Genotyping circulating tumor DNA of pediatric Hodgkin lymphoma.

We used hybrid capture-targeted next-generation sequencing of circulating cell-free DNA (ccfDNA) of pediatric Hodgkin lymphoma (PHL) patients to determine pathogenic mechanisms and assess the clinical utility of this method. Hodgkin-Reed/Sternberg (HRS) cell-derived single nucleotide variants, insertions/deletions, translocations and VH-DH-JH rearrangements were detected in pretherapy ccfDNA of 72 of 96 patients. Number of variants per patient ranged from 1 to 21 with allele frequencies from 0.6 to 42%. Nine translocation breakpoints were detected. Genes involved in JAK/STAT, NFkB and PI3K signaling and antigen presentation were most frequently affected. SOCS1 variants, mainly deletions, were found in most circulating tumor (ct) DNAs, and seven of the nine translocation breakpoints involved SOCS1. Analysis of VH-DH-JH rearrangements revealed an origin of PHL HRS cells from partially selected germinal center B cells. Amounts of pretherapy ctDNA were correlated with metabolic tumor volumes. Furthermore, in all ccfDNA samples of 43 patients with early response assessment quantitative qPET < 3, indicative of a favorable clinical course, ctDNA was not detectable. In contrast, in five of six patients with qPET > 3, indicative of an unfavorable clinical course, ctDNA remained detectable. ccfDNA analysis of PHL is thus a suitable approach to determine pathogenic mechanisms and monitor therapy response.

Importance of whole-body imaging with complete coverage of hands and feet in alveolar rhabdomyosarcoma staging.

BACKGROUND: Alveolar rhabdomyosarcoma commonly arises in the extremities and is characterized by aggressive biology and high frequency of metastases. Whole-body imaging is increasingly employed in pediatric oncology but not recommended as standard in the staging of soft-tissue sarcomas. OBJECTIVE: After observing patients with a large symptomatic alveolar rhabdomyosarcoma lesion and a smaller silent lesion in the more distal part of an extremity we sought to estimate the frequency of this constellation. MATERIALS AND METHODS: We retrospectively evaluated the data of prospectively registered paediatric patients (age <21 years) with alveolar rhabdomyosarcoma in the SoTiSaR (Soft Tissue Sarcoma Registry) of the Cooperative Weichteilsarkom Studiengruppe (CWS) 09/2011-04/2015 with regard to whole-body imaging. RESULTS: Seventy-five patients were eligible. Images of 57 patients had been submitted for reference consultation, including 80 whole-body examinations in 36 patients. Among them were 5 patients (14%, 95% confidence interval 3-25%) who had been diagnosed because of a symptomatic lesion while an additional silent lesion in the distal part of an extremity had remained unnoticed and had only been detected by later whole-body imaging. It is noteworthy that in 42 (53%) of all 80 whole-body examinations, the hands and feet had been only partially covered or completely excluded. CONCLUSION: In alveolar rhabdomyosarcoma silent lesions can be overlooked when the distal parts of the limbs are not thoroughly examined and not completely covered by imaging. Missing them influences treatment decisions and prognosis. Our results should be considered when evaluating the potential role of whole-body imaging in rhabdomyosarcoma.

Treatment and outcome of Ganglioneuroma and Ganglioneuroblastoma intermixed.

BACKGROUND: Ganglioneuroma (GN) and ganglioneuroblastoma intermixed (GNBI) are mature variants of neuroblastic tumors (NT). It is still discussed whether incomplete resection of GN/GNBI impairs the outcome of patients. METHODS: Clinical characteristics and outcome of localized GN/GNBI were retrospectively compared to localized neuroblastoma (NB) and ganglioneuroblastoma-nodular (GNBN) registered in the German neuroblastoma trials between 2000 and 2010. RESULTS: Of 808 consecutive localized NT, 162 (20 %) were classified as GN and 55 (7 %) as GNBI. GN/GNBI patients presented more often with stage 1 disease (68 % vs. 37 %, p < 0.001), less frequently with adrenal tumors (31 % vs. 43 %, p = 0.001) and positive mIBG-uptake (34 % vs. 90 %, p < 0.001), and had less often elevated urine catecholamine metabolites (homovanillic acid 39 % vs. 62 %, p < 0.001, vanillylmandelic acid 27 % vs. 64 %, p < 0.001). Median age at diagnosis increased with grade of differentiation (NB/GNBN: 9; GNBI: 61; GN-maturing: 71; GN-mature: 125 months, p < 0.001). Complete tumor resection was achieved at diagnosis in 70 % of 162 GN and 67 % of 55 GNBI, and after 4 to 32 months of observation in 4 GN (2 %) and 5 GNBI (9 %). Eleven patients received chemotherapy without substantial effect. Fifty-five residual tumors (42 GN, 13 GNBI) are currently under observation (median: 44 months). Five patients (3 GN, 2 GNBI) showed local progression; all had tumor residuals > 2 cm. No progression occurred after subtotal resection. Two patients died of treatment, none of tumor progression. CONCLUSIONS: GN/GNBI account for one quarter of localized NT and differ from immature tumors in their clinical features. Chemotherapy is not effective. Subtotal resection appears to be a sufficient treatment. TRIAL REGISTRATION: ClinicalTrials.gov identifiers - NB97 (NCT00017225; registered June 6, 2001); NB2004 (NCT00410631; registered December 11, 2006).

Stanniocalcin 2 promotes invasion and is associated with metastatic stages in neuroblastoma.

Stanniocalcin 2 (STC2) is a secreted glycoprotein of as yet unknown functions. We investigated STC2 in human neuroblastoma, the most common solid extra-cranial tumor of infancy. In primary tumor samples, we found that expression of STC2 is associated with the metastatic Stages 4 and 4s and MYCN expression. In vitro, however, we demonstrate that cell proliferation is reduced by STC2 due to an increase in the basal apoptosis rate of the transfected cells. On the other hand, in vitro assays showed that STC2-transfected neuroblastoma cells have an increased invasive potential and display higher activity of collagen-degrading matrix metalloproteinase 2 (MMP2). Using experimental tumors on the chick chorioallantoic membrane (CAM), we observed that STC2 expressing cells show signs of emigration from the solid tumor and destroy blood vessels of the CAM, giving rise to massively bleeding tumors. Erosion of blood vessels was also seen when purified STC2 protein was applied on the CAM. Taken together, we demonstrate a dual role for STC2 in neuroblastoma. It reduces proliferation of tumor cells in vitro, but increases the invasive potential and induces bleeding, and thereby may facilitate early metastasis. The potential of STC2 as a surrogate marker for metastatic neuroblastoma calls for further investigation.

Keratoepithelin reverts the suppression of tissue factor pathway inhibitor 2 by MYCN in human neuroblastoma: a mechanism to inhibit invasion.

Neuroblastoma is the most frequent solid malignancy of children. The most reliable prognostic factor in neuroblastoma is the amplification status of the MYCN oncogene, but exceptions from this rule have been observed. Recently we have demonstrated that keratoepithelin (BIGH3, TGFBI) expression significantly reduces proliferation and invasion of neuroblastomas in vitro and in vivo. In these experiments, we also observed that tissue factor pathway inhibitor 2 (TFPI2, PP5, MSPI), a potent inhibitor of matrix-metalloproteinases, is most prominently up-regulated. As MYCN-amplified neuroblastomas are highly invasive, we sought to determine the interaction between MYCN, keratoepithelin and TFPI2. In this study we provide initial evidence that i) keratoepithelin expression in neuroblastoma inversely correlates with MYCN expression; ii) TFPI2 expression in neuroblastoma also correlates inversely with MYCN expression but positively with keratoepithelin expression and iii) keratoepithelin induces elevated TFPI2 transcript levels in neuroblastoma cells without alterations of MYCN expression.

Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas.

BACKGROUND: Lymphangiomas are neoplasias of childhood. Their etiology is unknown and a causal therapy does not exist. The recent discovery of highly specific markers for lymphatic endothelial cells (LECs) has permitted their isolation and characterization, but expression levels and stability of molecular markers on LECs from healthy and lymphangioma tissues have not been studied yet. We addressed this problem by profiling LECs from normal dermis and two children suffering from lymphangioma, and also compared them with blood endothelial cells (BECs) from umbilical vein, aorta and myometrial microvessels. METHODS: Lymphangioma tissue samples were obtained from two young patients suffering from lymphangioma in the axillary and upper arm region. Initially isolated with anti-CD31 (PECAM-1) antibodies, the cells were separated by FACS sorting and magnetic beads using anti-podoplanin and/or LYVE-1 antibodies. Characterization was performed by FACS analysis, immunofluorescence staining, ELISA and micro-array gene analysis. RESULTS: LECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-alpha1 and -alpha9. However, LYVE-1 was down-regulated and VEGFR-2 and R-3 were up-regulated in lymphangiomas. Prox1 was constantly expressed in LECs but not in any of the BECs. CONCLUSION: LECs from different sources express slightly variable molecular markers, but can always be distinguished from BECs by their Prox1 expression. High levels of VEGFR-3 and -2 seem to contribute to the etiology of lymphangiomas.

The proepicardium delivers hemangioblasts but not lymphangioblasts to the developing heart.

The mass of the myocardium and endocardium of the vertebrate heart derive from the heart-forming fields of the lateral plate mesoderm. Further components of the mature heart such as the epicardium, cardiac interstitium and coronary blood vessels originate from a primarily extracardiac progenitor cell population: the proepicardium (PE). The coronary blood vessels are accompanied by lymph vessels, suggesting a common origin of the two vessel types. However, the origin of cardiac lymphatics has not been studied yet. We have grafted PE of HH-stage 17 (day 3) quail embryos hetero- and homotopically into chick embryos, which were re-incubated until day 15. Double staining with the quail endothelial cell (EC) marker QH1 and the lymphendothelial marker Prox1 shows that the PE of avian embryos delivers hemangioblasts but not lymphangioblasts. We have never observed quail ECs in lymphatics of the chick host. However, one exception was a large lymphatic trunk at the base of the chick heart, indicating a lympho-venous anastomosis and a 'homing' mechanism of venous ECs into the lymphatic trunk. Cardiac lymphatics grow from the base toward the apex of the heart. In murine embryos, we observed a basal to apical gradient of scattered Lyve-1+/CD31+/CD45+ cells in the subepicardium at embryonic day 12.5, indicating a contribution of immigrating lymphangioblasts to the cardiac lymphatic system. Our studies show that coronary blood and lymph vessels are derived from different sources, but grow in close association with each other.

Embryonic development and malformation of lymphatic vessels.

In the human, malformations of lymphatic vessels can be observed as lymphangiectasia, lymphangioma and lymphangiomatosis, with a prevalence of 1.2-2.8 per thousand. Their aetiology is unknown and a causal therapy does not exist. We investigated the origin of lymphatic endothelial cells (LECs) in avian and murine embryos, and compared the molecular profile of LECs from normal and malformed lymphatics of children. In avian embryos, Prox1+ lymphangioblasts are located in the confluence of the cranial and caudal cardinal veins, where the jugular lymph sac (JLS) forms. Cell lineage studies show that the JLS is of venous origin. In contrast, the lymphatics of the dermis are derived from mesenchymal lymphangioblasts located in the dermatomes, suggesting a dual origin of LECs in avian embryos. The same may hold true for murine embryos, where Lyve1+ LEC precursors are found in the cardinal veins, and in the mesenchyme. The mesenchymal cells express the pan-leukocyte marker CD45, indicating a cell type with lymphendothelial and leukocyte characteristics. In the human, such cells might give rise to Kaposi's sarcoma. Microarray analyses of LECs from lymphangiomas of children show a large number of regulated genes, such as VEGFR3. Our studies show that lymphvasculogenesis and lymphangiogenesis occur simultaneously in the embryo, and suggest a function for VEGFR3 in lymphangiomas.

Keratoepithelin suppresses the progression of experimental human neuroblastomas.

Neuroblastoma is the most common extracranial childhood tumor. High expression of activin A is associated with a favorable prognosis, but the contributing mechanisms have remained unclear. Our previous demonstration of the activin A-mediated up-regulation of keratoepithelin led to the consideration that keratoepithelin could modulate neuroblastoma growth and/or progression. We report here that enhanced keratoepithelin expression in human neuroblastoma cells suppresses neuroblastoma cell cohesion and adhesion to various extracellular matrix proteins and that it inhibits neuroblastoma cell proliferation and invasion in vitro and in vivo. Using microarray analysis, we identified several keratoepithelin-regulated genes that may contribute to these biological changes. Together with the observation that keratoepithelin is expressed in human neuroblastomas in vivo, our data suggest that keratoepithelin could play a beneficial role in neuroblastoma development and/or progression.

Dual origin of avian lymphatics.

The earliest signs of the lymphatic vascular system are the lymph sacs, which develop adjacent to specific embryonic veins. It has been suggested that sprouts from the lymph sacs form the complete lymphatic vascular system. We have studied the origin of the jugular lymph sacs (JLS), the dermal lymphatics and the lymph hearts of avian embryos. In day 6.5 embryos, the JLS is an endothelial-lined sinusoidal structure. The lymphatic endothelial cells (LECs) stain (in the quail) positive for QH1 antibody and soybean agglutinin. As early as day 4, the anlagen of the JLS can be recognized by their Prox1 expression. Prox1 is found in the jugular section of the cardinal veins, and in scattered cells located in the dermatomes along the cranio-caudal axis and in the splanchnopleura. In the quail, such cells are positive for Prox1 and QH1. In the jugular region, the veins co-express the angiopoietin receptor Tie2. Quail-chick-chimera studies show that the peripheral parts of the JLS form by integration of cells from the paraxial mesoderm. Intra-venous application of DiI-conjugated acetylated low-density lipoprotein into day 4 embryos suggests a venous origin of the deep parts of the JLS. Superficial lymphatics are directly derived from the dermatomes, as shown by dermatome grafting. The lymph hearts in the lumbo-sacral region develop from a plexus of Prox1-positive lymphatic capillaries. Both LECs and muscle cells of the lymph hearts are of somitic origin. In sum, avian lymphatics are of dual origin. The deep parts of the lymph sacs are derived from adjacent veins, the superficial parts of the JLS and the dermal lymphatics from local lymphangioblasts.

Mesenchymal cells with leukocyte and lymphendothelial characteristics in murine embryos.

The development of lymphatic endothelial cells (LECs) from deep embryonic veins or mesenchymal lymphangioblasts is controversially discussed. Studies employing quail-chick grafting experiments have shown that various mesodermal compartments of the embryo possess lymphangiogenic potential, whereas studies on murine embryos have been in favor of a venous origin of LECs. We have investigated NMRI mice from embryonic day (ED) 9.5 to 13.5 with antibodies against the leukocyte marker CD45, the pan-endothelial marker CD31, and the lymphendothelial markers Prox1 and Lyve-1. Early signs of the development of lymphatics are the Lyve-1- and Prox1-positive segments of the jugular and vitelline veins. Then, lymph sacs, which are found in the jugular region of ED 11.5 mice, express Prox1, Lyve-1, and CD31. Furthermore, scattered cells positive for all of the four markers are present in the mesenchyme of the dermatomes and the mediastinum before lymphatic vessels are present in these regions. Their number increases during development. A gradient of increasing CD31 expression can be seen the closer the cells are located to the lymph sacs. Our studies provide evidence for the existence of scattered mesenchymal cells, which up-regulate lymphendothelial and down-regulate leukocyte characteristics when they integrate into growing murine lymphatics. Such stem cells may also be present in the human and may be the cell of origin in post-transplantation Kaposi sarcoma.

The neurotrophin receptor TrkB cooperates with c-Met in enhancing neuroblastoma invasiveness.

Neuroblastoma is the most frequent extracranial solid malignancy of childhood with a high mortality in advanced tumour stages. The hallmark of neuroblastoma is its clinical and biological heterogeneity. The molecular mechanisms leading to favourable or unfavourable tumour behaviour are still speculative. However, amplification of the oncogene MYCN and expression of the neurotrophin receptor TrkB are known to contribute to a highly malignant phenotype. To define the mechanisms through which TrkB may mediate neuroblastoma progression, we stably expressed this receptor in the neuroblastoma cell lines SH-SY5Y and SK-N-AS. The transfectants, but not the controls, had an increased invasive potency both, in vitro and in vivo, as demonstrated by Matrigel-invasion and chorioallantoic membrane assays, respectively. The retinoic acid-induced TrkB expression in parental SH-SY5Y cells was also associated with enhanced cell invasiveness. The TrkB mediated invasiveness involved the upregulation of the hepatocyte growth factor (HGF) and its receptor c-Met, resulting in an autocrine loop. Inhibition of HGF activity by anti-HGF neutralizing antibodies or disabling the function of c-Met by small interfering RNA suppressed the TrkB-induced invasiveness. The enhanced TrkB expression was associated with a significant increase in the secretion of various matrix-degrading proteases. Immunostaining and real-time RT-PCR analysis of tumour specimens demonstrated coordinated expression of TrkB and HGF/c-Met in experimental and primary neuroblastomas. We conclude that TrkB expression in neuroblastoma cells results in an increase in their invasive capability via upregulated expression of HGF/c-Met and enhanced activity of proteolytic networks.

Sublethal irradiation promotes invasiveness of neuroblastoma cells.

Neuroblastoma is the most frequent extracranial solid tumour of childhood. Despite multiple clinical efforts, clinical outcome has remained poor. Neuroblastoma is considered to be radiosensitive, but some clinical studies including the German trial NB90 failed to show a clinical benefit of radiation therapy. The mechanisms underlying this apparent discrepancy are still unclear. We have therefore investigated the effects of radiation on neuroblastoma cell behaviour in vitro. We show that sublethal doses of irradiation up-regulated the expression of the hepatocyte growth factor (HGF) and its receptor c-Met in some neuroblastoma cell lines. The increase in HGF/c-Met expression was correlated with enhanced invasiveness and activation of proteases degrading the extracellular matrix. Thus, irradiation at sublethal doses may promote the metastatic dissemination of neuroblastoma cells through activating the HGF/c-Met pathway and triggering matrix degradation.

Altered expression patterns of EphrinB2 and EphB2 in human umbilical vessels and congenital venous malformations.

Vascular malformations cause discomfort and pain in children and are often associated with skeletal hypertrophy. Their molecular basis is poorly understood. Ephrin ligands and Eph receptor tyrosine kinases are involved in embryonic vascular development. In mice, some ephrin/Eph family members show a complementary expression pattern in blood vessels, with ephrinB2 being expressed on arterial and EphB4 on venous endothelium. Targeted deletions of the genes reveal their essential roles for conduit vessel development in mice, suggesting similar functions during human vascular development and deregulation in vascular malformations. Here, we have defined the expression patterns of human ephrinB2, EphB4, and EphB2 in normal vessels of neonates (i.e. umbilici) and adults and compared them with those in congenital venous malformations. In adults, normal vessels of the skin, muscle, and legs express ephrinB2 and EphB2 on arterial endothelial cells (ECs), whereas EphB4 is found in arteries and veins. In the umbilicus, EphB2 is a specific marker of arterial ECs, whereas ephrinB2 is additionally expressed in venous ECs, suggesting an arterial function of the veins. In venous malformations, the expression of EphB4 is not altered, but both ephrinB2 and EphB2 are ectopically expressed in venous ECs. This may reflect a nonphysiologic arterialization of malformed veins. Our study shows that the arterial markers ephrin B2 and EphB2 are expressed in a subset of veins, and it remains to be studied whether this is cause or consequence of an altered vascular identity.

Microarray analysis reveals differential gene expression patterns and regulation of single target genes contributing to the opposing phenotype of TrkA- and TrkB-expressing neuroblastomas.

Expression of neurotrophin receptors of the tyrosine kinase receptor (Trk) family is an important prognostic factor in solid tumors including neuroblastoma. High expression of TrkA (NTRK1) is associated with a favorable biology and outcome of neuroblastoma, whereas TrkB (NTRK2) is expressed on aggressive neuroblastomas with unfavorable outcome. To gain new insights into the global gene expression program resulting in these divergent biological phenotypes, we stably expressed either TrkA or TrkB in the human SH-SY5Y neuroblastoma cell line. Gene expression profiles were obtained from parental cells and transfectants activated by their ligands in a time course over 24 h using oligonucleotide microarrays. Basal activation of Trk receptors in the absence of exogenous ligand was sufficient to induce broad and divergent genetic changes. Global gene regulation following external ligand stimulation was surprisingly similar in SY5Y-TrkA and SY5Y-TrkB cells except for the differential expression of distinct novel target genes. Consistent with their divergent biological phenotype, SY5Y-TrkA cells were characterized by upregulation of proapoptotic genes and angiogenesis inhibitors, whereas SY5Y-TrkB cells demonstrated upregulation of genes involved in invasion or therapy resistance. We suggest that the transcriptional program of neuroblastoma cells is modulated by Trk-receptor expression and basal activation rather than by ligand-induced activation. Fine-tuning of the malignant phenotype may be achieved by additional ligand stimulation with subsequent activation of a few specific genes.

High activin A-expression in human neuroblastoma: suppression of malignant potential and correlation with favourable clinical outcome.

Amplification of the MYCN oncogene contributes to the malignant progression of human neuroblastomas, but the mechanisms have remained unclear. We have previously demonstrated that N-Myc facilitates angiogenesis by downregulating an angiogenesis inhibitor identified as the inhibin betaA homodimer activin A. Here, we have sought to define the molecular, biological and clinical consequences of activin A expression in human neuroblastoma. We report that enhanced activin A expression suppresses proliferation and colony formation of human neuroblastoma cells with amplified MYCN in vitro; that it inhibits neuroblastoma growth and angiogenesis in vivo; that it is highly expressed in differentiated, but not undifferentiated human neuroblastomas; and that it correlates with favourable outcome of neuroblastoma patients. Our results indicate that high activin A expression plays an important beneficial role in human neuroblastoma.

Hypoxia-induced erythropoietin expression in human neuroblastoma requires a methylation free HIF-1 binding site.

The glycoprotein hormone Erythropoietin (EPO) stimulates red cell production and maturation. EPO is produced by the kidneys and the fetal liver in response to hypoxia (HOX). Recently, EPO expression has also been observed in the central nervous system where it may be neuroprotective. It remained unclear, however, whether EPO is expressed in the peripheral nervous system and, if so, whether a neuronal phenotype is required for its regulation. Herein, we report that EPO expression was induced by HOX and a HOX mimetic in two cell lines derived from neuroblastoma (NB), a tumor of the peripheral nervous system. Both cell lines with inducible EPO expression, SH-SY5Y and Kelly cells, expressed typical neuronal markers like neuropeptide Y (NPY), growth-associated protein-43 (GAP-43), and neuron-specific enolase (ENO). NB cells with a more epithelial phenotype like SH-SHEP and LAN-5 did not show HOX inducible EPO gene regulation. Still, oxygen sensing and up-regulation of hypoxia-inducible factor-1 (HIF-1) were intact in all cell lines. We found that CpG methylation of the HIF binding site (HBS) in the EPO gene 3' enhancer was only present in the SH-SHEP and LAN-5 cells but not in SH-SY5Y and Kelly cells with regulated EPO expression. The addition of recombinant EPO to all NB cells, both under normoxic and hypoxic conditions, had no effect on cell proliferation. We conclude that the ability to respond to HOX with an increase in EPO expression in human NB may depend on CpG methylation and the differentiation status of these embryonic tumor cells but does not affect the proliferative characteristics of the cells.

Hepatocyte growth factor/c-Met signaling promotes the progression of experimental human neuroblastomas.

Neuroblastoma is the most frequent solid childhood malignancy. Despite aggressive therapy, mortality is high due to rapid tumor progression to advanced stages. The molecules and mechanisms underlying poor prognosis are not well understood. Here, we report that cultured human neuroblastoma cells express the hepatocyte growth factor (HGF) and its receptor c-Met. Binding of HGF to c-Met triggers receptor autophosphorylation, indicating functional relevance of this interaction. HGF activates several downstream effectors of c-Met such as the mitogen-activated protein kinases extracellular signal-regulated kinase 1/extracellular signal-regulated kinase 2 and phospholipase C-gamma, whereas signal transducer and activator of transcription 3 is constitutively activated in neuroblastoma cells expressing c-Met. In addition, HGF is able to stimulate expression and proteolytic activity of matrix metalloproteinase-2 and tissue-type plasminogen activator in neuroblastoma cells, thereby promoting degradation of extracellular matrix components. We show that HGF stimulates invasion of neuroblastoma cells in vitro and in vivo, and it promotes the formation of angiogenic neuroblastomas in vivo. These processes can be blocked by specific inhibitors of the mitogen-activated protein kinase cascade, by inhibitors of phospholipase C-gamma, and also by the expression of a dominant negative signal transducer and activator of transcription 3 mutant. Our data provide the first evidence that the HGF/c-Met pathway is essential for invasiveness and malignant progression of human neuroblastomas. They further suggest that specific inhibitors of this pathway may be suitable as therapeutic agents to improve clinical outcome of neuroblastomas.