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. The primary end point was best overall response (complete or partial) rate (ORR) during the first six courses, by RECIST or International Neuroblastoma Response Criteria for patients with measurable or evaluable disease, respectively. Safety, progression-free survival (PFS), and overall survival (OS) time were secondary end points. RESULTS: One hundred sixty patients with RR-HRNB were included. For B random assignment (n = 160), the ORR was 26% (95% CI, 17 to 37) with B and 18% (95% CI, 10 to 28) without B (risk ratio [RR], 1.52 [95% CI, 0.83 to 2.77]; P = .17). Adjusted hazard ratio for PFS and OS were 0.89 (95% CI, 0.63 to 1.27) and 1.01 (95% CI, 0.70 to 1.45), respectively. For irinotecan ([I]; n = 121) and topotecan (n = 60) random assignments, RRs for ORR were 0.94 and 1.22, respectively. A potential interaction between I and B was identified. For patients in the bevacizumab-irinotecan-temozolomide (BIT) arm, the ORR was 23% (95% CI, 10 to 42), and the 1-year PFS estimate was 0.67 (95% CI, 0.47 to 0.80). CONCLUSION: The addition of B met protocol-defined success criteria for ORR and appeared to improve PFS. Within this phase II trial, BIT showed signals of antitumor activity with acceptable tolerability. Future trials will confirm these results in the chemoimmunotherapy era.

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. This can be hijacked in malignancy for invasion and metastasis, with high expression predicting decreased survival in multiple cancers. In this study, we have examined the hypothesis that MT1-MMP is expressed by ES cells and explored the relationship between expression and outcomes. PROCEDURE: MT1-MMP expression in ES established cell lines, primary patient-derived cultures and daughter ES-CSCs was characterised by RNA sequencing, Western blotting, immunocytochemistry and flow cytometry. Immunohistochemistry was used to detect MT1-MMP in tumour biopsies, and the relationship between expression, event-free and overall survival examined. RESULTS: MT1-MMP was detected at both RNA and protein levels in five of six established cell lines, all primary cultures (n = 25) and all daughter ES-CSCs (n = 7). Immunohistochemistry of treatment-naïve biopsy tissue demonstrated that high MT1-MMP expression predicted decreased event-free and overall survival (p = .017 and .036, respectively; n = 47); this was not significant in multivariate analysis. CONCLUSIONS: MT1-MMP is expressed by ES cells, including ES-CSCs, making it a candidate therapeutic target. The level of MT1-MMP expression at diagnosis may be considered as a prognostic biomarker if validated by retrospective analysis of a larger cohort of clinical trial samples.

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 GD2 +/CD45- neuroblastoma CTCs from blood and disseminated tumor cells (DTCs) from bone marrow using the Imagestream Imaging flow cytometer (ISx). In six cases, circulating free DNA (cfDNA) extracted from plasma isolated from the CTC sample was analyzed by high-density single-nucleotide polymorphism (SNP) arrays. RESULTS: CTCs were detected in 26 of 42 blood samples (1-264/mL) and DTCs in 25 of 35 bone marrow samples (57-291,544/mL). Higher numbers of CTCs in patients with newly diagnosed, high-risk neuroblastoma correlated with failure to obtain a complete bone marrow (BM) metastatic response after induction chemotherapy (P < 0.01). Ex vivo Nutlin-3 (MDM2 inhibitor) treatment of blood and BM increased p53 and p21 expression in CTCs and DTCs compared with DMSO controls. In five of six cases, cfDNA analyzed by SNP arrays revealed copy number abnormalities associated with neuroblastoma. CONCLUSIONS: This is the first study to show that CTCs and DTCs are detectable in neuroblastoma using the ISx, with concurrently extracted cfDNA used for copy number profiling, and may be useful as pharmacodynamic biomarkers in early-phase clinical trials. Further investigation is required to determine whether CTC numbers are predictive biomarkers of BM response to first-line induction chemotherapy.

Macrophage-Derived IL1ß 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 in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1ß and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1ß and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1ß and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. SIGNIFICANCE: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

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. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. METHODS: qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. RESULTS: The wild type α+/ß+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/ß- hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (p = 0.05) and primary cultures (p = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/ß+ hTERT (p = 0.04), and a strong positive association between MCPH1/BRIT1 and both α-/ß+ hTERT and α-/ß- hTERT (both p = 0.02). A positive association was observed between BRCA1 and α-/ß+ hTERT and α-/ß- hTERT expression (p = 0.003 and p = 0.04, respectively). CONCLUSIONS: These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/ß+).

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. RESULTS: Bone marrow tyrosine hydroxylase (TH) or paired-like homeobox 2b (PHOX2B) levels in the highest tertile were associated with worse EFS; hazard ratios, adjusted for age and MYCN status, were 1.5 and 1.8 respectively. Expression of both TH and PHOX2B in the highest tertile predicted worse outcome (p = 0.015), and identified 20 (23%) infants/toddlers with 5-year EFS of 20% (95%CI: 4%-44%). Prognostic significance was maintained after adjusting for over-fitting bias (p = 0.038), age and MYCN status. In peripheral blood, PHOX2B levels in the highest tertile predicted a two-fold increased risk of an event (p = 0.032), and identified 23 (34%) infants/toddlers with 5-year EFS of 29% (95%CI: 12%-48%). Time-dependent receiver operating characteristic analysis confirmed the prognostic value of combined TH and PHOX2B in bone marrow and of PHOX2B in peripheral blood during the first year of follow-up. CONCLUSIONS: High levels of bone marrow TH and PHOX2B and of peripheral blood PHOX2B at diagnosis allow early identification of a group of high-risk infant and toddlers with neuroblastoma who may be candidates for alternative treatments. Integration with additional biomarkers, as well as validation in additional international trials is warranted.

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

BACKGROUND: The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. METHODS: A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. RESULTS: To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. CONCLUSIONS: The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society.

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. After induction therapy, high levels of these mRNAs predicted worse EFS and OS in BM but not in PB. Combinations of mRNAs in BM did not add to the predictive power of any single mRNA. However, in the original (n = 182) and validation (n = 137) PB cohorts, high TH (log10TH > 0.8) or high PHOX2B (log10PHOX2B > 0.28) identify 19% of children as ultrahigh risk, with 5-year EFS and OS rates of 0%; OS rate was 25% (95% CI, 16% to 36%) and EFS rate was 38% (95% CI, 28% to 49%) in the remaining children. The magnitude of reduction in mRNA level between diagnosis and postinduction therapy in BM or PB was not of additional predictive value. CONCLUSION: High levels of TH and PHOX2B mRNA in PB at diagnosis objectively identify children with ultrahigh-risk disease who may benefit from novel treatment approaches. The level of TH, PHOX2B, and DCX mRNA in BM and/or PB at diagnosis might contribute to an algorithm to improve stratification of children for treatment.

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. There was no correlation with storage time and variation of expression for any single miRNA (r < 0.50). The profile of miRNAs isolated as small RNAs or co-isolated with small/large RNAs was highly correlated (r=0.96). The mean expression of all miRNAs and the geNorm program identified miR-26a, miR-28-5p, and miR-24 as the most stable reference miRNAs. This study describes detailed methodologies for reliable miRNA isolation and profiling of PB and BM, including reference miRNAs for qPCR normalization, and demonstrates the suitability of clinical samples archived at -80 °C into PAXgene™ blood RNA tubes for miRNA expression studies.

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. Hypoxia significantly decreased the sensitivity of all ESFT cell lines to fenretinide-induced death, whereas the effect of doxorubicin or vincristine was marginal and cell-line-specific. The response of the GSH antioxidant system in ESFT cell lines to hypoxia was variable and also cell-line-specific, although the level of GSH appeared to be most dependent on de novo biosynthesis rather than recycling. RNAi-mediated knockdown of key GSH regulatory enzymes γ-glutamylcysteine synthetase or glutathione disulfide reductase partially reversed the hypoxia-induced resistance to fenretinide, and increasing GSH levels using N-acetylcysteine augmented the hypoxia-induced resistance in a cell line-specific manner. These observations are consistent with the conclusion that the role of the GSH antioxidant system in modulating the sensitivity of ESFT cells to fenretinide is heterogeneous depending on environment and cell type. This is likely to limit the value of targeting GSH as a therapeutic strategy to overcome hypoxia-induced drug resistance in ESFT. Whether targeting the GSH antioxidant system in conjunction with other therapeutics may benefit some patients with ESFT remains to be seen.

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. Many anticancer agents work by further increasing cellular levels of ROS, to overcome the antioxidant detoxification capacity of the cancer cell and induce cell death. However, adaptation of the level of cellular antioxidants can lead to drug resistance. The challenge for the design of effective cancer therapeutics exploiting oxidative stress is to tip the cellular redox balance to induce ROS-dependent cell death but without increasing the antioxidant activity of the cancer cell or inducing toxicity in normal cells.

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. Furthermore, we discuss how the p38(MAPK) pathway might be best exploited for the development of more effective therapeutics with minimal side effects in a range of specific disease settings.

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. Up-regulation of hTERT is most likely responsible for the high levels of telomerase activity in primary ESFT, although telomerase activity and expression of hTERT are not predictive of outcome. However levels of telomerase activity in peripheral blood may be useful to monitor response to some therapeutics. Despite high levels of telomerase activity, telomeres in ESFT are frequently shorter than those of matched normal cells. Uncertainty about the role that telomerase and regulators of its activity play in the maintenance of telomere length in normal and cancer cells, and lack of studies examining the relationship between telomerase activity, regulators of its activity and their clinical significance in patient samples have limited their introduction into clinical practice. Studies in clinical samples using standardised assays are critical to establish how telomerase and regulators of its activity might best be exploited for patient benefit.

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. However, a viable rim of proliferating cells remained, which repopulated the tumour within 10 days following the withdrawal of treatment. Combined treatment with doxorubicin 1h prior to administration of OXi4503/CA1P enhanced the effects of OXi4503/CA1P causing a synergistic delay in tumour growth (p<0.001). This study demonstrates that OXi4503/CA1P is a potent VDA in ESFT and in combination with conventional cytotoxic agents represents a promising treatment strategy for this tumour group.

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. The importance of GSH in ESFT physiology was demonstrated by the depletion of intracellular GSH using l-buthionine (S,R) sulphoximine (BSO), which decreased cell viability. Furthermore, pre-treatment of ESFT cells with BSO sensitised them to fenretinide-induced death. Overall, these results demonstrate that ESFT cells are sensitive to changes in intracellular redox environment, and that targeting specific cellular antioxidants might be a viable strategy in treating ESFT.

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. This was independent of the increase in reactive oxygen species, p38(MAPK) phosphorylation and expression of NF-kappaB target proteins. NF-kappaB knockdown did not induce death under normal growth conditions, but did reduce TNFalpha-dependent cell survival. Fenretinide-induced apoptosis was independent of NF-kappaB. BAY 11-7082-induced cell death through an NF-kappaB-independent mechanism and enhanced cell death when combined with fenretinide.

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. Secondary changes were identified in 80% (70/88) of tumors at diagnosis. Multivariate analysis showed a worse overall and relapse free survival (RFS) for those with a complex karyotype (overall survival, P = 0.005; RFS, P = 0.04), independent of metastatic disease. Univariate survival analysis showed that a chromosome number above 50 or a complex karyotype was associated with a worse overall survival (>50 chromosomes, P = 0.05; complex karyotype, P = 0.04). There was no association between type of cytogenetic abnormality and the presence of metastatic disease at diagnosis. Univariate and multivariate survival analysis of a small subgroup with trisomy 20 indicated that trisomy 20 was associated with a worse overall and RFS. There was no difference in outcome associated with other recurrent trisomies (2, 5, 7, 8, or 12) or the common recurrent secondary structural rearrangements (deletions of 1p36, 9p12, 17p13, and 16q, and gain of 1q), although numbers were small. These data demonstrate the continued value of cytogenetics as a genome-wide screen in ESFT and illustrates the potential importance of secondary chromosome changes for stratification of patients for risk. Specifically, karyotype complexity appears to be a powerful predictor of prognosis, and the presence of trisomy 20 may be a marker of a more aggressive subset of this group.

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. Haemopoietic samples are collected into PAXgene blood RNA tubes, which stabilise mRNA for 48 h at room temperature and more than 6 months at -80 degrees C. Tyrosine hydroxylase (TH) was selected as the target for NB cell detection, expression is normalised to beta2-microglobulin and reported using the DeltaDeltaCt method. The sensitivity of QRT-PCR increased from 58% to 90% following the development of SOPs. A robust, transferable, objective method for the detection of NB cells by QRT-PCR has been defined to improve the power and consistency of studies on MRD in children with NB.