Metabolic biomarker signature to differentiate pancreatic ductal adenocarcinoma from chronic pancreatitis.

OBJECTIVE: Current non-invasive diagnostic tests can distinguish between pancreatic cancer (pancreatic ductal adenocarcinoma (PDAC)) and chronic pancreatitis (CP) in only about two thirds of patients. We have searched for blood-derived metabolite biomarkers for this diagnostic purpose. DESIGN: For a case-control study in three tertiary referral centres, 914 subjects were prospectively recruited with PDAC (n=271), CP (n=282), liver cirrhosis (n=100) or healthy as well as non-pancreatic disease controls (n=261) in three consecutive studies. Metabolomic profiles of plasma and serum samples were generated from 477 metabolites identified by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. RESULTS: A biomarker signature (nine metabolites and additionally CA19-9) was identified for the differential diagnosis between PDAC and CP. The biomarker signature distinguished PDAC from CP in the training set with an area under the curve (AUC) of 0.96 (95% CI 0.93-0.98). The biomarker signature cut-off of 0.384 at 85% fixed specificity showed a sensitivity of 94.9% (95% CI 87.0%-97.0%). In the test set, an AUC of 0.94 (95% CI 0.91-0.97) and, using the same cut-off, a sensitivity of 89.9% (95% CI 81.0%-95.5%) and a specificity of 91.3% (95% CI 82.8%-96.4%) were achieved, successfully validating the biomarker signature. CONCLUSIONS: In patients with CP with an increased risk for pancreatic cancer (cumulative incidence 1.95%), the performance of this biomarker signature results in a negative predictive value of 99.9% (95% CI 99.7%-99.9%) (training set) and 99.8% (95% CI 99.6%-99.9%) (test set). In one third of our patients, the clinical use of this biomarker signature would have improved diagnosis and treatment stratification in comparison to CA19-9.

Integration of tissue metabolomics, transcriptomics and immunohistochemistry reveals ERG- and gleason score-specific metabolomic alterations in prostate cancer.

Integrated analysis of metabolomics, transcriptomics and immunohistochemistry can contribute to a deeper understanding of biological processes altered in cancer and possibly enable improved diagnostic or prognostic tests. In this study, a set of 254 metabolites was determined by gas-chromatography/liquid chromatography-mass spectrometry in matched malignant and non-malignant prostatectomy samples of 106 prostate cancer (PCa) patients. Transcription analysis of matched samples was performed on a set of 15 PCa patients using Affymetrix U133 Plus 2.0 arrays. Expression of several proteins was immunohistochemically determined in 41 matched patient samples and the association with clinico-pathological parameters was analyzed by an integrated data analysis. These results further outline the highly deregulated metabolism of fatty acids, sphingolipids and polyamines in PCa. For the first time, the impact of the ERG translocation on the metabolome was demonstrated, highlighting an altered fatty acid oxidation in TMPRSS2-ERG translocation positive PCa specimens. Furthermore, alterations in cholesterol metabolism were found preferentially in high grade tumors, enabling the cells to create energy storage. With this integrated analysis we could not only confirm several findings from previous metabolomic studies, but also contradict others and finally expand our concepts of deregulated biological pathways in PCa.

Tissue metabolite profiling identifies differentiating and prognostic biomarkers for prostate carcinoma.

Metabolomic research offers a deeper insight into biochemical changes in cancer metabolism and is a promising tool for identifying novel biomarkers. We aimed to evaluate the diagnostic and prognostic potential of metabolites in prostate cancer (PCa) tissue after radical prostatectomy. In matched malignant and nonmalignant prostatectomy samples from 95 PCa patients, aminoadipic acid, cerebronic acid, gluconic acid, glycerophosphoethanolamine, 2-hydroxybehenic acid, isopentenyl pyrophosphate, maltotriose, 7-methylguanine and tricosanoic acid were determined within a global metabolite profiling study using gas chromatography/liquid chromatography-mass spectrometry. The data were related to clinicopathological variables like prostate volume, tumor stage, Gleason score, preoperative prostate-specific antigen and disease recurrence in the follow-up. All nine metabolites showed higher concentrations in malignant than in nonmalignant samples except for gluconic acid and maltotriose, which had lower levels in tumors. Receiver -operating characteristics analysis demonstrated a significant discrimination for all metabolites between malignant and nonmalignant tissue with a maximal area under the curve of 0.86 for tricosanoic acid, whereas no correlation was observed between the metabolite levels and the Gleason score or tumor stage except for gluconic acid. Univariate Cox regression and Kaplan-Meier analyses showed that levels of aminoadipic acid, gluconic acid and maltotriose were associated with the biochemical tumor recurrence (prostate-specific antigen > 0.2 ng/mL). In multivariate Cox regression analyses, aminoadipic acid together with tumor stage and Gleason score remained in a model as independent marker for prediction of biochemical recurrence. This study proved that metabolites in PCa tissue can be used, in combination with traditional clinicopathological factors, as promising diagnostic and prognostic tools.

Clinical trials: considerations for researchers and hospital administrators.

BACKGROUND: Clinical studies play a pivotal role in the development of new pharmaceutical drugs. Before newly developed active substances can be put on the market, the law requires that they be tested in a large number of clinical trials. The initiators of these trials are usually study groups (publicly funded trials) or the pharmaceutical industry, which often supplies the drugs for clinical research in hospitals. There is reason to believe that hospital administrators could make cost savings on drugs. PURPOSE: The purpose of this article is to quantify drug cost savings in hospitals related to clinical trials and to examine the relationship between researchers and hospital administrators with respect to clinical trials. METHODOLOGY/APPROACH: We analyzed 88 clinical trials in oncology including 29 researchers in 11 hospitals in Germany from 2002 through 2005. We also interviewed researchers and hospital administrators concerning their attitude toward these clinical trials. We propose that hospital administrators tend to focus on the economics of conducting clinical trials. FINDINGS: The results showed a drug cost saving potential of US $6.7 million (euro5.1 million) in 11 hospitals from 2002 through 2005 and an actual cost saving of US $2.0 million (euro1.5 million). The hospital administrators underestimated the difficulties that researchers experienced because of lack of personnel resources when conducting clinical trials. PRACTICE IMPLICATIONS: The hospital administrator has a financial incentive to provide internal conditions in the hospital that facilitate incentives for researchers to become involved in clinical trials. We suggest supporting researchers actively in research with additional human resources (study nurses, etc.). We propose that this would result in a higher number of patients taking part and in more clinical trials. Consequently, higher drug cost savings could also be realized.

Intracavitary chemotherapy (paclitaxel/carboplatin liquid crystalline cubic phases) for recurrent glioblastoma -- clinical observations.

Human malignant brain tumors have a poor prognosis in spite of surgery and radiation therapy. Cubic phases consist of curved biocontinuous lipid bilayers, separating two congruent networks of water channels. Used as a host for cytotoxic drugs, the gel-like matrix can easily be applied to the walls of a surgical resection cavity. For human glioblastoma recurrences, the feasibility, safety, and short-term effects of a surgical intracavitary application of paclitaxel and carboplatin encapsulated by liquid crystalline cubic phases are examined in a pilot study. A total of 12 patients with a recurrence of a glioblastoma multiforme underwent re-resection and received an intracavitary application of paclitaxel and carboplatin cubic phases in different dosages. Six of the patients received more than 15 mg paclitaxel and suffered from moderate to severe brain edema, while the remaining patients received only a total of 15 mg paclitaxel. In the latter group, brain edema was markedly reduced and dealt medically. Intracavitary chemotherapy in recurrent glioblastoma using cubic phases is feasible and safe, yet the clinical benefit remains to be examined in a clinical phase II study.

Evaluation of gas-filled microparticles and sonoporation as gene delivery system: feasibility study in rodent tumor models.

PURPOSE: To evaluate the feasibility of gene delivery mediated with diagnostic ultrasound and plasmid DNA (pDNA) encapsulated in gas-filled microparticles (GFMP) in rodent tumor models. MATERIALS AND METHODS: This study was performed according to a protocol approved by the regional animal research committee. The model plasmid UT651 (pUT651) that contained the Escherichia coli LacZ gene for beta-galactosidase was used to demonstrate the feasibility of ultrasound-mediated gene delivery in CC531 liver tumors in rats. In preliminary experiments, a single injection of pUT651-containing GFMP was administered intraarterially (n=4) or intravenously (n=6) with simultaneous sonication (color Doppler mode, maximum mechanical index) of the GFMP passing through the capillaries of the tumors. All animals were sacrificed 2-5 days later, and liver tumors were examined for beta-galactosidase expression histochemically. Subsequently, potential medical usefulness of this delivery system was tested in nude mice bearing Capan-1 tumors (adenocarcinoma of the human pancreas) by using the plasmid RC/CMV-p16 (pRC/CMV-p16), which contains tumor suppressor gene p16. The tumor suppressor gene p16 is deleted in Capan-1 cells. Twenty-five tumor-bearing mice were classified into five groups (four to six mice per group, one treatment group, four control groups) at random. All mice were treated once weekly for 5 weeks with intravenous infusion of p16-containing GFMP or control substances with simultaneous tumor sonication with color Doppler mode ultrasound and maximum mechanical index or without ultrasound treatment. The therapeutic effect of p16 was measured as an increase in tumor volume doubling time. Data were analyzed with analysis of variance. Results were considered significant at the 5% critical level (P < .05). RESULTS: A clear expression of pDNA was found in tumors in rats treated with a combination of pUT651-containing GFMP and ultrasound; relevant controls showed a significantly lower expression of marker gene. The controlled ultrasound-triggered release of pRC/CMV-p16 from GFMP leads to a strong tumor growth inhibition, which is significant (P < .002), compared with that in controls. CONCLUSION: A combination of GFMP and ultrasound provides an effective approach for nonviral gene therapy-based cancer treatment.

Local chemotherapy of F98 rat glioblastoma with paclitaxel and carboplatin embedded in liquid crystalline cubic phases.

Implanted drug carrier systems for retarded chemotherapy against gliomas are mainly based upon polymers containing nitrosoureas. The authors have developed an intracavitary carrier system of biodegradable liquid crystalline cubic phases encapsulating carboplatin and paclitaxel and studied it for release kinetics, antitumor activity, and survival prolongation. A total of 61 Fisher rats with F98 tumors were divided into six treatment groups at day 12 post-inoculation, receiving either no treatment, surgery with partial tumor resection, or partial resection with implantation of cubic phases containing either paclitaxel and carboplatin, paclitaxel alone, carboplatin alone, or no drug. Animals were killed for tumor size analysis at day 21 post-inoculation (n=28) or were included in survival studies (n=33). Additional 12 animals received a paclitaxel/carboplatin application and were killed at different time intervals (6 h, 24 h, 48 h, 5 d, 7 d, 10 d post-agent application) for in vivo diffusion studies. Animals from the paclitaxel/carboplatin group showed a significantly smaller tumor (mean 3.25 mm2+/-SD 1.79 mm2) than animals from the control group (15.30+/-5.86 mm2; P=0.0031), animals having received the empty matrix (11.62+/-6.66 mm2; P=0.0241), and animals after tumor resection without implantation (20.87+/-3.56 mm2; P

Liposome-mediated suicide gene therapy in humans.

The LIPO-HSV-1-tk gene transfer system was developed for a 3-day pump application in a first prospective Phase I?II clinical study. Eight patients suffering from recurrent glioblastoma multiforme were treated intratumorally on the basis of convection-enhanced delivery using the nonviral vector system. It was possible to identify the target tissue together with assessment of vector distribution and gene product expression, as well as the metabolic effect of ganciclovir treatment, noninvasively, by the combination of magnetic resonance imaging and positron emission tomography as a multimodal molecular imaging system. The therapy was well tolerated without major side effects. In two of eight patients, we observed a greater than 50% reduction of tumor volume and in six of eight patients focal treatment effects. The noninvasive visualization of therapeutic effects on tumor metabolism and documentation of gene expression will be important for the further successful development and implementation of patient individual gene therapy.

Tissue monocytes/macrophages in inflammation: hyperalgesia versus opioid-mediated peripheral antinociception.

BACKGROUND: Opioid-containing leukocytes migrate to peripheral sites of inflammation. On exposure to stress, opioid peptides are released, bind to opioid receptors on peripheral sensory neurons, and induce endogenous antinociception. In later stages of Freund's complete adjuvant-induced local inflammation, monocytes/macrophages are a major opioid-containing leukocyte subpopulation, but these cells also produce proalgesic cytokines. In this study, the role of tissue monocytes/macrophages in hyperalgesia and in peripheral opioid-mediated antinociception was investigated. METHODS: After intraplantar injection of Freund's adjuvant, leukocyte subpopulations and opioid-containing leukocytes were analyzed by flow cytometry in the inflamed paw in the presence or absence of monocyte/macrophage depletion by intraplantar injection of clodronate-containing liposomes (phosphate-buffered saline and empty liposomes served as controls). Paw volume was measured with a plethysmometer. Hyperalgesia was determined by measuring heat-induced paw withdrawal latency and paw pressure threshold. Paw pressure threshold was also measured after swim stress and injection of fentanyl. RESULTS: At 48 and 96 h of inflammation, it was found that (1). monocytes/macrophages were the largest leukocyte subpopulation (> 55% of all leukocytes) and the predominant producers of opioid peptides (71-77% of all opioid-containing leukocytes in the paw), (2). clodronate-containing liposomes depleted monocytes/macrophages by 30-35% (P < 0.05), (3). hyperalgesia was unaltered by liposome injection (P > 0.05), and (4) opioid-containing leukocytes and swim stress but not fentanyl-induced antinociception were significantly decreased by clodronate-containing liposomes (P < 0.05, P > 0.05, all by t test; opioid-containing cells and swim stress-induced increase of paw pressure threshold were reduced by 35-42% and 20%, respectively). CONCLUSION: Partial depletion of tissue monocytes/macrophages impairs peripheral endogenous opioid-mediated antinociception without affecting hyperalgesia.

Imaging-guided convection-enhanced delivery and gene therapy of glioblastoma.

In a prospective phase I/II clinical study, we treated eight patients suffering from recurrent glioblastoma multiform with stereotactically guided intratumoral convection-enhanced delivery of an HSV-1-tk gene-bearing liposomal vector and systemic ganciclovir. Noninvasive identification of target tissue together with assessment of vector-distribution volume and the effects of gene therapy were achieved using magnetic resonance imaging and positron emission tomography. The treatment was tolerated well without major side effects. In two of eight patients, we observed a greater than 50% reduction of tumor volume and in six of eight patients focal treatment effects. Intracerebral infusion of contrast medium before vector application displayed substantial inhomogeneity of tissue staining indicating the need of test infusions to monitor the mechanical distribution of vectors. Visualization of therapeutic effects on tumor metabolism and documentation of gene expression using positron emission tomography indicated that molecular imaging technology appears to be essential for the further development of biological treatment strategies.

Ultrastructural analysis of DNA complexes during transfection and intracellular transport.

We present a simple method based on transmission electron microscopy that allows investigation of the early steps of polyplex-mediated transfection without the use of labeled DNA. The ultrastructural analysis showed internalization of 0.2-1-micro m aggregates composed of 30-50-nm subunits. In addition, new details of the internalization process were revealed, suggesting an unspecific cell entry mechanism of large DNA aggregates.

Efficiency of cytokine gene transfer in corneal endothelial cells and organ-cultured corneas mediated by liposomal vehicles and recombinant adenovirus.

PURPOSE: Gene transfer of immunoregulatory cytokines could contribute to reduce rejection of corneal grafts. The aim of our study was to examine the gene transfer efficiency of liposomal vehicles compared to adenoviral vectors for transferring the Epstein-Barr-virus-derived interleukin 10 homologue (viral IL-10, vIL-10) into corneal endothelial cells and organ-cultured human corneas (HC) in vitro. METHOD: To test liposomal efficiency, 2 lipid formulations (SP-Chol/DOPE 20/80 and DDAB/DOPE 30/70 in various concentrations) were complexed with a plasmid containing the vIL-10 cDNA in an eukaryotic expression vector (pcDSRalpha-BCRF-I). The complexes were transferred to (1) subconfluent bovine corneal endothelial cells (BCEC) after 1 passage and to (2) HC stored in organ culture. In addition, BCEC and HC were transduced with the recombinant adenoviral vector encoding for vIL-10 (AdvIL-10). Secretion of vIL-10 in the supernatants from both transfected BCEC and HC was measured by specific ELISA. RESULTS: For gene transfer in BCEC, both transfection methods (liposomes and adenovirus) led to high secretion of vIL-10 [>2 ng/ml (liposomes) and <150 ng/ml (adenovirus) per 5,000 initially planted BCEC]. Expression levels in BCEC were dependent on the concentration of applied liposomes. For gene transfer in HC, only the adenoviral transduction technique achieved a high production of vIL-10, whereas liposomal transfection led only to low vIL-10 secretion (4.8 microg/ml vs. 95 pg/ml per quarter of cornea). CONCLUSION: For transfection of corneal endothelial cells in culture, liposomes can be considered as a safe and useful alternative method of gene transfer avoiding side-effects of viral vectors. However, for transfection of organ-cultured HC, adenoviral vectors are superior to liposomal vehicles.

Pharmaceutical evaluation of gas-filled microparticles as gene delivery system.

PURPOSE: To produce and characterize a nonviral ultrasound-controlled release system of plasmid DNA (pDNA) encapsulated in gas-filled poly(D,L-lactide-co-glycolide) microparticles (PLGA-MPs). METHODS: Different cationic polymers were used to form pDNA/polymer complexes to enhance the stability of pDNA during microparticle preparation. The physico-acoustical properties of the microparticles, particle size, pDNA integrity, encapsulation efficiency and pDNA release behavior were studied in vitro. RESULTS: The microparticles had an average particle size of around 5 microm. More than 50% of all microparticles contained a gas core, and when exposed to pulsed ultrasound as used for color Doppler imaging create a signal that yields typical color patterns (stimulated acoustic emission) as a result of the ultrasound-induced destruction of the microparticles. Thirty percent of the pDNA used was successfully encapsulated and approximately 10% of the encapsulated pDNA was released by ultrasound within 10 min. CONCLUSIONS: Plasmid DNA can be encapsulated in biodegradable gas-filled PLGA-MPs without hints for a structural disintegration. A pDNA release by ultrasound-induced microparticle-destruction could be shown in vitro.