Darrell J. Yamashiro

Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma

Vascular endothelial growth factor (VEGF) plays a key role in human tumor angiogenesis. We compared the effects of inhibitors of VEGF with different specificities in a xenograft model of neuroblastoma. Cultured human neuroblastoma NGP-GFP cells were implanted intrarenally in nude mice. Three anti-VEGF agents were tested: an anti-human VEGF165 RNA-based fluoropyrimidine aptamer; a monoclonal anti-human VEGF antibody; and VEGF-Trap, a composite decoy receptor based on VEGFR-1 and VEGFR-2 fused to an Fc segment of IgG1. A wide range of efficacy was observed, with high-dose VEGF-Trap causing the greatest inhibition of tumor growth (81% compared with controls). We examined tumor angiogenesis and found that early in tumor formation, cooption of host vasculature occurs. We postulate that this coopted vasculature serves as a source of blood supply during the initial phase of tumor growth. Subsequently, control tumors undergo vigorous growth and remodeling of vascular networks, which results in disappearance of the coopted vessels. However, if VEGF function is blocked, cooption of host vessels may persist. Persistent cooption, therefore, may represent a novel mechanism by which neuroblastoma can partly evade antiangiogenic therapy and may explain why experimental neuroblastoma is less susceptible to VEGF blockade than a parallel model of Wilms tumor. However, more effective VEGF blockade, as achieved by high doses of VEGF-Trap, can lead to regression of coopted vascular structures. These results demonstrate that cooption of host vasculature is an early event in tumor formation, and that persistence of this effect is related to the degree of blockade of VEGF activity.

Regression of established tumors and metastases by potent vascular endothelial growth factor blockade

Vascular endothelial growth factor (VEGF) is a critical promoter of blood vessel growth during embryonic development and tumorigenesis. To date, studies of VEGF antagonists have primarily focused on halting progression in models of minimal residual cancer. Consistent with this focus, recent clinical trials suggest that blockade of VEGF may impede cancer progression, presumably by preventing neoangiogenesis. However, VEGF is also a key mediator of endothelial–vascular mural cell interactions, a role that may contribute to the integrity of mature vessels in advanced tumors. Here, we report that high-affinity blockade of VEGF, using the recently described VEGF-Trap, abolishes mature, preexisting vasculature in established xenografts. Eradication of vasculature is followed by marked tumor regression, including regression of lung micrometastases. Thus, the contribution of relatively low levels of VEGF to vessel integrity may be critical to maintenance of even very small tumor masses. Potent blockade of VEGF may provide a new therapeutic option for patients with bulky, metastatic cancers.

Biology and genetics of human neuroblastomas.

PURPOSE Neuroblastomas have a variety of clinical behaviors, from spontaneous regression or differentiation to early metastasis and death. We have examined a variety of genetic variables that might explain or predict the clinical behavior. PATIENTS AND METHODS We have studied DNA or RNA from a number of children enrolled in clinical trials with the major pediatric oncology cooperative groups. RESULTS We propose that neuroblastomas may be classified into three subsets with distinct biological features and clinical behavior. The first subset consists of those tumors with hyperdiploid modal karyotypes and high TRK-A expression. Patients with these tumors are usually infants with low stages of disease and a very favorable outcome. The second group consists of tumors that have a near-diploid DNA content, usually with 1p allelic loss or other structural changes, but they lack MYCN amplification, and TRK-A expression is low. The patients are generally older, with advanced stages of disease and an intermediate outcome. The third group is characterized by tumors with MYCN amplification, 1p allelic loss, and low or absent TRK-A expression. The patients are 1-5 years of age and have advanced stages of disease, rapid tumor progression, and a very poor prognosis. Current evidence suggests the tumor types are genetically distinct, and one type seldom if ever evolves into another. CONCLUSIONS Identification of these genetic and clinical subsets permits a more accurate prediction of outcome. This, in turn, allows more appropriate selection of therapeutic intensity to minimize side effects in those with a favorable outcome but optimize the chance of cure in those requiring aggressive treatment.

VEGF blocking therapy in the treatment of cancer

It is widely accepted that tumour growth beyond a few cubic millimetres cannot occur without the induction of a new vascular supply. Inhibiting the development of new blood vessels (antiangiogenesis) is a potential approach to cancer therapy that has attracted interest in recent years. In theory, this approach should be relatively selective for tumour cells. The endothelial cells which form new vascular networks in tumours are responding to angiogenic stimuli produced by the tumour, but are themselves genetically normal. Endothelium in normal tissue, by contrast, is usually quiescent. Vascular endothelial growth factor (VEGF) is the best-characterised pro-angiogenic factor. It is virtually ubiquitous in human tumours, and higher levels have been correlated with more aggressive disease. Effective blockade of the VEGF pathway has been demonstrated with multiple agents: neutralising antibody, receptor tyrosine kinase inhibitors, and ribozyme or antisense molecules targeting expression. Promising preclinical data document the potential of these agents for tumour growth inhibition and even tumour regression, yet translation of novel therapeutics targeting the VEGF pathway to the clinic has proved a substantial challenge in itself. While showing clear evidence of antitumour activity over a broad spectrum of experimental tumours, the proper selection, dose, timing and sequence of anti-VEGF treatment in human cancer is not at all obvious. Classic Phase I dose escalation trial design may need to be modified, as higher doses may not be optimal in all patients or for all tumours. In addition, alternate or secondary biological end points (e.g., non-progression) may be needed for early phase studies to document true activity, so as not to abandon effective agents. Recent studies of the neutralising antibody bevacizumab, and small molecule tyrosine kinase inhibitor SU5416, demonstrate that, while unlikely to be effective as monotherapy, incorporation of VEGF blockade into cytotoxic regimens may increase overall response rates. However, incorporation may also produce new toxicities, including thromboembolic complications and bleeding. Newer oral agents, such as SU6668, SU11248, PTK787/ZK222584 and ZD6474, are particularly interesting for their potential for chronic therapy. Future clinical trials are likely to build on past experience with stricter entry criteria, supportive care guidelines and the use of surrogate markers.

Expression of TrkA, TrkB and TrkC in human neuroblastomas.

There is considerable interest in the role of the TRK family of neurotrophin receptors in regulating the survival, growth and differentiation of normal and neoplastic nerve cells. Indeed, there is increasing evidence that TRK genes play an important role in the biology and clinical behavior of neuroblastomas, tumors of the peripheral nervous system. Evidence from several independent studies suggests that high expression of TrkA is an indicator of favorable outcome, and there is an inverse correlation between TrkA expression and N-myc amplification. In addition, some primary neuroblastomas differentiate in vitro in the presence of NGF but die in its absence. We have evidence that coexpression of full-length TrkB and BDNF is associated with N-myc amplification and may represent an autocrine survival pathway. Conversely, truncated TrkB is expressed predominantly in differentiated tumors. Finally, TrkC is expressed in favorable neuroblastomas, essentially all of which also express TrkA. In summary, the study of neurotrophin receptor expression and function in neuroblastomas may provide important insights into the role that these pathways play in the pathogenesis and clinical behavior of this tumor. Ultimately, these pathways may provide attractive targets for the development of therapy aimed at inducing differentiation or programmed cell death in these tumors.

A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis

Notch signaling is required for vascular development and tumor angiogenesis. Although inhibition of the Notch ligand Delta-like 4 can restrict tumor growth and disrupt neovasculature, the effect of inhibiting Notch receptor function on angiogenesis has yet to be defined. In this study, we generated a soluble form of the Notch1 receptor (Notch1 decoy) and assessed its effect on angiogenesis in vitro and in vivo. Notch1 decoy expression reduced signaling stimulated by the binding of three distinct Notch ligands to Notch1 and inhibited morphogenesis of endothelial cells overexpressing Notch4. Thus, Notch1 decoy functioned as an antagonist of ligand-dependent Notch signaling. In mice, Notch1 decoy also inhibited vascular endothelial growth factor-induced angiogenesis in skin, establishing a role for Notch receptor function in this process. We tested the effects of Notch1 decoy on tumor angiogenesis using two models: mouse mammary Mm5MT cells overexpressing fibroblast growth factor 4 (Mm5MT-FGF4) and NGP human neuroblastoma cells. Exogenously expressed FGF4 induced Notch ligand expression in Mm5MT cells and xenografts. Notch1 decoy expression did not affect tumorigenicity of Mm5MT-FGF4 cells in vitro but restricted Mm5MT-FGF4 xenograft growth in mice while markedly impairing neoangiogenesis. Similarly, Notch1 decoy expression did not affect NGP cells in vitro but disrupted vessels and decreased tumor viability in vivo. These results strongly suggest that Notch receptor signaling is required for tumor neoangiogenesis and provides a new target for tumor therapy.

A pilot study of reduced intensity conditioning and allogeneic stem cell transplantation from unrelated cord blood and matched family donors in children and adolescent recipients

Summary:Reduced intensity (RI) allogeneic stem cell transplantation (AlloSCT) was initially demonstrated in adults following HLA-matched family and unrelated adult donor AlloSCT. There is little information about RI AlloSCT in children. We report results of a pilot study of RI AlloSCT in 21 recipients (⩽21 years). Age: median 13 (0.5–21) years, 8F:13M, 14 unrelated cord blood units (UCB) (10 4/6, 4 5/6), two related BM (6/6, 5/6), four related PBSC (2 6/6, 2 5/6), and one related BM+PBSC (6/6). RI: fludarabine, busulfan (n=14); fludarabine, cyclophosphamide (n=4); fludarabine, melphalan (n=1); total body irradiation, fludarabine, cyclophosphamide (n=1); or fludarabine, cyclophosphamide, and etoposide (n=1). Graft-versus-host disease prophylaxis: FK506 0.03 mg/kg/day and mycophenolate mofetil 15 mg/kg/q 12 h. UCB median nuc/kg and CD34/kg was 4.3 × 107/kg (0.9–10.8) and 1.9 × 105/kg (0.3–6.9), and related BM/PBSC median nuc/kg and CD34/kg was 8.3 × 108 (4.7–18.9) and 5.0 × 106/kg (4.6–6.4). Maximal chimerism following unrelated cord blood transplantation, 100% × 7, 98% × 1, 95% × 2, 55% × 1, and 0% × 3; related PBSC/BM, 100% × 5, 65% × 1, and 55% × 1. Graft failure occurred in 5/21 (24%). In summary, RI AlloSCT in children is feasible and tolerable (⩽25% GF) and results in ⩾85% of recipients initially achieving ⩾50% donor chimerism.

Cloning and chromosomal localization of the human TRK-B tyrosine kinase receptor gene (NTRK2)

There is increasing evidence that neutrophins and their receptors play an important role in regulating development of both the central and the peripheral nervous systems. Human TRK-A (NTRK1) and TRK-C (NTRK3) have been cloned and sequenced, but only a truncated form of human TRK-B has been published. Therefore, we isolated complementary DNAs spanning the entire coding region of both human full-length and truncated forms of TRK-B from human brain cDNA libraries. Human full-length TRK-B codes for a protein of 822 amino acid residues. The putative mature peptide sequence is 49% homologous to human TRK-A and 55% to full-length human TRK-C, with 40% amino acid identify among TRK-A, -B, and -C. Nine of 13 cysteine residues, 4 of 12N-glycosylation sites in the extracellular domain, and 10 of 13 tyrosine residues in the intracellular domain are conserved among human TRK-A, -B, and -C. There is a cluster of 10 serine residues in the juxtamembrane region of TRK-B that is absent in TRK-A. Two major sizes of TRK-B transcripts were expressed in human brain. Northern blot analysis using probes specific for the extracellular or the tyrosine kinase domain revealed that the 9.5-kb band encodes the full-length TRK-B mRNA and the 8.0-kb band encodes the truncated form of TRK-B mRNA. By fluorescence in situ hybridization and somatic cell hybrid mapping, the human TRK-B gene was localized to chromosome 9q22.1.

Polyplex-microbubble hybrids for ultrasound-guided plasmid DNA delivery to solid tumors.

Microbubble ultrasound contrast agents are being developed as image-guided gene carriers for targeted delivery in vivo. In this study, novel polyplex-microbubbles were synthesized, characterized and evaluated for systemic circulation and tumor transfection. Branched polyethylenimine (PEI; 25 kDa) was modified with polyethylene glycol (PEG; 5 kDa), thiolated and covalently attached to maleimide groups on lipid-coated microbubbles. The PEI-microbubbles demonstrated increasingly positive surface charge and DNA loading capacity with increasing maleimide content. The in vivo ultrasound contrast persistence of PEI-microbubbles was measured in the healthy mouse kidney, and a two-compartment pharmacokinetic model accounting for free and adherent microbubbles was developed to describe the anomalous time-intensity curves. The model suggested that PEI loading dramatically reduced free circulation and increased nonspecific adhesion to the vasculature. However, DNA loading to form polyplex-microbubbles increased circulation in the bloodstream and decreased nonspecific adhesion. PEI-microbubbles coupled to a luciferase bioluminescence reporter plasmid DNA were shown to transfect tumors implanted in the mouse kidney. Site-specific delivery was achieved using ultrasound applied over the tumor area following bolus injection of the DNA/PEI-microbubbles. In vivo imaging showed over 10-fold higher bioluminescence from the tumor region compared to untreated tissue. Ex vivo analysis of excised tumors showed greater than 40-fold higher expression in tumor tissue than non-sonicated control (heart) tissue. These results suggest that the polyplex-microbubble platform offers improved control of DNA loading and packaging suitable for ultrasound-guided tissue transfection.

A multi-cancer mesenchymal transition gene expression signature is associated with prolonged time to recurrence in glioblastoma.

A stage-associated gene expression signature of coordinately expressed genes, including the transcription factor Slug (SNAI2) and other epithelial-mesenchymal transition (EMT) markers has been found present in samples from publicly available gene expression datasets in multiple cancer types, including nonepithelial cancers. The expression levels of the co-expressed genes vary in a continuous and coordinate manner across the samples, ranging from absence of expression to strong co-expression of all genes. These data suggest that tumor cells may pass through an EMT-like process of mesenchymal transition to varying degrees. Here we show that, in glioblastoma multiforme (GBM), this signature is associated with time to recurrence following initial treatment. By analyzing data from The Cancer Genome Atlas (TCGA), we found that GBM patients who responded to therapy and had long time to recurrence had low levels of the signature in their tumor samples (P = 3×10−7). We also found that the signature is strongly correlated in gliomas with the putative stem cell marker CD44, and is highly enriched among the differentially expressed genes in glioblastomas vs. lower grade gliomas. Our results suggest that long delay before tumor recurrence is associated with absence of the mesenchymal transition signature, raising the possibility that inhibiting this transition might improve the durability of therapy in glioma patients.