Analysis of Several Pathways for Efficient Killing of Prostate Cancer Stem Cells: A Central Role of NF-κB RELA.

Prostate cancer is a common cause of death worldwide. Here, we isolated cancer stem cells (CSCs) from four adenocarcinomas of the prostate (Gleason scores from 3 + 3 up to 4 + 5). CSCs were characterized by the expression of the stem cell markers TWIST, the epithelial cell adhesion molecule (EPCAM), the transcription factors SNAI1 (SNAIL) and SNAI2 (SLUG) and cancer markers such as CD44 and prominin-1 (CD133). All investigated CSC populations contained a fraction highly positive for aldehyde dehydrogenase (ALDH) function and displayed robust expressions of programmed cell death 1 (PD-1) ligands. Furthermore, we investigated immunotherapeutic approaches but had no success even with the clinically used PD-1 inhibitor pembrolizumab. In addition, we studied another death-inducing pathway via interferon gamma signaling and detected high-level upregulations of human leukocyte antigen A (HLA-A) and beta 2-microglobulin (B2M) with only moderate killing efficacy. To examine further killing mechanisms in prostate cancer stem cells (PCSCs), we analyzed NF-κB signaling. Surprisingly, two patient-specific populations of PCSCs were found: one with canonical NF-κB signaling and another one with blunted NF-κB activation, which can be efficiently killed by tumor necrosis factor (TNF). Thus, culturing of PCSCs and analysis of respective NF-κB induction potency after surgery might be a powerful tool for optimizing patient-specific treatment options, such as the use of TNF-inducing chemotherapeutics and/or NF-κB inhibitors.

Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition.

Cancer stem cells (CSC) are crucial mediators of cancer relapse. Here, we isolated two primary human colorectal cancer cell lines derived from a rectal neuroendocrine carcinoma (BKZ-2) and a colorectal adenocarcinoma (BKZ-3), both containing subpopulations with potential stem-like properties. Protein expression of CSC-markers prominin-1 and CD44 antigen was significantly higher for BKZ-2 and BKZ-3 in comparison to well-established colon carcinoma cell lines. High sphere-formation capacity further confirmed the existence of a subpopulation with potential stem-like phenotype. Epithelial-mesenchymal transition markers as well as immune checkpoint ligands were expressed more pronounced in BKZ-2. Both cell populations demonstrated N-myc proto-oncogene (NMYC) copy number gain. Myc proto-oncogene (MYC)/NMYC activity inhibitor all-trans retinoic acid (ATRA) significantly reduced the number of tumor spheres for both and the volume of BKZ-2 spheres. In contrast, the sphere volume of ATRA-treated BKZ-3 was increased, and only BKZ-2 cell proliferation was reduced in monolayer culture. Treatment with KJ-Pyr-9, a specific inhibitor of MYC/NMYC-myc-associated factor X interaction, decreased survival by the induction of apoptosis of both. In summary, here, we present the novel colorectal cancer cell lines BKZ-2 and BKZ-3 as promising cellular in vitro models for colorectal carcinomas and identify the MYC/NMYC molecular pathway involved in CSC-induced carcinogenesis with relevant therapeutic potential.

Expression of TRAIL-splice variants in gastric carcinomas: identification of TRAIL-γ as a prognostic marker.

BACKGROUND: TNF-related apoptosis inducing ligand (TRAIL) belongs to the TNF-superfamily that induces apoptotic cell death in a wide range of neoplastic cells in vivo as well as in vitro. We identified two alternative TRAIL-splice variants, i.e. TRAIL-ß and TRAIL-γ that are characterized by the loss of their proapoptotic properties. Herein, we investigated the expression and the prognostic values of the TRAIL-splice variants in gastric carcinomas. METHODS: Real time PCR for amplification of the TRAIL-splice variants was performed in tumour tissue specimens and corresponding normal tissues of 41 consecutive patients with gastric carcinoma. Differences on mRNA-expression levels of the TRAIL-isoforms were compared to histo-pathological variables and correlated with survival data. RESULTS: All three TRAIL-splice variants could be detected in both non-malignant and malignant tissues, irrespective of their histological staging, grading or tumour types. However, TRAIL-ß exhibited a higher expression in normal gastric tissue. The proapoptotic TRAIL-α expression was increased in gastric carcinomas when compared to TRAIL-ß and TRAIL-γ. In addition, overexpression of TRAIL-γ was associated with a significant higher survival rate. CONCLUSIONS: This is the first study that investigated the expression of TRAIL-splice variants in gastric carcinoma tissue samples. Thus, we provide first data that indicate a prognostic value for TRAIL-γ overexpression in this tumour entity.

High EpCAM expression is linked to proliferation and lauren classification in gastric cancer.

BACKGROUND: The association of EpCAM expression with the progression of gastric cancer remains unclear. Here, we investigated the expression of EpCAM in gastric cancer subtypes and correlated the data to tumor cell proliferation and clinicopathologic factors. METHODS: The intratumoral expression of EpCAM was assessed in 163 primary gastric cancers (61 diffuse-, 62 intestinal-, 32 mixed-type and 8 unclassified tumors) by immunohistochemistry, using the monoclonal antibody Ber-EP4. Intensity of staining was classified according the HercepTest-score using a standardized scoring system. Ki-67 was used to examine the proliferation in tumor tissue. RESULTS: Strong EpCAM expression was observed in 77% of the tumors and in 85% of the corresponding lymph nodes. Of the primary tumors, 58% (n=74) presented a homogeneous intratumoral EpCAM expression while 42% were characterised by a heterogenous expression pattern. Tumors with high EpCAM expression at the invasive front were associated with significantly (p=0.03) higher proportion of lymph node metastases and lower median overall survival (p=0.001). Diffuse type tumors presented a significantly higher EpCAM expression at the invasion front compared with the tumor centre (p=0.036). Multivariate survival analysis identified high EpCAM expression at the invasive front as an independent prognostic factor.We observed a significant (p=0.001) correlation between high EpCAM expression and higher tumor cell proliferation. CONCLUSION: High EpCAM expression associates with proliferation and progression of gastric cancer, especially in the diffuse type. Considering the discontenting results of the current adjuvant concepts for gastric cancer patients, EpCAM might be target in the adjuvant therapy of this malignant disease.

Infusion of CD133+ bone marrow-derived stem cells after selective portal vein embolization enhances functional hepatic reserves after extended right hepatectomy: a retrospective single-center study.

OBJECTIVE: This study was designed to evaluate the clinical outcome of patients undergoing portal vein embolization (PVE) and autologous CD133 bone marrow-derived stem cell (CD133+ BMSC) application before extended right hepatectomy. BACKGROUND: We have previously shown that portal venous infusion of CD133+ BMSCs substantially increases hepatic proliferation, when compared with PVE alone. METHODS: : Among 40 consecutive patients with a median follow-up of 28 months (7.4-57.2) scheduled for extended right hepatectomy, we compared a preconditioned group with PVE and CD133+ BMSC cotreatment (PVE+SC group, n = 11) and a group pretreated only with PVE (PVE group, n = 11). Functional and overall outcomes after extended right hepatectomy were evaluated. Patients without presurgical treatment served as controls (n = 18). RESULTS: In preconditioned patients, mean hepatic growth of segments II/III 14 days after PVE in the PVE+SC group was significantly higher (138.66 mL ± 66.29) when compared with that of PVE group patients (62.95 mL ± 40.03; P = 0.004). There were no significant differences among all 3 groups regarding general and oncological characteristics and functional parameters on postoperative day (POD) 7. Lack of hepatic preconditioning, extrahepatic extension of resection, and postoperative complications were of negative prognostic value, using univariate analysis (P < 0.05). In multivariate analysis, freedom from postoperative major complications (P = 0.012), coagulation status on POD 7 (international normalized ratio < 1.4; P = 0.027), and presurgical expansion of the future liver remnant volume (P = 0.048) were positively associated with overall survival. Post hoc analysis revealed a better survival for the PVE+SC group (P = 0.028) compared with the PVE group (P = 0.094) and compared with controls. CONCLUSION: Promising data from this survival analysis suggest that PVE, together with CD133+ BMSC pretreatment, could positively impact overall outcomes after extended right hepatectomy.

GPIbalpha-selective activation of platelets induces platelet signaling events comparable to GPVI activation events.

Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbalpha (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbalpha-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrand's Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbalpha- and alpha(IIb)beta(3)-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbalpha and anti-alpha(IIb)beta(3) antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbalpha-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbalpha engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbalpha can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.

Non-colorectal, non-neuroendocrine, and non-sarcoma metastases of the liver: resection as a promising tool in the palliative management.

PURPOSE: Hepatic resection is established as a safe procedure for colorectal, neuroendocrine, and sarcoma liver metastases. The present study evaluates whether liver resection is an option for patients with non-colorectal, non-neuroendocrine, and non-sarcoma metastases of the liver. METHODS: According to data from our prospective clinical tumor registry, we reviewed the medical records of 44 consecutive patients with non-colorectal, non-neuroendocrine, and non-sarcoma liver metastases, who underwent hepatic resection from January 2000 to December 2008. Univariate Kaplan-Meier analysis and a stepwise multivariable Cox regression model were applied. RESULTS: Following hepatic resection, mean overall survival was 21 months, and 5-year survival was 20%. Following hepatic resection, gender, histology, and chemotherapy were of prognostic value in our patient cohort in univariate analysis (p < 0.05). Multivariate survival analysis confirmed chemotherapy (p = 0.002) as an independent prognostic variable. Following initial resection of the primary tumor, synchrone occurrence of metastases, histology, localization of primary, perioperative complications, interval between initial resection of the primary tumor and resection of the metastases, and metastases in follow-up after hepatic resection were of prognostic value in univariate analysis (p < 0.05). Histology (p = 0.017) and interval between resection of the primary and resection of the metastases (p = 0.030) were confirmed as independent prognostic variables in multivariate survival analysis. CONCLUSIONS: Hepatic resection seems to be a safe and promising additive for a selective group of patients with non-colorectal, non-neuroendocrine, and non-sarcoma metastases of the liver.

Intrahepatic cholestasis without jaundice.

BACKGROUND: Cholangiocarcinoma (CC), the most common biliary tract malignancy, is frequently seen in advanced unresectable stages and is typically localized extrahepatically. Early diagnosis is unusual because of nonspecific symptoms. Painless jaundice is usually the first sign of tumor. METHOD: We present a patient with a CC (Klatskin tumor) with a complete biliary drainage by an aberrant bile duct without jaundice. RESULTS: A 67-year-old woman presented with persisting elevation of liver parameters. Diagnostic tests showed a Klatskin tumor type II. A curative right hepatic trisegmentectomy was performed after liver volume augmentation by preoperative vein embolization. CONCLUSIONS: A direct drainage of the right posterior bile duct into the common bile duct as an aberrant hepatic duct is a rare variation and is present in less than 5% of the population. In case of persistently perturbed liver function tests, an aberrant bile duct can cover up severe intrahepatic cholestasis and even obscure the diagnosis of a Klatskin tumor. Up to now it has not been described in the literature.

Surgical access to jejunal veins for local thrombolysis and stent placement in portal vein thrombosis.

Portal vein thrombosis is an infrequent entity, which may cause high morbidity and mortality. We report a case of portal vein thrombosis due to benign stenosis following partial pancreatoduodenectomy with segmental replacement of the portal vein by a Gore-tex graft. Using a surgical access to jenunal veins, local thrombolysis, mechanical fragmentation of thrombus, and stent placement were successfully performed.

Recipient levels and function of von Willebrand factor prior to liver transplantation and its consumption in the course of grafting correlate with hepatocellular damage and outcome.

Von Willebrand factor (vWF) is a major platelet adhesion molecule at sites of vascular injury, such as observed in ischemia/reperfusion injury following orthotopic liver transplantation (OLT). Thirty-three OLT patients were divided into groups with elevated or low markers of hepatocellular damage (high and low-HD). Whole-blood aggregometry was performed to evaluate platelet function. Multimeric analysis was utilized to evaluate functional vWF levels in the course of OLT. Donor and recipient demographics were comparable among groups. Low-HD patients demonstrated better preserved coagulation parameters on POD 1-6 if contrasted to high-HD patients. One year graft survival for the high-HD group was lower than low-HD patients (P = 0.037). Preoperative vWF-dependent platelet aggregation and functional vWF plasma levels correlated directly with alanine transaminase levels early after OLT as did the decrease of functional vWF to reperfusion. In summary, these data suggest that vWF may serve as a significant mediator of platelet recruitment and hepatocellular injury in the graft following reperfusion.

Portal application of autologous CD133+ bone marrow cells to the liver: a novel concept to support hepatic regeneration.

The liver has a large capacity for regeneration after resection. However, below a critical level of future liver remnant volume (FLRV), partial hepatectomy is accompanied by a significant increase of postoperative liver failure. There is accumulating evidence for the contribution of bone marrow stem cells (BMSCs) to participate in liver regeneration. Here we report on three patients subjected to intraportal administration of autologous CD133(+) BMSCs subsequent to portal venous embolization of right liver segments, used to expand left lateral hepatic segments as FLRV. Computerized tomography scan volumetry revealed 2.5-fold increased mean proliferation rates of left lateral segments compared with a group of three consecutive patients treated without application of BMSCs. This early experience with portovenous application of CD133(+) BMSCs could suggest that this novel therapeutic approach bears the potential of enhancing and accelerating hepatic regeneration in a clinical setting.

Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure.

OBJECTIVE: The HepatAssist liver support system is an extracorporeal porcine hepatocyte-based bioartificial liver (BAL). The safety and efficacy of the BAL were evaluated in a prospective, randomized, controlled, multicenter trial in patients with severe acute liver failure. SUMMARY BACKGROUND DATA: In experimental animals with acute liver failure, we demonstrated beneficial effects of the BAL. Similarly, Phase I trials of the BAL in acute liver failure patients yielded promising results. METHODS: A total of 171 patients (86 control and 85 BAL) were enrolled. Patients with fulminant/subfulminant hepatic failure and primary nonfunction following liver transplantation were included. Data were analyzed with and without accounting for the following confounding factors: liver transplantation, time to transplant, disease etiology, disease severity, and treatment site. RESULTS: For the entire patient population, survival at 30 days was 71% for BAL versus 62% for control (P = 0.26). After exclusion of primary nonfunction patients, survival was 73% for BAL versus 59% for control (n = 147; P = 0.12). When survival was analyzed accounting for confounding factors, in the entire patient population, there was no difference between the 2 groups (risk ratio = 0.67; P = 0.13). However, survival in fulminant/subfulminant hepatic failure patients was significantly higher in the BAL compared with the control group (risk ratio = 0.56; P = 0.048). CONCLUSIONS: This is the first prospective, randomized, controlled trial of an extracorporeal liver support system, demonstrating safety and improved survival in patients with fulminant/subfulminant hepatic failure.