Jochen Rössler

Dissecting the genomic complexity underlying medulloblastoma

Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour–normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.

The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues.

Detection of lymphatic endothelal cells (LECs) has been problematic because of the lack of specific markers. The homeobox transcription factor Prox1 is expressed in LECs of murine and avian embryos. We have studied expression of Prox1 in human tissues with immunofluorescence. In 19‐wk‐old human fetuses, Prox1 and vascular endothelial growth factor receptor‐3 (VEGFR‐3) are coexpressed in LECs of lymphatic trunks and lymphatic capillaries. Prox1 is located in the nucleus, and its expression is mutually exclusive with that of the blood vascular marker PAL‐E. Prox1 is a constitutive marker of LECs and is found in tissues of healthy adults and lymphedema patients. Blood vascular endothelial cells (BECs) of hemangiomas express CD31 and CD34, but not Prox1. A subset of these cells is positive for VEGFR‐3. Lymphatics in the periphery of hemangiomas express Prox1 and CD31, but not CD34. In lymphangiomas, LECs express Prox1, CD31, and VEGFR‐3, but rarely CD34. In the stroma, spindle‐shaped CD34‐positive cells are present. We show that Prox1 is a reliable marker for LECs in normal and pathologic human tissues, coexpressed with VEGFR‐3 and CD31. VEGFR‐3 and CD34 are less reliable markers for LECs and BECs, respectively, because exceptions from their normal expression patterns are found in pathologic tissues.

Efficacy of Propranolol in Hepatic Infantile Hemangiomas with Diffuse Neonatal Hemangiomatosis

We report the rapid and dramatic efficacy of propranolol in 8 infants with infantile hepatic hemangiomas. The degree of response varied from a significant improvement to a complete resolution of hepatic lesions. Heart failure and hypothyroidism resolved, and hepatomegaly decreased. No side-effects of the drug were noted.

Vascular endothelial growth factor expression in human neuroblastoma: up-regulation by hypoxia.

Enhanced angiogenesis apparently contributes to the poor clinical outcome of human neuroblastoma, but the mechanisms have remained unclear. We report here that cultured human neuroblastoma cells express a bioactive endothelial cell growth factor indistinguishable from the angiogenesis stimulator vascular endothelial growth factor (VEGF). VEGF is present in neuroblastoma but not vascular endothelial cells, whereas the corresponding VEGF receptors (Flt‐1 and Flk‐1/KDR) are expressed in endothelial but not neuroblastoma cells. Exposure of neuroblastoma cells to hypoxia induces a marked increase in bioactive VEGF. VEGF is also present in human neuroblastoma specimens, with substantial amounts in apparently hypoxic neuroblastoma cells, eventually accumulating in tumor microvessels. Our results indicate that VEGF (i) is present in human neuroblastomas, (ii) is up‐regulated by tumor hypoxia and (iii) may stimulate neuroblastoma angiogenesis by paracrine mechanisms, thereby contributing to the progression of human neuroblastomas. We suggest that inhibition of VEGF activity may represent a novel approach for the therapy of human neuroblastoma. Int. J. Cancer 81:113–117, 1999.

[18F]Fluorodeoxyglucose Positron Emission Tomography for Detection of Bone Marrow Involvement in Children and Adolescents With Hodgkin's Lymphoma

PURPOSE Currently, a routine bone marrow biopsy (BMB) is performed to detect bone marrow (BM) involvement in pediatric Hodgkins lymphoma (HL) stage greater than IIA. [(18)F]fluorodeoxyglucose positron emission tomography (FDG-PET) is increasingly used for the initial staging of HL. The value of using FDG-PET to detect BM involvement has not been sufficiently defined. We compared the results of BMBs and FDG-PET for the diagnosis of BM involvement in a large pediatric group with HL. PATIENTS AND METHODS The initial staging of 175 pediatric patients with newly diagnosed classical HL stage greater than IIA was determined by using BMB, FDG-PET, chest computed tomography (CT), and magnetic resonance imaging (MRI) or CT of the neck, abdomen, and pelvis. Staging images were prospectively evaluated by a central review board. Skeletal regions that were suggestive of BM involvement by either method were re-evaluated by using different imaging modalities. In suspicious cases, bone scintigraphy was performed. If follow-up FDG-PET scans were available, the remission of skeletal lesions during treatment was evaluated. RESULTS BMB results were positive in seven of 175 patients and were identified by FDG-PET. FDG-PET scans showed BM involvement in 45 patients. In addition, the lesions of 32 of these 45 patients had a typical multifocal pattern. In 38 of 39 follow-up positron emission tomography scans, most of the skeletal lesions disappeared after chemotherapy. There was no patient with skeletal findings suggestive of BM involvement by MRI or CT with a negative FDG-PET. CONCLUSION FDG-PET is a sensitive and specific method for the detection of BM involvement in pediatric HL. The sensitivity of a BMB appears compromised by the focal pattern of BM involvement. Thus, FDG-PET may safely be substituted for a BMB in routine staging procedures.

High Levels of Circulating VEGFR2+ Bone Marrow–Derived Progenitor Cells Correlate with Metastatic Disease in Patients with Pediatric Solid Malignancies

Purpose: Pediatric solid malignancies display important angiogenic potential, and blocking tumor angiogenesis represents a new therapeutic approach for these patients. Recent studies have evidenced rare circulating cells with endothelial features contributing to tumor neovascularization and have shown the pivotal role of bone marrow–derived (BMD) progenitor cells in metastatic disease progression. We measured these cells in patients with pediatric solid malignancies as a prerequisite to clinical trials with antiangiogenic therapy. Patients and Methods: Peripheral blood was drawn from 45 patients with localized (n = 23) or metastatic (n = 22) disease, and 20 healthy subjects. Subsets of circulating vascular endothelial growth factor receptor (VEGFR)2+-BMD progenitor cells, defined as CD45−CD34+VEGFR2(KDR)+7AAD− and CD45dimCD34+VEGFR2+7AAD− events, were measured in progenitor-enriched fractions by flow cytometry. Mature circulating endothelial cells (CEC) were measured in whole blood as CD31+CD146+CD45−7AAD− viable events. Data were correlated with VEGF and sVEGFR2 plasma levels. Results: The CD45−CD34+VEGFR2(KDR)+7AAD− subset represented <0.003% of circulating BMD progenitor cells (≤0.05 cells/mL). However, the median level (range) of the CD45dimCD34+VEGFR2+7AAD− subset was higher in patients compared with healthy subjects, 1.5% (0%-10.3%) versus 0.3% (0%-1.6%) of circulating BMD progenitors (P < 0.0001), and differed significantly between patients with localized and metastatic disease, 0.7% (0%-8.6%) versus 2.9% (0.6%-10.3%) of circulating BMD progenitors (P < 0.001). Median CEC value was 7 cells/mL (0-152 cells/mL) and similar in all groups. Unlike VEGFR2+-BMD progenitors, neither CECs, VEGF, or sVEGFR2 plasma levels correlated with disease status. Conclusion: High levels of circulating VEGFR2+-BMD progenitor cells correlated with metastatic disease. Our study provides novel insights for angiogenesis mechanisms in pediatric solid malignancies for which antiangiogenic targeting of VEGFR2+-BMD progenitors could be of interest.

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.

Angiogenesis as a target in neuroblastoma

Several research investigations on neuroblastoma (NB) have shown the important dependency of this embryonic tumour on angiogenesis, especially in the advanced and aggressive stages. However, the first pre-clinical data on anti-angiogenic drugs in NB have not been published until recently and clinical trials with anti-angiogenic agents in NB treatment protocols are still missing. Here, we summarise current knowledge on the important role and mechanisms of angiogenesis in NB, and report available pre-clinical results of anti-angiogenic agents used to treat NB. This review clearly shows that angiogenesis is a target in NB and that clinical trials are urgently needed to bring forward promising anti-angiogenesis treatment strategies into NB therapy.

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

BackgroundLymphangiomas 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.MethodsLymphangioma 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.ResultsLECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-α1 and -α9. 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.ConclusionLECs 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.

Higher vessel densities in retinoblastoma with local invasive growth and metastasis.

In this study, the importance of angiogenesis (the growth of new blood vessels from existing ones) for the growth of retinoblastoma was investigated by a retrospective immunohistochemical analysis. An individual vessel index for each tumor was determined using the endothelial-specific antibody CD 31 for vessel staining. The obtained data were correlated with clinical features, pathohistological characteristics, and the presence of metastasis. In 107 retinoblastomas collected between 1980 and 1990, we found no difference in the vessel densities between uni- and bilateral retinoblastomas (P = 0.41). However, tumors that had invaded the chorioid and/or the optic nerve statistically showed higher vessel densities than tumors without local invasive growth (P = 0.05 and P = 0.024). A tendency of higher vessel densities in retinoblastomas presenting with metastasis at the time of diagnosis was observed (P = 0.11). Based on this observation, we proceeded to examine all retinoblastomas presenting with metastasis at the time of diagnosis. These included patients that were treated between 1968 and 1993. The 18 investigated retinoblastomas had significantly higher vessel densities than all other retinoblastomas presenting without metastasis (P = 0.025). Our data indicate that in retinoblastoma, blood vessels are essential for local and systemic invasive growth. Therefore, an anti-angiogenic therapy could be considered in the multimodal therapy concept for retinoblastomas with invasive growth, both locally and systemically.