EEC syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts. We have mapped the genetic defect in several EEC syndrome families to a region of chromosome 3q27 previously implicated in the EEC-like disorder, limb mammary syndrome (LMS). Analysis of the p63 gene, a homolog of p53 located in the critical LMS/EEC interval, revealed heterozygous mutations in nine unrelated EEC families. Eight mutations result in amino acid substitutions that are predicted to abolish the DNA binding capacity of p63. The ninth is a frameshift mutation that affects the p63alpha, but not p63beta and p63gamma isotypes. Transactivation studies with these mutant p63 isotypes provide a molecular explanation for the dominant character of p63 mutations in EEC syndrome.

Hay-Wells syndrome, also known as ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome (OMIM 106260), is a rare autosomal dominant disorder characterized by congenital ectodermal dysplasia, including alopecia, scalp infections, dystrophic nails, hypodontia, ankyloblepharon and cleft lip and/or cleft palate. This constellation of clinical signs is unique, but some overlap can be recognized with other ectodermal dysplasia syndromes, for example ectrodactyly--ectodermal dysplasia--cleft lip/palate (EEC; OMIM 604292), limb--mammary syndrome (LMS; OMIM 603543), acro-dermato-ungual-lacrimal-tooth syndrome (ADULT; OMIM 103285) and recessive cleft lip/palate--ectodermal dysplasia (CLPED1; OMIM 225060). We have recently demonstrated that heterozygous mutations in the p63 gene are the major cause of EEC syndrome. Linkage studies suggest that the related LMS and ADULT syndromes are also caused by mutations in the p63 gene. Thus, it appears that p63 gene mutations have highly pleiotropic effects. We have analysed p63 in AEC syndrome patients and identified missense mutations in eight families. All mutations give rise to amino acid substitutions in the sterile alpha motif (SAM) domain, and are predicted to affect protein--protein interactions. In contrast, the vast majority of the mutations found in EEC syndrome are amino acid substitutions in the DNA-binding domain. Thus, a clear genotype--phenotype correlation can be recognized for EEC and AEC syndromes.

Purpose: In the brain, tumors may grow without inducing angiogenesis, via co-option of the dense pre-existent capillary bed. The purpose of this study was to investigate how this phenomenon influences the outcome of antiangiogenic therapy.Experimental Design: Mice carrying brain metastases of the human, highly angiogenic melanoma cell line Mel57-VEGF-A were either or not treated with different dosages of ZD6474, a vascular endothelial growth factor (VEGF) receptor 2 tyrosine kinase inhibitor with additional activity against epidermal growth factor receptor. Effect of treatment was evaluated using contrast-enhanced magnetic resonance imaging (CE-MRI) and (immuno)morphologic analysis.Results: Placebo-treated Mel57-VEGF-A brain metastases evoked an angiogenic response and were highlighted in CE-MRI. After treatment with ZD6474 (100 mg/kg), CE-MRI failed to detect tumors in either prevention or therapeutic treatment regimens. However, (immuno)histologic analysis revealed the presence of numerous, small, nonangiogenic lesions. Treatment with 25 mg/kg ZD6474 also resulted in efficient blockade of vessel formation, but it did not fully inhibit vascular leakage, thereby still allowing visualization in CE-MRI scans.Conclusions: Our data show that, although angiogenesis can be effectively blocked by ZD6474, in vessel-dense organs this may result in sustained tumor progression via co-option, rather than in tumor dormancy. Importantly, blocking VEGF-A may result in undetectability of tumors in CE-MRI scans, leading to erroneous conclusions about therapeutic efficacy during magnetic resonance imaging followup. The maintenance of VEGF-A-induced vessel leakage in the absence of neovascularization at lower ZD6474 doses may be exploited to improve delivery of chemotherapeutic agents in combined treatment regimens of antiangiogenic and chemotherapeutic compounds.

Recent studies have indicated that TNF can promote activation of the coagulation mechanism by modulating coagulant properties of endothelial cells. In this report, we demonstrate that infusion of low concentrations of TNF (3 micrograms/animal) into mice bearing meth A fibrosarcomas leads to localized fibrin deposition with formation of occlusive intravascular thrombi in close association with the endothelial cell surface. Studies with 125I-fibrinogen showed tenfold enhanced accumulation of radioactivity in tumor within 2 h after TNF infusion. Western blots of tumor extracts subjected to SDS-PAGE and visualized with a fibrin-specific mAb indicated that fibrin forms in the tumor after the TNF infusion. Electron microscopic studies demonstrated fibrin strands, based on the characteristic 21-nm periodicity, which appeared to be adherent to the endothelial cell surface. Further ultrastructural studies indicated that fibrin formation, first evident within 30 min of the TNF infusion, led to occlusive thrombi limited to the tumor vascular bed (i.e., not in the normal mouse vasculature) within 2 h and was associated with an 80% reduction in tumor perfusion based on studies with Evans blue. In view of previous work concerning TNF induction of endothelial cell procoagulant activity, the hypothesis that tumor cell products prime the response of endothelium to this cytokine was tested. Supernatants of cultured meth A fibrosarcomas obtained serum-free conditions, which had no intrinsic procoagulant activity, considerably enhanced tissue factor induction in endothelium in response to submaximal concentrations of TNF. The factor(s) in the tumor-conditioned medium appeared to be distinct from IL-1, fibroblast growth factor, IFN-gamma, TNF, endotoxin, TGF-alpha, and TGF-beta. These studies delineate a novel model of localized clot formation in which thrombosis is initiated by a pathophysiologic mediator, TNF, and provides an opportunity to examine mechanisms in the microenvironment directing clot formation to the tumor vascular bed.

Tumour metastasis is the result of a complex sequence of events, including migration of tumour cells through stroma, proteolytic degradation of stromal and vessel wall elements, intravasation, transport through the circulation, extravasation and outgrowth at compatible sites in the body (the 'seed and soil' hypothesis). However, the high incidence of metastasis from various tumour types in liver and lung may be explained by a stochastic process as well, based on the anatomical relationship of the primary tumour with the circulation and mechanical entrapment of metastatic tumour cells in capillary beds. We previously reported that constitutive VEGF-A expression in tumour xenografts facilitates this type of metastatic seeding by promoting shedding of multicellular tumour tissue fragments, surrounded by vessel wall elements, into the circulation. After transport through the vena cava, such fragments may be trapped in pulmonary arteries, allowing them to expand to symptomatic lesions. Here we tested whether this process has clinical relevance for clear cell renal cell carcinoma (ccRCC), a prototype tumour in the sense of high constitutive VEGF-A expression. To this end we collected and analysed outflow samples from the renal vein, directly after tumour nephrectomy, in 42 patients diagnosed with ccRCC. Tumour fragments in venous outflow were observed in 33% of ccRCC patients and correlated with the synchronous presence or metachronous development of pulmonary metastases (p < 0.001, Fisher's exact test). In patients with tumours that, in retrospect, were not of the VEGF-A-expressing clear cell type, tumour fragments were never observed in the renal outflow. These data suggest that, in ccRCC, a VEGF-A-induced phenotype promotes a release of tumour cell clusters into the circulation that may contribute to pulmonary metastasis.