Bevacizumab, Irinotecan, or Topotecan Added to Temozolomide for Children With Relapsed and Refractory Neuroblastoma: Results of the ITCC-SIOPEN BEACON-Neuroblastoma Trial.

Purpose: Outcomes for children with relapsed and refractory high-risk neuroblastoma (RR-HRNB) remain dismal. The BEACON Neuroblastoma trial (EudraCT 2012-000072-42) evaluated three backbone chemotherapy regimens and the addition of the antiangiogenic agent bevacizumab (B).

Materials And Methods: Patients age 1-21 years with RR-HRNB with adequate organ function and performance status were randomly assigned in a 3 × 2 factorial design to temozolomide (T), irinotecan-temozolomide (IT), or topotecan-temozolomide (TTo) with or without B.

Exploiting the Stemness and Chemoresistance Transcriptome of Ewing Sarcoma to Identify Candidate Therapeutic Targets and Drug-Repurposing Candidates.

Outcomes for most patients with Ewing sarcoma (ES) have remained unchanged for the last 30 years, emphasising the need for more effective and tolerable treatments. We have hypothesised that using small-molecule inhibitors to kill the self-renewing chemotherapy-resistant cells (Ewing sarcoma cancer stem-like cells; ES-CSCs) responsible for progression and relapse could improve outcomes and minimise treatment-induced morbidities. For the first time, we demonstrate that ABCG1, a potential oncogene in some cancers, is highly expressed in ES-CSCs independently of CD133.

Membrane-type 1 matrix metalloproteinase as predictor of survival and candidate therapeutic target in Ewing sarcoma.

Background: Ewing sarcoma (ES) is the second most common primary bone malignancy, with an urgent need for new treatments. ES is associated with high rates of progression and relapse, driven by drug-resistant cells capable of migration, self-renewal and single-cell tumorigenesis, termed cancer stem-like cells (CSCs). Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteolytic enzyme, which, via direct and indirect mechanisms, digests four of the main types of collagen.

RNA sequencing and functional studies of patient-derived cells reveal that neurexin-1 and regulators of this pathway are associated with poor outcomes in Ewing sarcoma.

Purpose: The development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets.

Methods: Thirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods.

Multimodal analysis of cell-free DNA whole-genome sequencing for pediatric cancers with low mutational burden.

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.

Detection of Circulating and Disseminated Neuroblastoma Cells Using the ImageStream Flow Cytometer for Use as Predictive and Pharmacodynamic Biomarkers.

Purpose: Circulating tumor cells (CTCs) serve as noninvasive tumor biomarkers in many types of cancer. Our aim was to detect CTCs from patients with neuroblastoma for use as predictive and pharmacodynamic biomarkers.

Experimental Design: We collected matched blood and bone marrow samples from 40 patients with neuroblastoma to detect GD /CD45 neuroblastoma CTCs from blood and disseminated tumor cells (DTCs) from bone marrow using the Imagestream Imaging flow cytometer (ISx).

ABCG1 and Pgp identify drug resistant, self-renewing osteosarcoma cells.

Patients with osteosarcoma (OST) frequently relapse with drug resistant disease, consistent with the hypothesis that tumours contain a cancer stem-like cell (CSCs) population that survives chemotherapy to re-populate the tumour at local or metastatic sites. We describe a dual functional approach to isolate OST-CSCs and identify the ABC transporter proteins driving this population to reveal potential targets for the development of new treatments. OST-CSCs were isolated by selection in doxorubicin (OST-EC50 cells) and based on the ability to produce progeny from a single cell (HOS-EC50.

Macrophage-Derived IL1β and TNFα Regulate Arginine Metabolism in Neuroblastoma.

Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival.

Expression analysis of the MCPH1/BRIT1 and BRCA1 tumor suppressor genes and telomerase splice variants in epithelial ovarian cancer.

Aims: The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples.

Background: Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear.

Event-free survival of infants and toddlers enrolled in the HR-NBL-1/SIOPEN trial is associated with the level of neuroblastoma mRNAs at diagnosis.

Background: The purpose of this study was to evaluate whether levels of neuroblastoma mRNAs in bone marrow and peripheral blood from stage M infants (≤12 months of age at diagnosis, MYCN amplified) and toddlers (between 12 and 18 months, any MYCN status) predict event-free survival (EFS).

Methods: Bone marrow aspirates and peripheral blood samples from 97 infants/toddlers enrolled in the European High-Risk Neuroblastoma trial were collected at diagnosis in PAXgene blood RNA tubes. Samples were analyzed by reverse transcription quantitative polymerase chain reaction according to standardized procedures.

Neuroblastoma mRNAs predict outcome in children with stage 4 neuroblastoma: a European HR-NBL1/SIOPEN study.

Purpose: To evaluate the hypothesis that detection of neuroblastoma mRNAs by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) in peripheral blood (PB) and bone marrow aspirates (BM) from children with stage 4 neuroblastoma are clinically useful biomarkers of risk.

Methods: RTqPCR for paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), and doublecortin (DCX) mRNA in PB and BM of children enrolled onto the High-Risk Neuroblastoma Trial-1 of the European Society of Pediatric Oncology Neuroblastoma Group (HR-NBL1/SIOPEN) was performed at diagnosis and after induction therapy.

Results: High levels of TH, PHOX2B, or DCX mRNA in PB or BM at diagnosis strongly predicted for worse event-free survival (EFS) and overall survival (OS) in a cohort of 290 children.

Heterogeneous role of the glutathione antioxidant system in modulating the response of ESFT to fenretinide in normoxia and hypoxia.

Glutathione (GSH) is implicated in drug resistance mechanisms of several cancers and is a key regulator of cell death pathways within cells. We studied Ewing's sarcoma family of tumours (ESFT) cell lines and three mechanistically distinct anticancer agents (fenretinide, doxorubicin, and vincristine) to investigate whether the GSH antioxidant system is involved in the reduced sensitivity to these chemotherapeutic agents in hypoxia. Cell viability and death were assessed by the trypan blue exclusion assay and annexin V-PI staining, respectively.

Identification of reference microRNAs and suitability of archived hemopoietic samples for robust microRNA expression profiling.

In many cancers, including neuroblastoma, microRNA (miRNA) expression profiling of peripheral blood (PB) and bone marrow (BM) may increase understanding of the metastatic process and lead to the identification of clinically informative biomarkers. The quality of miRNAs in PB and BM samples archived in PAXgene™ blood RNA tubes from large-scale clinical studies and the identity of reference miRNAs for standard reporting of data are to date unknown. In this study, we evaluated the reliability of expression profiling of 377 miRNAs using quantitative polymerase chain reaction (qPCR) in PB and BM samples (n=90) stored at -80 °C for up to 5 years in PAXgene™ blood RNA tubes.

Oxidative stress and therapeutic opportunities: focus on the Ewing's sarcoma family of tumors.

Reactive oxygen species (ROS) are highly reactive by-products of energy production that can have detrimental as well as beneficial effects. Unchecked, high levels of ROS result in an imbalance of cellular redox state and oxidative stress. High levels of ROS have been detected in most cancers, where they promote tumor development and progression.

Gene expression profiling for discovery of novel markers of minimal disease.

Statement of clinical relevance: Disseminated disease is a predictor of poor outcome in children with neuroblastoma, and remains the most difficult challenge for successful patient management and cure. In many children at diagnosis this disease can be detected using current methods. However for a small percentage of children at diagnosis and for those on treatment and during follow up, more sensitive and specific methods for the detection of minimal disease are required.

p38(MAPK): stress responses from molecular mechanisms to therapeutics.

The p38(MAPK) protein kinases affect a variety of intracellular responses, with well-recognized roles in inflammation, cell-cycle regulation, cell death, development, differentiation, senescence and tumorigenesis. In this review, we examine the regulatory and effector components of this pathway, focusing on their emerging roles in biological processes involved in different pathologies. We summarize how this pathway has been exploited for the development of therapeutics and discuss the potential obstacles of targeting this promiscuous protein kinase pathway for the treatment of different diseases.

The promise of telomere length, telomerase activity and its regulation in the translocation-dependent cancer ESFT; clinical challenges and utility.

The Ewing's sarcoma family of tumours (ESFT) are diagnosed by EWS-ETS gene translocations. The resulting fusion proteins play a role in both the initiation and maintenance of these solid aggressive malignant tumours, suppressing cellular senescence and increasing cell proliferation and survival. EWS-ETS fusion proteins have altered transcriptional activity, inducing expression of a number of different target genes including telomerase.

Preclinical evaluation of vascular-disrupting agents in Ewing's sarcoma family of tumours.

The effects of the tubulin-binding vascular-disrupting agents (VDAs), combretastatin A4 phosphate (CA4P), OXi4503/CA1P and OXi8007, in subcutaneous mouse models of the Ewing's sarcoma family of tumours (ESFTs) have been investigated alone and in combination with doxorubicin. Delay in subcutaneous tumour growth was observed following treatment of mice with multiple doses of OXi4503/CA1P but not with CA4P or OXi8007. A single dose of OXi4503/CA1P caused complete shutdown of vasculature by 24h and extensive haemorrhagic necrosis by 48h.

Molecular abnormalities in Ewing's sarcoma.

Ewing's sarcoma is one of the few solid tumors for which the underlying molecular genetic abnormality has been described: rearrangement of the EWS gene on chromosome 22q12 with an ETS gene family member. These translocations define the Ewing's sarcoma family of tumors (ESFT) and provide a valuable tool for their accurate and unequivocal diagnosis. They also represent ideal targets for the development of tumor-specific therapeutics.

Manipulation of oxidative stress to induce cell death in Ewing's sarcoma family of tumours.

Ewing's sarcoma family of tumours (ESFT) are childhood cancers whose aggressive behaviour and propensity to relapse prompts the need for new treatment approaches. In this study, the role of cellular antioxidants in determining the sensitivity of ESFT cell lines to the cytotoxicity of the antineoplasic agent fenretinide was investigated with a view to identifying targets for the development of new treatment strategies. ESFT cell lines differentially express cellular antioxidants, although cellular glutathione (GSH) was identified as the major determinant of sensitivity to fenretinide.

BAY 11-7082 induces cell death through NF-kappaB-independent mechanisms in the Ewing's sarcoma family of tumours.

The role of NF-kappaB in the Ewing's sarcoma family of tumours (ESFT) and their response to fenretinide has been investigated. Basal levels of phosphorylated NF-kappaB were low in all ESFT cells. BAY 11-7082 decreased cell viability, which was accompanied by caspase-3 cleavage.

Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group.

Ewing's sarcoma family tumors (ESFT) are characterized by the presence of EWSR1-ETS fusion genes. Secondary chromosome changes are frequently described, although their clinical significance is not clear. In this study, we have collected and reviewed abnormal karyotypes from 88 patients with primary ESFT and a rearrangement of 22q12.

Standardisation of operating procedures for the detection of minimal disease by QRT-PCR in children with neuroblastoma: quality assurance on behalf of SIOPEN-R-NET.

The clinical utility of detecting minimal residual disease (MRD) in children with neuroblastoma (NB) by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) is not clear. This in part reflects the lack of uniform methodology for analysis and reporting. Reference laboratories across Europe have therefore established standard operating procedures (SOPs) for the detection of NB cells by QRT-PCR.