Feasibility and tolerability of bevacizumab in children with primary CNS tumors.

BACKGROUND: Bevacizumab, an antibody to the vascular endothelial growth factor, has demonstrated anti-cancer activity in a number of solid tumors. Fear of intratumoral hemorrhage, however, has slowed its introduction into the treatment of central nervous system (CNS) tumors. Currently, only a small number of children with gliomas received bevacizumab. METHODS: We retrospectively analyzed 30 patients who received bevacizumab between January 2007 and August 2009. The median age at start of bevacizumab treatment was 9.9 years (range: 1.5-18). Most patients had recurrent/progressive disease, 25 high-grade and 5 low-grade tumors. The median dose of bevacizumab was 9.5 mg/kg (range 5-15 mg/kg) every 2-3 weeks. In total, 478 courses were administered (median/patient 15.9, range: 2-52). The median duration of bevacizumab treatment was 10.0 months (range: 1.6-30.4). Twenty-nine of 30 patients received additional therapy concomitant to bevacizumab. RESULTS: No bevacizumab related intratumoral hemorrhage occurred in any of our 30 patients. Grade III hypertension was seen in two patients. One patient developed nephrotic syndrome requiring cessation of treatment. Grade III and I proteinuria were observed in one and five patients, respectively. New onset lymphopenia occurred in 12/30 and new onset hypothyroidism in 7/30 patients. Impaired wound healing was manageable. No immediate bevacizumab-related cardiotoxicity was observed as evidenced by echocardiography. CONCLUSIONS: Bevacizumab appears to be safe for children with primary CNS tumors. Adverse effects did occur but were manageable. No treatment-related death occurred. Long-term monitoring is advisable to detect lymphopenia and hypothyroidism. Hypertension occurred less frequently than in adult patients. Further prospective studies including more patients are warranted.

Pharmacokinetics and safety of intrathecal liposomal cytarabine in children aged <3 years.

BACKGROUND AND OBJECTIVE: Liposomal cytarabine (DepoCyte) is a slow-release formulation for intrathecal application, ensuring prolonged drug exposure. Although there is an urgent need for new treatment options for infants with leptomeningeal dissemination of a malignant brain tumour, there are no clinical and pharmacokinetic data available on this drug for children aged <3 years. The objective of this pilot study was to determine the feasibility, safety and pharmacokinetics of cytarabine after intrathecal administration of liposomal cytarabine 25 mg in patients aged <3 years. PATIENTS AND METHODS: Six male patients with a mean age of 21 months and CNS primitive neuroectodermal tumours (n = 3) or atypical teratoid/rhabdoid tumours (n = 3) were included. Liposomal cytarabine (25 mg) was administered intraventricularly. One patient also received the drug by lumbar puncture. Dexamethasone was used concomitantly for 3-5 days to prevent arachnoiditis. Cerebrospinal fluid (CSF) and plasma samples were collected before administration of liposomal cytarabine and 1 hour, 12 hours, 24 hours, 1 week and 2 weeks post-dosing. Noncompartmental pharmacokinetic analysis of CSF and plasma was performed. RESULTS: Liposomal cytarabine was generally well tolerated; only grade 2 headache occurred in one patient. After intraventricular administration of cytarabine 25 mg, free and encapsulated drug concentrations above the cytotoxic drug level of 0.1 microg/mL were detectable in the CSF for at least 7 days and up to 14 days post-dosing. The average elimination half-lives were 56.7 hours for encapsulated cytarabine and 59.3 hours for free cytarabine. After intralumbar administration, the elimination half-life of free cytarabine, measured in the ventricular CSF during two courses in one patient, was significantly shorter (32.7 hours). CONCLUSION: Application of liposomal cytarabine with concomitant dexamethasone appears to be safe and well tolerated in children aged <3 years. Drug exposure in infants aged <3 years after an intraventricular dose of 25 mg is comparable to that after administration of 50 mg in adult patients and 35 mg in older children.

Neurotrophin 3/TrkC-regulated proteins in the human medulloblastoma cell line DAOY.

Medulloblastoma (MB) is the most common malignant childhood brain tumor and high neurotrophin (NP) receptor TrkC mRNA expression was identified as a powerful independent predictor of favorable survival outcome. In order to determine downstream effector proteins of TrkC signaling, the MB cell line DAOY was stably transfected with a vector containing the full-length TrkC cDNA sequence or an empty vector control. A proteomic approach was used to search for expressional changes by two mass spectrometric methods and immunoblotting for validation of significant results. Multiple time points for up to 48 h following NP-3-induced TrkC receptor activation were chosen. Thirteen proteins from several pathways (nucleoside diphosphate kinase A, stathmin, valosin-containing protein, annexin A1, dihydropyrimidinase-related protein-3, DJ-1 protein, glutathione S-transferase P, lamin A/C, fascin, cofilin, vimentin, vinculin, and moesin) were differentially expressed and most have been shown to play a role in differentiation, migration, invasion, proliferation, apoptosis, drug resistance, or oncogenesis. Knowledge on effectors of TrkC signaling may represent a first useful step for the identification of marker candidates or reflecting probable pharmacological targets for specific treatment of MB.

Tumor stabilization under treatment with imatinib in progressive hypothalamic-chiasmatic glioma.

BACKGROUND: Hypothalamic-chiasmatic gliomas (HCG) account for up to 20% of tumors in patients under the age of 3 years. While most children respond to chemotherapy, alternative treatment approaches are needed for those with progressive disease refractory to chemotherapy. PROCEDURE: Six patients (median age: 5.5 years) with progressive HCG were treated with imatinib for 3-29 months at a median daily oral dose of 270 mg/m(2). All patients initially presented with extensive tumors during infancy and had undergone two to three surgical resections and two to three prior chemotherapies with multiple agents. RESULTS: The best response achieved was stable disease in all six patients. Disease control lasted from 5 to 46 months and was sustained longer in comparison to their last prior chemotherapy. Toxicities possibly related to imatinib included edema, elevated liver enzymes and bowel problems. Immunohistochemistry in our patients' tumor cells revealed focal expression of arg and PDGFR-alpha in one patient, in the remaining five patients no expression of any of the five known targets of imatinib could be detected. Expression of PDGFR-alpha and PDGFR-beta was detected in endothelial cells of tumor capillaries of all six patients. CONCLUSIONS: We conclude that imatinib has possible activity in progressive HCG and may present an additional therapeutic option for patients who are too young or whose tumor is too extensive to receive radiotherapy. However, the optimal use of imatinib in this disease, its mechanism of action, and possible long-term effects remain unclear and will require additional study.

The medulloblastoma cell line DAOY but not eleven other tumor cell lines expresses minichromosome maintenance protein 4.

Minichromosome maintenance proteins (MCM) are required for initiation and elongation of chromosomal DNA, ensuring that DNA replication takes place only once. Although MCMs are considered of utmost importance in tumor biology and as potential marker proteins, they were not unambiguously identified at the protein level and we therefore aimed to characterize MCM 4 in a medulloblastoma cell line and provide a protein chemical analytical tool. In addition, we searched for this protein in twelve tumor cell lines and a series of non-tumor cells. The DAOY medulloblastoma cell line was cultivated, lysed, proteins extracted and run on two-dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical (MS/MS) analysis of protein spots. One spot at pI 6.2 with an observed molecular weight of 98 kDa was identified as minichromosome maintenance protein 4 by peptide fingerprinting. Sequence coverage of 35% along with 25 matched peptides and MS/MS analyses of three matching peptides warranted unambiguous identification. The use of mass spectrometrical identification unequivocally allowed determination of MCM 4 expression in a medulloblastoma cell line exclusively. Given the biological and probable clinical importance of this molecule as a tentative marker protein, a fair analytical tool, independent of antibody availability and specificity is mandatory and determinations at the transcriptional level cannot be extrapolated to protein levels per se, as there is a long and unpredictable way from mRNA to protein.

Live or let die: CCM2 provides the link.

TrkA receptors are well known for promoting neuronal cell survival. However, in some neuroblastic tumors, TrkA activation can instead induce apoptosis. In this issue of Neuron, Harel et al. identify CCM2 as a mediator of TrkA-dependent cell death, suggesting that CCM2 is a distinctive type of tumor suppressor that modulates tyrosine kinase signaling.

Synthesis, chaperoning, and metabolism of proteins are regulated by NT-3/TrkC signaling in the medulloblastoma cell line DAOY.

The human medulloblastoma cell line DAOY was transfected with Tropomyosin receptor kinase (TrkC), a marker for good prognostic outcome. Following TrkC-activation by its ligand neurotrophin-3, protein extracts from DAOY cells were run on 2DE with subsequent MALDI-TOF-TOF analysis and quantification in order to detect downstream effectors. Protein levels of translational, splicing, processing, chaperone, protein handling, and metabolism machineries were shown to depend on neurotrophin-3-induced TrkC activation probably representing pharmacological targets.

Mitosis-dependent protein expression in neuroblastoma cell line N1E-115.

Systematic work on differential protein expression in mitosis is limited, and we therefore used neuroblastoma cells (N1E-115) incubated with either colcemid or nocodazole to arrest mitosis. Proteins were identified by MALDI-TOF/TOF and nano-LC-ESI-MS/MS with subsequent quantification of spot volumes with specific software. Immunoblotting was used for verification of selected proteins. Levels of 10 individual proteins were increased and levels of 6 proteins were decreased concordantly by both treatments. These proteins were constituents of heat shock and chaperone, cytoskeleton, proteasomal, heterochromatin, and DNA replication signaling as well as housekeeping and metabolic systems. Identification of mitosis-dependent proteins is of importance for the interpretation of previous work and for designing future experiments.

Proteomic Determination of Metabolic Protein Expression in Ten Different Tumor Cell Lines.

Alterations in different metabolic pathways are a prerequisite to facilitate malignant features such as invasiveness, metastasis, progression and resistance mechanisms to therapy in tumor cells. To generate metabolic protein expression patterns of tumor cells, and in order to provide an analytical tool for establishing metabolic maps, "metabolomes", we applied two-dimensional electrophoresis (2-DE) followed by mass spectrometry (MALDI-TOF-TOF with LIFT technology) in ten individual tumor cell lines (Saos-2; SK-N-SH; HCT-116; Caov3; A-549; HL60; A-673; A-375; MCF-7; Hela) widely used in tumor research. A series of 124 metabolic proteins, represented by 432 spots, was unambiguously identified by this proteomic approach. The proteins detected comprise a multitude of pathways including intermediary, energy, lipid, nucleic acid, amino acid, carbohydrate, redox, phosphate, iron and folate metabolism. Fifty-six enzymes were present in a single tumor cell line exclusively, whereas only enolase-1, a key component of the glycolytic cascade, was found in all cell lines investigated, thereby underscoring the heterogeneous protein expression profile in different cancer types. Construction of individual protein maps provides an analytical tool and reference base for studying the "metabolome" of tumor cells, forming the basis for designing studies in tumor metabolism, and reveals proteins that can be used as pharmaceutical targets in experimental therapies.