be maintained despite of counteracting stimulation of tumoral VEGF secretion due to hypoxia. The direct impact on tumor cells further underscores the attractiveness of an anti-VEGF based siRNA treatment.

Plasminogen derivatives encoding kringles 1-4 and kringles 1-5 exert indirect antiangiogenic and direct antitumoral effects in experimental lung cancer.

Recently, increasing evidence has been found demonstrating direct effects of angiostatin on tumor cells themselves. We have applied the plasminogen derivatives K1-4 and K1-5 to a lung cancer model to analyse indirect angiostatic effects against endothelial and direct effects against tumor cells. In accordance with preceding findings both derivatives inhibited endothelial cell functions in vitro. Additionally K1-4 and K1-5 have also shown substantial anti-proliferative and pro-apoptotic effects in tumor cells and have inhibited tumor growth. In addition our data supports the recent conclusion that plasminogen derivatives have a dual antitumor mechanism affecting both tumor angiogenesis and tumor cells.

Plasminogen fragment K1-5 improves survival in a murine hepatocellular carcinoma model.

BACKGROUND: The prognosis of patients with hepatocellular carcinoma (HCC) remains poor, and new alternative treatments are needed. AIMS: To comparatively test the angiostatic and antitumour effects of adenoviral gene transfer of angiostatin (PlgK1-4, amino acids 1-440) and full kringles 1-5 (PlgK1-5, amino acids 1-546) in a model of subcutaneously transferred HCC in mice. METHODS: PlgK1-4 and PlgK1-5 were generated from human WtPlg cDNA and used for adenovirus construction. Vector function and angiostatic effects were confirmed in vitro and in vivo. Antitumoral efficacies of intratumoral vector injections were studied in a model of subcutaneously transferred HCC model. RESULTS: Cell supernatants containing PlgK1-4 and PlgK1-5 reduced endothelial tube formation in vitro by about 30%, whereas WtPlg exerted no inhibitory effect. Endothelial cell infiltration in vivo was decreased by about 60%, but not in AdWtPlg-treated animals. Intratumoral treatment of subcutaneous HCC tumours inhibited growth by 40% for AdPlgK1-4 and 63% for AdPlgK1-5 in surviving mice 12 days after initiation of treatment, whereas treatment with AdWtPlg even led to accelerated growth. Although PlgK1-4 and PlgK1-5 have similar inhibitory effects on intratumoral microvessels, PlgK1-5 markedly improved the survival time compared with PlgK1-4. CONCLUSION: PlgK1-5 and PlgK1-4 effectively inhibited HCC growth. As PlgK1-5 could also prolong the survival time, inducing complete tumour elimination in half of the AdPlgK1-5-treated mice, PlgK1-5 might be the most potential plasminogen fragment for treatment of experimental HCC.

Hammerhead ribozymes with cleavage site specificity for NUH and NCH display significant anti-hepatitis C viral effect in vitro and in recombinant HepG2 and CCL13 cells.

BACKGROUND/AIMS: Four different ribozymes (Rz) targeting the hepatitis C virus (HCV) 5'-non-coding region (NCR) at nucleotide (nt) positions GUA 165 (Rz1), GUC 270 (Rz2), GUA 330 (Rz3) and GCA 348 (Rz1293) were compared for in vitro cleavage using a 455 nt HCV RNA substrate. The GUA 330 (Rz3) and GCA 348 (Rz1293) ribozymes, both targeting the HCV loop IV region, were found to be the most efficient, and were further analyzed in an in vitro translation system. METHODS: For this purpose RNA transcribed from a construct encoding a HCV-5'-NCR-luciferase fusion protein was used. Cleavage-inactive (Rz1426), mismatch (Rz1293m) or unrelated ribozymes (Rz1437) were synthesized as controls for Rz-1293. HCV specificity was analysed by competition experiments using sense and mismatch oligodeoxynucleotides HCVrzCI and HCVrzMM, respectively. RESULTS: A chemically modified nuclease-resistant variant of the GCA 348 cleaving ribozyme was selected for cell culture experiments using recombinant HepG2 or CCL13 cell lines stably transfected with a HCV-5'-NCR-luciferase target construct. CONCLUSIONS: This ribozyme (Rz1293) showed an inhibitory activity of translation of more than 70% thus verifying that the GCA 348 cleavage site in the HCV loop IV is an accessible target site in vivo and may be suitable for the development of novel optimized hammerhead structures.

Keeping central venous lines open: a prospective comparison of heparin, vitamin C and sodium chloride sealing solutions in medical patients.

OBJECTIVE: To prevent catheter occlusion, intermittently used central venous catheters are frequently sealed with vitamin C solution or heparin solution between use. The present study was designed to test the effectiveness of this approach and to compare the efficiency of sealing solutions. DESIGN AND SETTING: Prospective randomized study performed on a 9-bed medical ICU and on medical wards of an academic tertiary care center. PARTICIPANTS. Ninety-nine central venous line placements were prospectively included in the study and randomized into three treatment groups: sodium chloride 0.9%, vitamin C (200 mg/ml) and heparin (5000 IU/ml) sealing solutions. INTERVENTIONS AND MEASUREMENTS: Catheters were filled with the respective sealing solution and patency was tested once every 2 days using a standardized routine. Catheter patency was compared among the three groups using Kaplan-Meier statistics and log-rank testing. RESULTS: There was a significant difference in catheter patency between the three groups (p<0.03, log-rank test). A comparison of catheter survival between the catheters filled with heparin and those filled with sodium chloride, but not between those filled with vitamin C solution and with sodium chloride solution, exhibited significant differences in catheter patency (p<0.04, log-rank test). CONCLUSIONS: Local anticoagulation of intermittently used central venous catheters prolongs catheter patency. High-dose (5000 IU/ml) heparin solution is a useful anticoagulant for this purpose, while vitamin C solution does not prolong catheter patency.