Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers.

No abstract

MetastasisReceptor tyrosine kinase ROR1 Therapeutic target A B S T R A C TReceptor tyrosine kinases (RTKs) have provided molecular targets for the development of novel, prognosis-improving agents in many cancers; however, resistances to these therapies occur. On the cellular level, one resistance mechanism is attributed to functional RTK redundancies and compensatory cross-signaling, leading to perception of RTKs as signaling and target networks. To provide a basis for better exploitation of this network in Ewing sarcoma, we generated comprehensive qPCR gene expression profiles of RTKs in Ewing sarcoma cell lines and 21 untreated primary tumors. Key findings confirm broad-spectrum RTK expressions with potential for signaling redundancy. Profile analyses with regard to patient risk-group further revealed several individual RTKs of interest.Among them, VEGFR3 and TIE1 showed high-level expressions and also were suggestive of poor prognosis in localized tumors; underscoring the relevance of angiogenic signaling pathways and tumor-stroma interactions in Ewing sarcoma. Of note, compared to localized disease, tumors derived from metastatic disease were marked by global high-level RTK expressions. Nine individual RTKs were significantly over-expressed, suggesting contributions to molecular mechanisms of metastasis. Of these, ROR1 is being pursued as therapeutic target in leukemias and carcinomas, but un-characterized in sarcomas. We demonstrate expression of ROR1 and its putative ligand Wnt5a in Ewing sarcomas, and of an active ROR1 protein variant in cell lines. ROR1 silencing impaired cell migration in vitro. Therefore, ROR1 calls for further evaluation as a therapeutic target in metastatic Abbreviations: MSC, mesenchymal stem cell; qPCR, real-time quantitative PCR; ROR1, receptor tyrosine kinase-like orphan receptor 1; RTK, receptor tyrosine kinase, for a listing of RTK abbreviations see

No abstract

BackgroundThe CXCR4 receptor antagonist plerixafor (AMD3100) is raising interest as an anti-cancer agent that disrupts the CXCL12-CXCR4 chemokine – receptor interaction between neoplastic cells and their microenvironment in tumor progression and metastasis. Here, we investigated plerixafor for anti-cancer activity in Ewing sarcoma, a rare and aggressive cancer of bone and soft tissues.MethodsWe used a variety of methods such as cell viability and migration assays, flow cytometry, phospho-tyrosine arrays and western blotting to determine plerixafor effects on five characterized Ewing sarcoma cell lines and a low-passage culture in vitro.ResultsUnexpectedly, plerixafor led to an increase in cell viability and proliferation in standard cell growth conditions, and to chemotactic migration towards plerixafor. Exploring potential molecular mechanisms underlying this effect, we found that Ewing sarcoma cell lines divided into classes of high- and low-level CXCR4 surface expression. Proliferative plerixafor responses were observed in both groups, maintained despite significant CXCR4 down-regulation or inhibition of Gαi-protein signal transduction, and involved activation of multiple receptor tyrosine kinases (DDR2, MERTK, MST1R, NTRK1, RET), the most prominent being platelet-derived growth factor receptor beta (PDGFRB). PDGFRB was activated in response to inhibition of the CXCL12-CXCR4 axis by plerixafor and/or pertussis toxin (Gαi-protein inhibitor). Dasatinib, a multi-kinase inhibitor of both PDGFRB and the CXCR4 downstream kinase SRC, counteracted this activation in some but not all cell lines.ConclusionThese data suggest a feedback interaction between the CXCR4 chemokine receptor and RTK signaling cascades that elicits compensatory cell survival signaling and can shift the net effect of plerixafor towards proliferation. PDGFRB was identified as a candidate driver RTK and potential therapeutic co-target for CXCR4 in Ewing sarcoma. Although as yet limited to in vitro studies, these findings call for further investigation in the cancer – microenvironment interplay in vivo.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0233-2) contains supplementary material, which is available to authorized users.

The superfamily of G-protein-coupled receptors (GPCR) conveys signals in response to various endogenous and exogenous stimuli. Consequently, GPCRs are the most important drug targets. CCR10, the receptor for the chemokines CCL27/CTACK and CCL28/MEC, belongs to the chemokine receptor subfamily of GPCRs and is thought to function in immune responses and tumour progression. However, there is only limited information on the intracellular regulation of CCR10. We find that S100A10, a member of the S100 family of Ca2+ binding proteins, binds directly to the C-terminal cytoplasmic tail of CCR10 and that this interaction regulates the CCR10 cell surface presentation. This identifies S100A10 as a novel interaction partner and regulator of CCR10 that might serve as a target for therapeutic intervention.

In the search for novel therapeutic targets, RNA interference screening has become a valuable tool. High-throughput technologies are now broadly accessible but their assay development from baseline remains resource-intensive and challenging. Focusing on this assay development process, we here describe a target discovery screen using pooled shRNA libraries and next-generation sequencing (NGS) deconvolution in a cell line model of Ewing sarcoma. In a strategy designed for comparative and synthetic lethal studies, we screened for targets specific to the A673 Ewing sarcoma cell line. Methods, results and pitfalls are described for the entire multi-step screening procedure, from lentiviral shRNA delivery to bioinformatics analysis, illustrating a complete model workflow. We demonstrate that successful studies are feasible from the first assay performance and independent of specialized screening units. Furthermore, we show that a resource-saving screen depth of 100-fold average shRNA representation can suffice to generate reproducible target hits despite heterogeneity in the derived datasets. Because statistical analysis methods are debatable for such datasets, we created ProFED, an analysis package designed to facilitate descriptive data analysis and hit calling using an aim-oriented profile filtering approach. In its versatile design, this open-source online tool provides fast and easy analysis of shRNA and other count-based datasets to complement other analytical algorithms.

Osteosarcoma is the most common primary bone cancer in children and is a highly malignant disease, in which 25% of patients present with metastasis at diagnosis. Considerable advances in the treatment of localized disease have been achieved since the introduction of combined modality treatment, increasing the prognosis of overall survival to 70%. Yet, established therapies have only limited success in treating both metastatic disease and nonresponders to primary chemotherapy. Therefore, new therapeutic approaches are required, particularly for the control of osteosarcoma in these patient groups. Epigenetically modifying substances are a class of emerging drugs that have shown therapeutic potential in various hematological and solid cancers. We examined the cytotoxic effects of 5-azacitidine, 3-deazaneplanocin A, and suberanilohydroxamic acid (SAHA) on osteosarcoma cell lines HOS, MG-63, MNNG, and ZK-58. SAHA was the only chemical agent that exerted a strong, growth-limiting effect in all cell lines tested. The growth-limiting effect of SAHA was accompanied by features characteristic of apoptotic death. We found that cotreatment with SAHA and cisplatin showed strong synergism in all cell lines. The effect of cotreatment with SAHA and doxorubicin was cell line dependent. In the cell lines HOS, MG-63, and MNNG, the combined effect was synergistic, whereas in the cell line ZK-58, SAHA antagonized doxorubicin. The strong synergism of SAHA indicated that in combination with cisplatin, it might enable a promising add-on to current therapy regimens. However, considering the cell line-dependent effect that was found when SAHA was combined with doxorubicin, further experimentation is needed.