Detection of circulating tumour cell clusters in human glioblastoma.

Human glioblastoma (GBM) is a highly aggressive, invasive and hypervascularised malignant brain cancer. Individual circulating tumour cells (CTCs) are sporadically found in GBM patients, yet it is unclear whether multicellular CTC clusters are generated in this disease and whether they can bypass the physical hurdle of the blood-brain barrier.  Here, we assessed CTC presence and composition at multiple time points in 13 patients with progressing GBM during an open-label phase 1/2a study with the microtubule inhibitor BAL101553. We observe CTC clusters ranging from 2 to 23 cells and present at multiple sampling time points in a GBM patient with pleomorphism and extensive necrosis, throughout disease progression. Exome sequencing of GBM CTC clusters highlights variants in 58 cancer-associated genes including ATM, PMS2, POLE, APC, XPO1, TFRC, JAK2, ERBB4 and ALK. Together, our findings represent the first evidence of the presence of CTC clusters in GBM.

Prediction of Safety Margin and Optimization of Dosing Protocol for a Novel Antibiotic using Quantitative Systems Pharmacology Modeling.

Elevations of liver enzymes have been observed in clinical trials with BAL30072, a novel antibiotic. In vitro assays have identified potential mechanisms for the observed hepatotoxicity, including electron transport chain (ETC) inhibition and reactive oxygen species (ROS) generation. DILIsym, a quantitative systems pharmacology (QSP) model of drug-induced liver injury, has been used to predict the likelihood that each mechanism explains the observed toxicity. DILIsym was also used to predict the safety margin for a novel BAL30072 dosing scheme; it was predicted to be low. DILIsym was then used to recommend potential modifications to this dosing scheme; weight-adjusted dosing and a requirement to assay plasma alanine aminotransferase (ALT) daily and stop dosing as soon as ALT increases were observed improved the predicted safety margin of BAL30072 and decreased the predicted likelihood of severe injury. This research demonstrates a potential application for QSP modeling in improving the safety profile of candidate drugs.

Mechanisms of hepatotoxicity associated with the monocyclic ß-lactam antibiotic BAL30072.

BAL30072 is a new monocyclic ß-lactam antibiotic under development which provides a therapeutic option for the treatment of severe infections caused by multi-drug-resistant Gram-negative bacteria. Despite the absence of liver toxicity in preclinical studies in rats and marmosets and in single dose clinical studies in humans, increased transaminase activities were observed in healthy subjects in multiple-dose clinical studies. We, therefore, initiated a comprehensive program to find out the mechanisms leading to hepatocellular injury using HepG2 cells (human hepatocellular carcinoma cell line), HepaRG cells (inducible hepatocytes derived from a human hepatic progenitor cell line), and human liver microtissue preparations. Our investigations demonstrated a concentration- and time-dependent reduction of the ATP content of BAL30072-treated HepG2 cells and liver microtissues. BAL30072 impaired oxygen consumption by HepG2 cells at clinically relevant concentrations, inhibited complexes II and III of the mitochondrial electron transport chain, increased the production of reactive oxygen species (ROS), and reduced the mitochondrial membrane potential. Furthermore, BAL 30072 impaired mitochondrial fatty acid metabolism, inhibited glycolysis, and was associated with hepatocyte apoptosis. Co-administration of N-acetyl-L-cysteine partially protected hepatocytes from BAL30072-mediated toxicity, underscoring the role of oxidative damage in the observed hepatocellular toxicity. In conclusion, BAL30072 is toxic for liver mitochondria and inhibits glycolysis at clinically relevant concentrations. Impaired hepatic mitochondrial function and inhibition of glycolysis can explain liver injury observed in human subjects receiving long-term treatment with this compound.

Adaptive Resistance to Immunotherapy Directed Against p53 Can be Overcome by Global Expression of Tumor-Antigens in Dendritic Cells.

Immunotherapy of cancer utilizes dendritic cells (DCs) for antigen presentation and the induction of tumor-specific immune responses. However, the therapeutic induction of anti-tumor immunity is limited by tumor escape mechanisms. In this study, immortalized dendritic D2SC/1 cells were transduced with a mutated version of the p53 tumor suppressor gene, p53M234I, or p53C132F/E168G, which are overexpressed in MethA fibrosarcoma tumor cells. In addition, D2SC/1 cells were fused with MethA tumor cells to generate a vaccine that potentially expresses a large repertoire of tumor-antigens. Cellular vaccines were transplanted onto Balb/c mice and MethA tumor growth and anti-tumor immune responses were examined in vaccinated animals. D2SC/1-p53M234I and D2SC/1-p53C132F/E168G cells induced strong therapeutic and protective MethA tumor immunity upon transplantation in Balb/c mice. However, in a fraction of immunized mice MethA tumor growth resumed after an extended latency period. Analysis of these tumors indicated loss of p53 expression. Mice, pre-treated with fusion hybrids generated from D2SC/1 and MethA tumor cells, suppressed MethA tumor growth and averted adaptive immune escape. Polyclonal B-cell responses directed against various MethA tumor proteins could be detected in the sera of D2SC/1-MethA inoculated mice. Athymic nude mice and Balb/c mice depleted of CD4(+) or CD8(+) T-cells were not protected against MethA tumor cell growth after immunization with D2SC/1-MethA hybrids. Our results highlight a potential drawback of cancer immunotherapy by demonstrating that the induction of a specific anti-tumor response favors the acquisition of tumor phenotypes promoting immune evasion. In contrast, the application of DC/tumor cell fusion hybrids prevents adaptive immune escape by a T-cell dependent mechanism and provides a simple strategy for personalized anti-cancer treatment without the need of selectively priming the host immune system.

First identification of Ewing's sarcoma-derived extracellular vesicles and exploration of their biological and potential diagnostic implications.

BACKGROUND INFORMATION: Exosomes are small RNA- and protein-containing extracellular vesicles (EVs) that are thought to mediate hetero- and homotypic intercellular communication between normal and malignant cells.Tumour-derived exosomes are believed to promote re-programming of the tumour-associated stroma to favour tumour growth and metastasis. Currently, exosomes have been intensively studied in carcinomas. However, little is known about their existence and possible role in sarcomas. RESULTS: Here, we report on the identification of vesicles with exosomal features derived from Ewing's sarcoma(ES), the second most common soft-tissue or bone cancer in children and adolescents. ES cell line-derived EV shave been isolated by ultracentrifugation and analysed by flow-cytometric assessment of the exosome-associated proteins CD63 and CD81 as well as by electron microscopy. They proved to contain ES-specific transcripts including EWS-FLI1, which were suitable for the sensitive detection of ES cell line-derived exosomes by qRT-PCRin a pre-clinical model for patient plasma. Microarray analysis of ES cell line-derived exosomes revealed that they share a common transcriptional signature potentially involved in G-protein-coupled signalling, neurotransmitter signalling and stemness. CONCLUSIONS: In summary, our results imply that ES-derived exosomes could eventually serve as biomarkers for minimal residual disease diagnostics in peripheral blood and prompt further investigation of their potential biological role in modification of the ES-associated microenvironment

Important role of caspase-8 for chemosensitivity of ALL cells.

PURPOSE: Sensitivity of tumor cells toward chemotherapy mainly determines the prognosis of patients suffering from acute lymphoblastic leukemia (ALL); nevertheless, underlying mechanisms regulating chemosensitivity remain poorly understood. Here, we aimed at characterizing the role of caspase-8 for chemosensitivity of B- and T-ALL cells. EXPERIMENTAL DESIGN: Primary tumor cells from children with ALL were evaluated for expression levels of the caspase-8 protein, were amplified in nonobese diabetic/severe combined immunodeficient mice, transfected with siRNA, and evaluated for their chemosensitivity in vitro. RESULTS: Effective cell death in B- and T-ALL cells depended on the presence of caspase-8 for the majority of cytotoxic drugs routinely used in antileukemia treatment. Caspase-8 was activated independently from extrinsic apoptosis signaling. Accordingly in primary ALL cells, the expression level of caspase-8 protein correlated with cell death sensitivity toward defined cytotoxic drugs in vitro. In the subgroup of primary ALL cells, with low expression of caspase-8, methotrexate (MTX) upregulated the expression of caspase-8 mediated by the transcription factor p53, suggesting epigenetic silencing of caspase-8. RNA interference in patient-derived B- and T-ALL cells revealed that effective cell death induction by most routine drug combinations involving MTX depended on the presence of caspase-8. CONCLUSION: Our results indicate that caspase-8 is crucial for the high antileukemic efficiency of numerous routine cytotoxic drugs. Reexpression of epigenetically downregulated caspase-8 represents a promising approach to increase efficiency of antileukemic therapy.

DNA vaccination with a mutated p53 allele induces specific cytolytic T cells and protects against tumor cell growth and the formation of metastasis.

PURPOSE: Genetic vaccination by expression plasmids that encode mutant p53 was conducted to characterize exogenously induced anti-tumoral immunity in mice. METHODS: Gene transfer was evaluated by reporter gene expression assays. The efficacy of genetic immunization was addressed by analysis of solid tumor outgrowth and the formation of metastases. Cell mediated immunity was determined by (51)Cr-release cytotoxicity assays and adoptive lymphocyte transfer experiments. RESULTS: Genetic vaccination resulted in a persistent protection against the growth and metastasis of transplanted tumor cells. Immunoprotection was based on the induction of cytolytic T cells (CTLs) able to recognize mutant but not wild type p53. Mice were not protected from tumor cell growth when the tumor cells expressed alternate p53 mutations or overexpressed wild type p53. No p53 specific humoral immune response was detected. T-lymphocyte transfer experiments demonstrated that resistance to tumor growth depended both on tumor size and a time-dependent induction of protective immunity. Small tumors (Ø < 0.4 cm(3)) went into remission or remained stable upon adoptive transfer of T-lymphocytes from mice immunized with mutant p53 DNA; larger tumors progressed. A time course of immunization was evaluated and showed that DNA vaccination must precede tumor cell inoculation in order to induce an efficient anti-tumoral response. CONCLUSION: DNA vaccination against the mutated form of p53 can elicit a specific adaptive immune response and has anti-tumoral activity. Tumor burden and the time necessary to acquire tumor immunity were recognized as critical parameters for immunization; however, tumors may evade specific immunotherapy.

Gene signature-based prediction of tumor response to cyclophosphamide.

Cyclophosphamide (CY) is a clinically used cytotoxic agent that is effective in a wide range of tumor types including breast and small cell lung cancers. However, by far not all patients benefit from CY therapy. We used patient tumor explants grown in nude mice as an experimental model system to identify a gene signature that, based on a tumor's gene expression profile, predicts its CY response. Forty-nine human tumor xenografts of different histologies were defined as the training set. Correlation of the gene expression profiles of untreated tumors to the sensitivity of the same tumors to CY led to the identification of 129 transcripts as predictive biomarkers for CY response. Interestingly, the products of 12 of these genes were known to interact at least indirectly with CY. A leave-one-out cross-validation approach led to a correct prediction of the CY response of the training set tumors in 15 out of 18 cases (83%) as compared to a response rate of 18 out of 49 (32%), following random testing. For an independent set of 25 previously untested tumors with known gene expression profiles (validation set) CY sensitivity was predicted correctly for 6 out of 8 tumors (75%), and CY resistance for 15 out of 17 tumors (88%). In comparison, random testing of the same tumors resulted in a response rate of 8 out of 25 (32%). For the same 25 tumors, the median minimum T/C value for predicted responders was 1% as compared to 49% for predicted non-responders. Finally, for tumor types considered as CY sensitive such as small cell lung and breast cancers as well as melanoma, the combined real and predicted response rates for 37 tested and 26 untested tumors was 49%. In contrast, for tumor types considered as CY resistant, including colon and renal cancer, the combined real and predicted response rate for 37 tested and 75 untested tumors was only 13%. Taken together, we identified a gene signature that can predict tumor response to CY and warrants clinical validation.

Arugosins G and H: prenylated polyketides from the marine-derived fungus Emericellanidulans var. acristata.

The fungus Emericella nidulans var. acristata was isolated as an endophyte from a Mediterranean green alga. Cultivation of this fungus yielded two new compounds, arugosins G (1) and H (2), together with the known metabolites 3-9. Arugosins (1-4) are benzophenone derivatives, biosynthetically related to the xanthones 5, 6, and 9. The indole alkaloid 7 displayed antitumor activity in a panel of 36 human tumor cell lines, exhibiting a mean IC(50) value of 5.5 microg/mL in an in vitro survival and proliferation assay. Furthermore, compounds 3 and 4 showed moderate antitumor activity toward individual tumor cell lines. None of compounds 1-8 exhibited any immunostimulatory activity assessed as the capacity to induce cytokines in PBMCs from healthy donors.

Correlation of ErbB2 gene status, mRNA and protein expression in a panel of >100 human tumor xenografts of different origin.

INTRODUCTION: The receptor tyrosine kinase ErbB2 is an important prognostic marker and therapeutic target in breast cancer. The aim of this study was to investigate the correlation between ErbB2 gene amplification, mRNA and protein expression in a panel of >100 patient-derived nude mouse tumor xenografts of different histological origin. MATERIALS AND METHODS: Data were obtained using fluorescence in situ hybridization, GenChip expression analysis, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Tumors included the following types: urinary bladder, breast, colon, stomach, kidney, liver, lung, melanoma, ovary, pancreas, prostate, uterus/cervix uteri and others. RESULTS: All tumors with high-level ErbB2 gene amplification expressed ErbB2 mRNA at a level >10-fold above average and protein at a level >20-fold above average (ELISA). Correlation was found between ErbB2 mRNA and protein expression. CONCLUSION: Based on expression data, cervical, gastric and adenocarcinomas of the lung emerged as new potential indications for ErbB2-directed cancer therapies.