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Additional file 8: Figure S5. A distinct pattern of TRF southern blot analysis dependent on the TMM subgroup. Southern blot for telomere restriction fragment analyses of neuroblastoma samples and cell lines. ALT-positive cell line SK-N-FI and ALT-negative but telomerase-positive cell line Kelly were used on every blot with tumor samples as controls on every blot.

Additional file 4: Figure S1. TERT expression and telomerase activity in neuroblastoma with activated telomere maintenance mechanism. (A) Distribution of TERT log2 expression values, determined by RNA sequencing, in neuroblastomas harboring TERT rearrangements, and/or MNA or none of these alterations. The threshold at 7.58 was defined as the lowest expression value having a posterior probability ≥95% to fall within the distribution on the right (i.e., the group of tumors with TERT/MYCN alteration). (B) Log2 TERT mRNA expression levels, dependent on telomere maintenance subgroup. ANOVA, Tukey’s multiple comparison test. n=55, RNA sequencing data was available for 64 cases, however ambiguous cases (n=7) as well as cases that show MNA and TERT RA in the same tumor (n=3) were excluded (see also Fig. 1B). Whiskers are limited to 1.5x interquartile range. (C) Relative telomerase activity dependent on underlying alteration, determined by TRAP assay. Unpaired t-test. n=15. Whiskers are limited to 1.5x interquartile range.

Aspergillus fumigatus (A. fumigatus) ist der häufigste Erreger der invasiven Aspergillose, welche vornehmlich immunsupprimierte Patientinnen und Patienten betrifft und mit einer hohen Letalität einhergeht. Zur Entwicklung neuer diagnostischer sowie therapeutischer Ansätze ist ein besseres Verständnis der Interaktion von A. fumigatus mit dem humanen Immunsystem zwingend erforderlich. Zur Erforschung dieser Interaktion werden häufig Mausmodelle herangezogen, welche aufgrund der unterschiedlichen Biologie des Wirts jedoch nicht direkt übertragbar sind. Ziel dieser Studie war es, einen funktionellen in vitro Vergleich zwischen humanen und murinen Makrophagen, neutrophilen Granulozyten (PMNs) und dendritischen Zellen (DCs) in ihrer Interaktion mit A. fumigatus Konidien, Keimschläuchen sowie depletiertem Zymosan zu erstellen, um eine bessere Beurteilung und Übertragbarkeit des Mausmodells bei der invasiven Aspergillose zu ermöglichen. Dabei wurden die verschiedenen Zellen des Immunsystems auf standardisierte und reproduzierbare Weise generiert und Stimulationsversuche durchgeführt. Hierbei zeigten humane und murine Zellen in vitro eine unterschiedliche Antwort auf die Stimulation mit A. fumigatus: Murine Makrophagen und neutrophile Granulozyten zeigten im Vergleich zu den humanen Zellen eine stärkere primäre Immunantwort mit einer vermehrten Ausschüttung reaktiver Sauerstoffspezies (ROS). Humane DCs hingegen, welche als Bindeglied zwischen angeborenem und adaptivem Immunsystem fungieren, zeigten nach Stimulation mit A. fumigatus eine vermehrte Oberflächenexpression von Maturationsmarkern sowie eine höhere Phagozytoserate als die murinen DCs. Weiterhin konnte eine inverse Dectin-1-Expression auf humanen und murinen DCs nach Stimulation mit A. fumigatus nachgewiesen werden. Es konnte gezeigt werden, dass es für alle untersuchten Zelltypen Unterschiede zwischen humanen und murinen Zellen in der basalen und der Zytokinausschüttung nach Stimulation mit A. fumigatus gab. In Zusammenschau der Ergebnisse dieser Arbeit zeigt das murine Immunsystem eine stärkere angeborene Immunantwort mit vermehrter ROS-Ausschüttung, jedoch auch eine anti-inflammatorische Zytokinantwort, um möglicherweise eine überschießende Inflammation zu verhindern. Dies könnte durch die stärkere Exposition der Maus gegenüber A. fumigatus durch den bodennahen Lebensraum sowie ihrer kurzen Lebensdauer bedingt sein. Im humanen System kommt hingegen der Aktivierung des adaptiven Immunsystems über die DCs eine übergeordnete Rolle zu. So zeigen beide Spezies distinkte Unterschiede in ihrer in vitro Immunantwort gegenüber A. fumigatus, welche bei der Übertragung von experimentellen Daten von der Maus auf den Menschen beachtet werden sollten., Aspergillus fumigatus (A. fumigatus) is the main causative organism of invasive aspergillosis which occurs almost exclusively in immunocompromised patients and is still associated with a high lethality. For the development of new diagnostic and therapeutical approaches a better understanding of the in vitro interaction of human and murine innate immune cell populations with A. fumigatus is urgently needed. To study host-pathogen interactions mice are frequently used as infection model. However, little is known about functional differences in the innate immune response of human and murine cell populations in the pathogenesis of invasive aspergillosis. Therefore, the aim of our study was to conduct a functional and highly standardized comparison of human and murine macrophages, polymorphonuclear cells (PMNs) and dendritic cells (DCs) regarding their interaction with A. fumigatus conidia, germtubes and depleted zymosan to improve extrapolation from data achieved in mice experiments. Human and murine innate immune cells displayed a differential behavior after the exposure to A. fumigatus. Murine macrophages and PMNs exhibited a significantly stronger release of reactive oxygen species (ROS) after exposure to A. fumigatus. Human DCs, which function as an important link between the innate and adaptive immune system, displayed a stronger cell surface expression of maturation markers after stimulation with A. fumigatus as well as a higher phagocytosis rate than their murine counterparts. Furthermore, human and murine DCs showed an inverse Dectin-1 surface expression after stimulation with A. fumigatus. Human and murine innate immune cells showed a differential basal as well as stimulated cytokine release. Our data indicate that the murine immune system may have a stronger innate immune response after stimulation with A. fumigatus with an increased release of ROS. However, murine cells also showed a strong anti-inflammatory cytokine release to potentially inhibit excessive inflammation. This effect in mice might be due to the higher exposure to A. fumigatus caused by near-ground habitat as well as the shorter life span. In humans the activation of the adaptive immune system by activation of DCs seems to predominate. To summarize, immune cells of both species display distinct differences in their in vitro immune response after stimulation with A. fumigatus, which should be considered when conducting host-pathogen interaction studies in mice.

Additional file 5: Figure S2. Dependency of C-circle assay on threshold and reference cell line. (A) Southern blot of C-circle assay of different cell lines (CHLA-90: ALT[+], SK-N-BE(2): TEL[+], SK-N-FI: ALT[+], LM-216-J: ALT[+]), normal human tissue and neuroblastoma samples with high telomerase activity (all ALT-negative). Left columns: sample without polymerase, right columns: samples with polymerase. (B) Neuroblastoma samples for which APB and C-circle assay was available (n=67). Different thresholds applied to the same samples reveal different results. (th1) C-circle signal intensity ≥5% relative to the signal of CHLA-90. (th2) C-circle signal intensity ≥20% relative to the signal of CHLA-90 and at least fourfold the area under the curve of polymerase-free dot-blot. Number of cases (n) classified as ALT-positive according to the respective threshold are indicated. (C) Representative image of southern blot of C-Circle assay of different cell lines and neuroblastoma samples. Left columns: sample without polymerase, right columns: samples with polymerase. ALT-positive cell line CHLA-90 and ALT-negative cell line SK-N-BE are depicted at the top, ALT status according to threshold th1 as indicated.

Additional file 11: Figure S8. Raw data of CCA and combined Immunofluorescence/FISH of ambiguous neuroblastoma cases. (A) Images of CCA and (B) combined Immunofluorescence/FISH of ambiguous neuroblastoma samples with contrasting results as revealed by the two methods. Images of Immunofluorescence/FISH show ultrabright telomeric signals (green) and associated APBs (red) in ALT-positive cases, whereas the ALT-negative sample (NB05) does not show prominent telomeric signals and only subtle PML bodies. (C) Example of one unambiguous ALT-negative and one unambiguous ALT-positive neuroblastoma.

Additional file 9: Figure S6. Features for prediction of TMM status in neuroblastoma. (A) Combined ROC for TC and TRF. After binary logistic regression and calculation of predicted probabilities, used for combined ROC, n=36. (B) ROC for normalized TERRA read count as a classifier for ALT, n=58.

Additional file 7: Figure S4. Detection of telomeric reads and calculation of telomere content on WGS data. (A) Number of detected telomeric reads, i.e., reads containing at least four t-type repeats, in tumor and normal WGS data in relation to the mean read depth. Pearson correlation displayed for normal controls only. (B) Normalized telomere content calculated from single tumor WGS data, dependent on telomere maintenance subgroups. Mean telomere content: TMM[-] 67.43, ALT[+] 339.33, TEL[+] 54.19. Kruskal-Wallis test and Dunn’s multiple comparison test, n=61, ambiguous cases were excluded from statistical analysis. Whiskers are limited to 1.5x interquartile range. (C) Correlation analysis of telomere content calculated from tumor/normal ratios and the respective single tumor samples.