BackgroundCirculating tumour cells (CTCs) have shown prognostic relevance in metastatic breast, prostate, colon and pancreatic cancer. For further development of CTCs as a biomarker, we compared the performance of different protocols for CTC detection in murine breast cancer xenograft models (MDA-MB-231, MDA-MB-468 and KPL-4). Blood samples were taken from tumour bearing animals (20 to 200 mm2) and analysed for CTCs using 1. an epithelial marker based enrichment method (AdnaTest), 2. an antibody independent technique, targeting human gene transcripts (qualitative PCR), and 3. an antibody-independent approach, targeting human DNA-sequences (quantitative PCR). Further, gene expression changes associated with epithelial-to-mesenchymal transition (EMT) were determined with an EMT-specific PCR assay.MethodsWe used the commercially available Adna Test, RT-PCR on human housekeeping genes and a PCR on AluJ sequences to detect CTCs in xenografts models. Phenotypic changes in CTCs were tested with the commercially available “Human Epithelial to Mesenchymal Transition RT-Profiler PCR Array”.ResultsAlthough the AdnaTest detects as few as 1 tumour cell in 1 ml of mouse blood spiking experiments, no CTCs were detectable with this approach in vivo despite visible metastasis formation. The presence of CTCs could, however, be demonstrated by PCR targeting human transcripts or DNA-sequences - without epithelial pre-enrichment. The failure of CTC detection by the AdnaTest resulted from downregulation of EpCAM, whereas mesenchymal markers like Twist and EGFR were upregulated on CTCs. Such a change in the expression profile during metastatic spread of tumour cells has already been reported and was linked to a biological program termed epithelial-mesenchymal transition (EMT).ConclusionsThe use of EpCAM-based enrichment techniques leads to the failure to detect CTC populations that have undergone EMT. Our findings may explain clinical results where low CTC numbers have been reported even in patients with late metastatic cancers. These results are a starting point for the identification of new markers for detection or capture of CTCs, including the mesenchymal-like subpopulations.

We examined the prognostic value of tumour-infiltrating lymphocytes (TILs) in patients with squamous cell carcinoma of the head and neck (SCCHN) after surgery and postoperative cisplatin-based chemoradiotherapy. FFPE-tissue originating from the surgery of 161 patients treated in 8 DKTK partner sites was immunohistochemically stained for CD3 and CD8. Their expression was correlated with clinicopathological characteristics as well as overall survival (OS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS), also in the context of the HPV16-DNA/p16 status. After a median follow-up of 48 months (range: 4100 months), OS at 4 years was 46.5% for the entire cohort. In multivariate analysis, high CD8 expression was confirmed as an independent prognostic parameter for OS (p 5 0.002), LPFS (p 5 0.004) and DMFS (p 5 0.006), while CD3 expression lacked significance. In multivariate analysis HPV16 DNA positivity was associated with improved OS (p 5 0.025)

PC12 cells respond to a variety of external stimuli such as growth factors, neurotransmitters, and membrane depolarization by activating the Ras/mitogen-activated protein kinase pathway. Here we demonstrate that both depolarization-induced calcium influx and treatment with bradykinin stimulate tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Using a tetracycline-controlled expression system in conjunction with a dominant-negative EGFR mutant, we demonstrate that depolarization and bradykinin triggered signals involve EGFR function upstream of SHC and MAP kinase. Furthermore, bradykinin-stimulated EGFR transactivation is critically dependent on the presence of extracellular calcium, and when triggered by ionophore treatment, calcium influx is already sufficient to induce EGFR tyrosine phosphorylation. Taken together, our results establish calcium-dependent EGFR transactivation as a signaling mechanism mediating activation of the Ras/mitogen-activated protein kinase pathway in neuronal cell types.In neurons, the cytosolic calcium concentration is tightly regulated and represents a critical parameter for a variety of intracellular signaling processes. Intracellular calcium levels are modulated either by release of calcium from internal stores or by calcium entry across the plasma membrane through ligand-or voltage-gated calcium channels (1-3). Stimuli such as membrane depolarization result in activation of L-type voltage-sensitive calcium channels, leading to calcium-mediated induction of a specific set of genes and thereby contributing to physiological responses such as neuronal differentiation and survival (4) .Before altering gene expression, elevation of intracellular calcium levels can trigger various signaling events, among them the activation of the small G-protein Ras resulting in stimulation of the mitogen-activated protein kinase (MAPK) 1 pathway (5). In PC12 cells, a rat pheochromocytoma cell line widely used as a model system for neuronal differentiation, calcium influx rapidly induces tyrosine phosphorylation of the adaptor protein SHC and SHC-Grb2 complex formation, steps known to couple cell surface receptors such as receptor tyrosine kinases to Ras (6). Using a PC12 subline overexpressing a dominant-negative mutant of the cytoplasmatic tyrosine kinase Src, Rusanescu et al. found that inhibiton of membrane depolarization induced SHC tyrosine phosphorylation and MAPK activation (7). Moreover calcium influx following membrane depolarization was recently reported to mediate ligandindependent epidermal growth factor receptor (EGFR) tyrosine phosphorylation in this system (8). Although direct evidence is lacking regarding whether this represents an essential signaling event for activation of the MAPK pathway, this finding raises the possibility that in PC12 cells calcium may play a role in the EGFR transactivation mechanism as previously demonstrated for signaling through G-protein coupled receptor (GPCR) in Rat-1 fibroblasts (9). In addition to membrane depolarization-induced acti...

Purpose: To investigate the impact of hypoxia-induced gene expression and cancer stem cell (CSC) marker expression on outcome of postoperative cisplatin-based radiochemotherapy (PORT-C) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC).Experimental Design: Expression of the CSC markers CD44, MET, and SLC3A2, and hypoxia gene signatures were analyzed in the resected primary tumors using RT-PCR and nanoString technology in a multicenter retrospective cohort of 195 patients. CD44 protein expression was further analyzed in tissue microarrays. Primary endpoint was locoregional tumor control.Results: Univariate analysis showed that hypoxia-induced gene expression was significantly associated with a high risk of locoregional recurrence using the 15-gene signature (P ¼ 0.010) or the 26-gene signature (P ¼ 0.002). In multivariate analyses, in patients with HPV16 DNA-negative but not with HPV16 DNA-positive tumors the effect of hypoxia-induced genes on locoregional control was apparent (15-gene signature: HR 4.54, P ¼ 0.006; 26-gene signature: HR 10.27, P ¼ 0.024). Furthermore, MET, SLC3A2, CD44, and CD44 protein showed an association with locoregional tumor control in multivariate analyses (MET: HR 3.71, P ¼ 0.016; SLC3A2: HR 8.54, P ¼ 0.037; CD44: HR 3.36, P ¼ 0.054; CD44 protein n/a because of no event in the CD44-negative group) in the HPV16 DNA-negative subgroup.Conclusions: We have shown for the first time that high hypoxia-induced gene expression and high CSC marker expression levels correlate with tumor recurrence after PORT-C in patients with HPV16 DNA-negative HNSCC. After validation in a currently ongoing prospective trial, these parameters may help to further stratify patients for individualized treatment de-escalation or intensification strategies.

Radiomics applies machine learning algorithms to quantitative imaging data to characterise the tumour phenotype and predict clinical outcome. For the development of radiomics risk models, a variety of different algorithms is available and it is not clear which one gives optimal results. Therefore, we assessed the performance of 11 machine learning algorithms combined with 12 feature selection methods by the concordance index (C-Index), to predict loco-regional tumour control (LRC) and overall survival for patients with head and neck squamous cell carcinoma. The considered algorithms are able to deal with continuous time-to-event survival data. Feature selection and model building were performed on a multicentre cohort (213 patients) and validated using an independent cohort (80 patients). We found several combinations of machine learning algorithms and feature selection methods which achieve similar results, e.g., MSR-RF: C-Index = 0.71 and BT-COX: C-Index = 0.70 in combination with Spearman feature selection. Using the best performing models, patients were stratified into groups of low and high risk of recurrence. Significant differences in LRC were obtained between both groups on the validation cohort. Based on the presented analysis, we identified a subset of algorithms which should be considered in future radiomics studies to develop stable and clinically relevant predictive models for time-to-event endpoints.

HPV16 DNA status appears to be a strong prognosticator of loco-regional tumour control after postoperative cisplatin-based radiochemotherapy of locally advanced oropharyngeal carcinoma and is now being explored in a prospective validation trial.

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We report that resveratrol (3,5,4'-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other plant food, induced a breakdown of the mitochondrial transmembrane potential (∆Ψ m ) in T-acute lymphoblastic leukemia cells and swelling of isolated rat mitochondria. The breakdown of ∆Ψ m was accompanied by the production of reactive oxygen species (ROS), and preceded phosphatidylserine exposure and DNA fragmentation. Breakdown of ∆Ψ m was not caused by the activation of caspase-8 or Bid, as no significant cleavage of these proteins could be detected in the induction phase of resveratrol-induced apoptosis. Though loss of ∆Ψ m was not followed by cytochrome c translocation to the cytosol, the mitochondrial changes triggered significant activation of caspase-9, -2, -3, and -6. Inhibition of ∆Ψ m breakdown and of ROS generation by N-acetylcysteine, or by overexpression of Bcl-2 protein, prevented apoptosis induction by resveratrol. The Bcl-2 expression status of tumor cells should therefore be considered relevant for potential clinical application of resveratrol as anticancer agent.Key words: cell death • antioxidant • cytochrome c-independent • lymphoblastic leukemia esveratrol, a polyphenolic compound present in grapes and in red wine, has been found to inhibit cellular events that increase the risk of carcinogenesis, a finding that has initiated numerous studies on its molecular mechanisms. These studies revealed antioxidant activities such as the inhibition of free radical formation following TPA stimulation (1) and of peroxidation of lipids in microsomes and of plasma LDL (2). Further mechanisms of action comprise modulation of lipoprotein metabolism and inhibition of platelet aggregation and coagulation (3). Its chemopreventive properties have been demonstrated in vitro, where R resveratrol lowers the mutagenic responses induced in salmonella by the aryl hydrocarbon dimethylbenz[a]anthracene treatment and induces quinone reductase activity (an enzyme capable of detoxifying carcinogens) (4). Moreover, it strongly inhibits P450-1A1 activity (an enzyme that transforms environmental toxins and procarcinogens into ultimate carcinogens), and it prevents dioxin toxicity by antagonistic activity on the aryl hydrocarbon receptor (5).More recently, its antioxidant and chemopreventive effects have been demonstrated in vivo by showing that it prevents the development of preneoplastic lesions in carcinogen-treated mouse mammary glands and inhibits tumorigenesis in a mouse skin cancer model (6). These results may be explained by its growth-inhibitory effects on tumor cell lines; by its capacity to directly induce apoptosis in various human tumor models, including promyelocytic leukemia (7), prostate cancer (8), and breast cancer (9, 10); and by its tumor specificity, because it does not affect human peripheral blood lymphocytes from healthy donors (9). The growth-inhibitory effect of resveratrol might result from its influence on the expression levels or phosphorylation status of cell cycle regulators such as cyclin E, c...