Ingeborg M. Ambros

Role of Ploidy, Chromosome 1p, and Schwann Cells in the Maturation of Neuroblastoma

BACKGROUND Neuroblastoma is a heterogeneous disease, with manifestations ranging from spontaneous regression to lethal spread. Sometimes the tumor spontaneously differentiates toward a benign ganglioneuroma (maturing neuroblastoma). The prognosis is frequently related to ploidy, deletions in the short arm of chromosome 1, and amplifications of the N-myc oncogene. Maturing neuroblastomas consist of both neuronal cells and Schwann cells. We investigated the genetic composition of both cell types in maturing neuroblastomas, to determine the relation between genetic abnormalities and maturation. METHODS We studied 20 maturing and mature neuroblastomas by in situ hybridization to count the chromosomes and evaluate possible deletions in the short arm of chromosome 1 in neuronal and Schwann cells. The DNA content of the cells was measured by flow cytometry. RESULTS Neuroblastic and ganglionic cells showed aberrations in the number of chromosomes. In situ hybridization and flow cytometry demonstrated near-trip-loidy in 18 of 19 tumors and pentaploidy in the remaining tumor. The Schwann cells in all 20 neuroblastomas contained normal numbers of chromosomes. In 18 tumors studied, there were no chromosome 1 deletions in either type of cell. CONCLUSIONS The Schwann cells in maturing neuroblastomas differ genetically from the neuronal cells. The normal number of chromosomes in Schwann cells and the abnormal number in neuroblastic ganglionic cells suggests that Schwann cells are a reactive population of normal cells that invade the neuroblastoma. Near-trip-loidy of neuroblastoma cells and intact chromosome 1 are presumably genetic prerequisites for spontaneous organoid maturation, because we found no diploidy or chromosome 1 depletions in the neuronal cells of spontaneously maturing neuroblastomas.

DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma

Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine.

Detection of Disseminated Tumor Cells in Neuroblastoma : 3 Log Improvement in Sensitivity by Automatic Immunofluorescence plus FISH (AIPF) Analysis Compared with Classical Bone Marrow Cytology

The sensitive detection of bone marrow involvement is crucial for tumor staging at diagnosis and for monitoring of the therapeutic response in the patients follow-up. In neuroblastoma, only conventional cytomorphological techniques are presently accepted for the detection of bone marrow involvement, yet since the therapeutic consequences of the bone marrow findings may be far-reaching, the need for highly reliable detection methods has become evident. For this purpose, we developed an automatic immunofluorescence plus FISH (AIPF) device which allows the exact quantification of disseminated tumor cells and the genetic verification in critical cases. In this study, the power of the immunofluorescence technique is compared with conventional cytomorphology. 198 samples from 23 neuroblastoma patients (stages 4 and 4s) at diagnosis and during follow-up were investigated. At diagnosis, 45.6% of the samples (26 of 57) which were positive by AIPF investigation were negative by cytomorphology. During follow-up, 74.2% (49 of 66) of AIPF-positive samples showed no cytological signs of tumor cell involvement. False negative morphological results were found in up to 10% of tumor cell content. A tumor cell infiltrate below 0.1% was virtually not detectable by conventional cytomorphology. Using the sensitive immunofluorescence technique, the analysis of only two instead of four puncture sites did not lead to false negative results. Thus, the immunofluorescence technique offers an excellent tool for reliable detection and quantification of disseminated tumor cells at diagnosis and during the course of the disease.

Induction of senescence in MYCN amplified neuroblastoma cell lines by hydroxyurea

Recently, it was shown that MYCN amplified cells spontaneously expulse extrachromosomally amplified gene copies by micronuclei formation. Furthermore, it was shown that these cells lose their malignant phenotype and start to age. We tested whether it is possible to encourage neuroblastoma tumor cells to enter the senescence pathway by low concentrations of the micronuclei‐inducing drug hydroxyurea (HU). We studied the effect of HU on 12 neuroblastoma cell lines with extra‐ or intrachromosomally amplified MYCN copies and without amplification. Two extrachromosomally amplified neuroblastoma cell lines (with double minutes) were investigated in detail. Already after 3 weeks of HU treatment, the BrdU uptake dropped to 25% of the starting cells. After 4 weeks, enlarged and flattened cells (F‐cells) and increased granularity in the majority of cells were observed. A drastic reduction of the MYCN copy number—down to one copy per cell—associated with CD44 and MHCI upregulation in up to 100% of the HU treated neuroblastoma cells was found after 5–8 weeks. Telomere length was reduced to half the length within 8 weeks of HU treatment, and telomerase activity was not detectable at this time, while being strongly expressed at the beginning. All these features and the expression of senescence‐associated‐β‐galactosidase (SA‐β‐GAL) in up to 100% of the cells support the hypothesis that these cells entered the senescence pathway. Thus, low‐dose HU is a potent senescence elicitor for tumor cells with gene amplification, possibly representing an attractive additional strategy for treatment of this subset of tumors.

Unequivocal identification of disseminated tumor cells in the bone marrow by combining immunological and genetic approaches--functional and prognostic information.

Unequivocal identification of disseminated tumor cells in the bone marrow by combining immunological and genetic approaches n functional and prognastic information

Abstract PR13: DNA methylation mapping and computational modeling in a large Ewing sarcoma cohort identifies principles of tumor heterogeneity and their impact on clinical phenotypes

Ewing sarcoma is an excellent model for studying the role of epigenetic deregulation and tumor heterogeneity, given its low mutation rates and the well-defined oncogenic driver. We have recently shown that the fusion oncogene EWS-FLI1 induces widespread epigenetic rewiring in proximal and distal enhancers (Tomazou et al. Cell Reports 2015). In the current study, we validate the clinical relevance of our results in a large cohort of primary tumors, and we explore the prevalence, characteristics, and clinical impact of epigenetic tumor heterogeneity in Ewing sarcoma. We used reduced representation bisulfite sequencing (RRBS) to generate genome-wide profiles of DNA methylation in 141 Ewing sarcoma primary tumors, 17 Ewing sarcoma cell lines, and 32 primary mesenchymal stem cell (MSC) samples. Deep sequencing resulted in DNA methylation measurements for an average of 3.5 million unique CpGs per sample with excellent data quality (>98% bisulfite conversion efficiency). In addition, for three primary tumors we generated comprehensive reference epigenome maps using whole genome bisulfite sequencing (WGBS) and ChIP-seq for seven histone marks (H3K4me3, H3K4me1, H3K27me3, H3K27ac, H3K56ac, H3K36me3, and H3K9me3). We show that DNA methylation data can be used to infer enhancer activity differences among tumors, allowing us to exploit our large primary tumor dataset to systematically compare the regulation of EWS-FLI1 correlated and anticorrelated enhancers. We also identified Ewing-specific DNA methylation patterns. For example, Ewing sarcoma samples consistently show higher DNA methylation than MSCs at AP-1 binding sites, but lower DNA methylation at EWS-FLI1 binding sites. To explore epigenetic heterogeneity within individual tumors, we developed a bioinformatic algorithm that quantifies DNA methylation disorder. Using individual reads containing multiple DNA methylation measurements from single cells, we assign scores at single-nucleotide resolution. This method uses a probabilistic model to account for overall methylation rate and expected disorder levels. By evaluating the likelihood of the data in a model that assumes that the DNA methylation status of a CpG is independent of the methylation status of a nearby CpG, we identify extremely heterogeneous as well as highly epigenetically conserved genomic elements. These different region types show distinct patterns of enrichment for regulatory modes and transcription factor binding. We also compared the observed DNA methylation disorder in 141 Ewing tumors to those observed in 17 Ewing sarcoma cell lines, 32 primary MSC samples, and several hundred additional tumor and normal samples that are unrelated to Ewing sarcoma. This analysis identified Ewing-specific patterns of DNA methylation heterogeneity and stratifies patients based on epigenetic heterogeneity. Our dataset constitutes the largest available resource of genome-scale DNA methylation maps in a solid pediatric tumor. It strongly confirms the relevance of enhancer reprogramming and tumor heterogeneity in Ewing sarcoma, and it constitutes a starting point to develop DNA methylation biomarkers for prognosis and patient stratification. This study is supported by the Austrian National Bank (OeNB project #15714) and the Kapsch group (https://www.kapsch.net/). This abstract is also presented as Poster A24. Citation Format: Nathan C. Sheffield, Franck Tirode, Sandrine Grossetete-Lalami, Paul Datlinger, Andreas Schonegger, Johanna Hadler, Diana Walder, Ingeborg M. Ambros, Ana Teresa Amaral, Enrique de Alava, Katharina Schallmoser, Dirk Strunk, Beate Rinner, Bernadette Liegl-Atzwanger, Berthold Huppertz, Andreas Leithner, Uta Dirksen, Peter Ambros, Olivier Delattre, Heinrich Kovar, Christoph Bock, Eleni M. Tomazou. DNA methylation mapping and computational modeling in a large Ewing sarcoma cohort identifies principles of tumor heterogeneity and their impact on clinical phenotypes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR13.

235 Genomic analyses of circulating tumor DNA in the plasma of neuroblastoma patients

Die genetische Heterogenitat innerhalb des Tumors sowie oftmals nicht verfugbares Biopsiematerial limitieren die genaue Analyse des Tumorgenoms, welche speziell beim Neuroblastom von groser Bedeutung ist. Die Genotypisierung des Tumors ist, neben dem Therapie-Monitoring und der Fruherkennung von Tumorrezidiven und minimalen Resterkrankungen, eine der wichtigsten potentiellen Anwedungen der Analyse von zirkulierender Tumor DNA. Das Vorkommen von zirkulierender Tumor DNA, welche den Tumor-spezifischen Anteil der zell-freien DNA darstellt, wurde im Plasma von Krebspatienten, vor allem von jenen mit einem metastasierten Krankheitsbild, von einer Reihe von Autoren umfassend beschrieben. Da bisher keine Daten von genom-weiten Analysen der zirkulierenden Tumor DNA im padiatrischen Bereich publiziert wurden, sollte in dieser Studie getestet werden, ob zell-freie DNA von 1 ml Plasma mittels SNP-Array Analyse untersucht werden kann, und ob Tumorgenom-spezifische Aberrationen identifizierbar sind. Von 22 Stadium M Neuroblastom-Patienten wurden 123 Plasmaproben untersucht, 52 waren aus dem peripheren Blut (PB) und 71 aus dem Knochenmark (KM). Eine auf Magnetic Beads basierende Methode, die sich im durchgefuhrten direkten Vergleich zu einer herkommlichen Extraktionsmethode in Bezug auf die Output-Menge an DNA als uberlegen herausgestellt hat, wurde verwendet um zell-freie DNA aus den Plasmaproben zu isolieren. Mittels SNP-Array Analyse konnten Tumor-spezifische Aberrationen in 11 von 16 analysierten Proben nachgewiesen werden. Die Moglichkeit, genomische Aberrationen des Tumors in 1 ml zell-freien Plasma zu detektieren, konnte dabei sowohl fur PB- als auch KM-Plasmaproben gezeigt werden. Die Detektierbarkeit von Tumor-spezifischen DNA-Fragmenten war weder von der DNA-Konzentration im Plasma noch von der DNA-Input Menge fur die Analyse abhangig, sondern vielmehr von der Tumorzell-Infiltrationsrate im KM. Die Resultate der SNP-Array Analysen wurden mit Daten von anderen Geweben bzw. anderen Korperflussigkeiten desselben Patienten verglichen. In dem dabei analysierten Patientenkollektiv wurde eine hohe Ubereinstimmung der genomischen Aberrationen zwischen den untersuchten Geweben bzw. Plasmaproben festgestellt. Doch wahrend die zell-freie DNA von KM-Plasmaproben mit den disseminierten Tumorzellen des KM in allen 3 Fallen eine 100%ige Ubereinstimmung aufwies, waren die Aberrationen zwischen den zell-freien DNAs von PB- und KM-Plasmaproben in einem weiteren Fall nicht komplett deckungsgleich. Die genom-weite Analyse von zirkulierender Tumor DNA aus Plasmaproben birgt hohes Potential, vor allem in Fallen, in denen eine Biopsie ein hohes Risiko fur den Patienten darstellt. Die Erkenntnisse dieser Studie stehen im Widerspruch zu der gegenwartigen Ansicht, dass die identen Genomveranderungen in allen Korperflussigkeiten vorhanden sind. Ein systematischer Vergleich verschiedener Datensatze innerhalb einzelner Patienten ware notwendig um ein besseres Verstandnis der Tumorheterogenitat sowie der Tumorevolution im Krankheitsverlauf zu erlangen.