High-risk Neuroblastoma Patients Research Articles

Iron-Chelation Treatment by Novel Thiosemicarbazone Targets Major Signaling Pathways in Neuroblastoma.

Despite constant advances in the field of pediatric oncology, the survival rate of high-risk neuroblastoma patients remains poor. The molecular and genetic features of neuroblastoma, such as MYCN amplification and stemness status, have established themselves not only as potent prognostic and predictive factors but also as intriguing targets for personalized therapy. Novel thiosemicarbazones target both total level and activity of a number of proteins involved in some of the most important signaling pathways in neuroblastoma. In this study, we found that di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) potently decreases N-MYC in MYCN-amplified and c-MYC in MYCN-nonamplified neuroblastoma cell lines. Furthermore, DpC succeeded in downregulating total EGFR and phosphorylation of its most prominent tyrosine residues through the involvement of NDRG1, a positive prognostic marker in neuroblastoma, which was markedly upregulated after thiosemicarbazone treatment. These findings could provide useful knowledge for the treatment of MYC-driven neuroblastomas that are unresponsive to conventional therapies.

Autologous or allogeneic hematopoietic stem cells transplantation combined with high-dose chemotherapy for refractory neuroblastoma: A protocol for systematic review and meta-analysis.

Background:Neuroblastoma is a common solid malignant tumor in children. Despite the development of new treatment options, the prognosis of high-risk neuroblastoma patients is still poor. High-dose chemotherapy and hematopoietic stem cell (HSC) transplantation might improve survival of patients with refractory neuroblastoma. In this study, we aimed to summarize the efficacy of autologous or allogeneic HSC transplantation combined with high-dose chemotherapy for patients with refractory neuroblastoma through the meta-analysis.Methods and analysis:Relevant clinical trials of autologous or allogeneic HSC transplantation for the treatment refractory neuroblastoma patients will be searched in Web of Science, Cochrane Library, PubMed, Google Scholar, Embase, Medline, China National Knowledge Infrastructure, China Scientific Journal Database, Chinese Biomedical Literature Database and Wanfang Database from their inception to December 2020. Two researchers will perform data extraction and risk of bias assessment independently. The clinical outcomes including tumor response, overall survival, event-free survival (EFS), quality of life (QoL) and adverse events, were systematically evaluated by using Review Manager 5.3 and Stata 14.0 statistical software.Results:The results of this study will provide high-quality evidence for the effect of autologous or allogeneic HSC transplantation combined with high-dose chemotherapy on tumor response, survival, and QoL in patients with refractory neuroblastoma.Conclusions:The conclusions of this meta-analysis will be published in a peer-reviewed journal, and provide more evidence-based guidance in clinical practice.

Hemorrhage During Induction Chemotherapy in Neuroblastoma: Additional Risk Factors in High-Risk Patients.

Background: Neuroblastoma is the most common solid extracranial tumor in children. Patients affected by neuroblastoma are stratified into low, intermediate, and high risk in terms of event-free and overall survival. Some high-risk patients have an additional risk of acute hemorrhagic complications during induction chemotherapy.Aim: To find easily and rapidly assessed parameters that help clinicians identify those patients affected by high-risk neuroblastoma who have an additional risk of hemorrhagic complications.Methods: The clinical notes of patients diagnosed with high-risk neuroblastoma from January 2013 until February 2021 were retrospectively reviewed. Clinical, demographic and laboratory data, biological characteristics of the tumor, and information about treatment and hospital stay were identified.Results: In the examined period, 44 patients were diagnosed with high-risk neuroblastoma. Four of these patients had hemorrhagic complications within 2–7 days after the initiation of induction chemotherapy; two patients had hemothorax, one patient had hemoperitoneum and one patient had hemothorax and hemoperitoneum. The patient with isolated hemoperitoneum was treated with blood components transfusions, clotting factors and colloids infusions; the three patients with hemothorax underwent thoracostomy tube placement and respiratory support. At initial presentation, patients who suffered from hemorrhagic complications had a higher degree of hypertension (stage 2, p = 0.0003), higher levels of LDH (median 3,745 U/L, p = 0.009) and lower levels of hemoglobin (mean 7.6 gr/dl, p = 0.0007) compared to other high-risk patients.Conclusions: A subgroup of “additional” high-risk patients can be identified within the high-risk neuroblastoma patients based on mean arterial pressure, LDH levels and hemoglobin levels at presentation. Further studies to define cut-off values and optimal management strategies for these patients are needed.

Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis.

The inflammatory factor IL6 secreted by bone marrow mesenchymal stem cells (BMSCs) in the tumor microenvironment (TME) facilitates the survival and therapeutic resistance of neuroblastoma (NB). Here, we found that IL6 expression in primary tumor tissues or bone marrow (BM) metastases was closely associated with the disease risk and prognosis of NB patients. IL6 secretion from immortalized BMSC (iBMSC) was directly regulated by NB cells and is involved in promoting the proliferation and metastasis of NB cells. Beta-Lapachone (ARQ-501, LPC), an ortho-naphthoquinone natural product, significantly prevented the iBMSC-induced malignant transformation effect on NB cells through suppressing the expression and secretion of IL6 from iBMSC in vitro and in vivo. Mechanistically, LPC disrupted the crosstalk between NB cells and iBMSC in an NQO1-dependent manner through blocking the Gal-3/Gal-3BP/IL6 axis. Our results reveal the effect of iBMSC-derived IL6 on TME-induced malignant transformation of NB cells, and provide theoretical basis for the clinical application of LPC as a potential IL6 inhibitor in high-risk refractory NB patients.

Loss of p53 suppresses replication stress-induced DNA damage in ATRX-deficient neuroblastoma

Genetic aberrations are present in the ATRX gene in older high-risk neuroblastoma (NB) patients with very poor clinical outcomes. Its loss-of-function (LoF) facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells and is strongly linked to replication stress (RS) and DNA damage through G-quadruplex (G4) DNA secondary structures. However, limited information is available on ATRX alteration-related NB tumorigenesis. We herein knocked out (KO) ATRX in MYCN-amplified (NGP) and MYCN single copy (SK-N-AS) NB cells with wild-type (wt) and truncated TP53 at the C terminus, respectively, using CRISPR/Cas9 technologies. The loss of ATRX increased DNA damage and G4 formation related to RS in TP53 wt isogenic ATRX KO NGP cells, but not in SK-N-AS clones. A gene set enrichment analysis (GSEA) showed that the gene sets related to DNA double-strand break repair, negative cell cycle regulation, the G2M checkpoint, and p53 pathway activation were enriched in NGP clones. The accumulation of DNA damage activated the ATM/CHK2/p53 pathway, leading to cell cycle arrest in NGP clones. Interestingly, ATRX loss did not induce RS related to DNA damage response (DDR) in TP53-truncated SK-N-AS cells. p53 inactivation abrogated cell cycle arrest and reduced G4 accumulation in NGP clones. The loss of p53 also induced G4 DNA helicases or Fanconi anemia group D2 protein (FANCD2) with ATRX deficiency, suggesting that ATRX maintained genome integrity and p53 deficiency attenuated RS-induced DNA damage in NB cells featuring inactivated ATRX by regulating DNA repair mechanisms and replication fork stability.

The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

Simple SummaryA clinical test for alternative-lengthening-of-telomeres (ALT) could assist with cancer diagnosis and monitoring of disease progression. ALT-targeted anticancer treatments are being developed; however, there is no appropriate companion ALT diagnostic. The C-Circle biomarker is the only known ALT specific molecule and the C-Circle Assay the only quantitative ALT assay that is amenable to clinical use. We show here that C-Circles are secreted by ALT+ cancer cell lines inside the exosomes and are protected from nucleases. We also show that secreted C-Circles, like intracellular C-Circles, are an ALT-specific biomarker, and in high-risk neuroblastoma, the blood-based C-Circle Assay has the potential to be an accurate diagnostic for ALT cancer activity. Therefore, the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and, potentially, a clinical diagnostic for ALT activity, which is required for the development of ALT-targeted therapies as well as for the diagnosis and monitoring of ALT+ cancer.C-Circles, self-primed telomeric C-strand templates for rolling circle amplification, are the only known alternative-lengthening-of-telomeres (ALT)-specific molecule. However, little is known about the biology of C-Circles and if they may be clinically useful. Here we show that C-Circles are secreted by ALT+ cancer cells inside exosomes, and that a blood-based C-Circle Assay (CCA) can provide an accurate diagnostic for ALT activity. Extracellular vesicles were isolated by differential centrifugation from the growth media of lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma, and soft tissue sarcoma cell lines, and C-Circles were detected in the exosome fraction from all eleven ALT+ cancer cell lines and not in any extracellular fraction from the eight matching telomerase positive cancer cell lines or the normal fibroblast strain. The existence of C-Circles in ALT+ exosomes was confirmed with exosomes isolated by iodixanol gradient separation and CD81-immunoprecipitation, and C-Circles in the exosomes were protected from nucleases. On average, 0.4% of the total ALT+ intracellular C-Circles were secreted in the exosomes every 24 h. Comparing the serum-based and tumor-based CCAs in 35 high risk neuroblastoma patients divided randomly into ALT+ threshold derivation and validation groups, we found the serum-based CCA to have 100% sensitivity (6/6), 70% specificity (7/10), and 81% concordance (13/16). We conclude that the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and a potential clinical diagnostic for ALT activity.

Immunotherapy by anti-GD2 antibodies in patients with primary high-risk neuroblastoma, primary resistant and relapsed disease: Raisa Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, First Pavlov State Medical University of St. Petersburg experience

Introduction. The long-term event-free survival of patients with high-risk neuroblastoma (NB) receiving intensive complex therapy according to current russian standard do not exceed 40 %. Also, there is no standard tactics in patients with primary resistant and relapsed disease, most of them die due to disease progression. While, anti-GD2 immunotherapy (IT) proved to be effective in patients with high-risk NB, in Russian Federation this method is not generally available. There are currently two pilot studies ongoing in Raisa Gorbacheva Memorial Institute aimed to evaluate the effectiveness of anti-GD2 antibodies in high-risk NB patients.Aim of the study – describing a single-center experience of anti-GD2 IT in primary high-risk NB patients and patients with primary resistant and relapsed disease.Materials and methods. A total of 20 patients received anti-GD2 antibodies, 16 of them were included into pilot trials. The median age at IT initiation was 5 (3–17) years. In 13 cases the therapy was initiated in patients with high-risk disease after auto-HSCT, in 3 cases – in patients with 1st systemic relapse of primary resistant disease after 2nd-line therapy and haplo-HSCT, in 1 case – in patient with 2nd chemosensitive relapse after haplo-HSCT. Also, 3 patients with progressive chemoresistant disease received anti-GD2 antibodies as monotherapy (n = 1) or in combination with chemotherapy (n = 2) as salvage regimen.Results. Patients receiving anti-GD2 antibodies after auto-HSCT retain response to therapy in 11 of 13 cases with a median follow-up period of 15 (6–27) months, in 2 cases there was disease progression during or immediately after IT cessation. Both patients with disease progression responded well to salvage therapy. Two of 3 haplo-HSCT recipients with prior good response to 2nd-line therapy are currently in remission 16 and 36 months past haplo-HSCT, one patient progressed 55 months after transplantation. A patient with 2nd late relapse after haplo-HSCT currently maintains remission on IT. Both patients with chemorefractory progressive disease did not respond to IT and died due to disease progression. IT was characterized by acceptable toxicity. In most cases it was complicated by Gr 1–2 fever, rash or neuropathic pain effectively controlled by supportive therapy. However, three patients had signs of neurotoxicity requiring therapy termination in one case.Conclusion. Dinutuximab beta IT is characterized by acceptable toxicity. With a median follow-up of 18 (6–59) months the majority (14 of 17) patients receiving anti-GD2 antibodies as maintenance therapy after auto- or allogeneic HSCT retain response. However, we did not observe any response in patients with progressive chemorefractory disease.

Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis

BackgroundGraft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products.MethodsNeuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture.ResultsPDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft.ConclusionThese results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.

MMP-9 reinforces radiation-induced delayed invasion and metastasis of neuroblastoma cells through second-signaling positive feedback with NFκB via both ERK and IKK activation.

Neuroblastoma (NB) progression is branded with hematogenous metastasis and frequent relapses. Despite intensive multimodal clinical therapy, outcomes for patients with progressive disease remain poor, with negligible long-term survival. Therefore, understanding the acquired molecular rearrangements in NB cells with therapy pressure and developing improved therapeutic strategies is a critical need to improve the outcomes for high-risk NB patients. We investigated the rearrangement of MMP9 in NB with therapy pressure, and unveiled the signaling that facilitates NB evolution. Radiation-treatment (RT) significantly increased MMP9 expression/activity, and the induced enzyme activity was persistently maintained across NB cell lines. Furthermore, RT-triggered NFκB transcriptional activity and this RT-induced NFκB were required/adequate for MMP9 maintenance. RT-triggered NFκB-dependent MMP9 actuated a second-signaling feedback to NFκB, facilitating a NFκB-MMP9-NFκB positive feedback cycle (PFC). Critically, MMP9-NFκB feedback is mediated by MMP9-dependent activation of IKKβ and ERK phosphotransferase activity. Beyond its tumor invasion/metastasis function, PFC-dependent MMP9 lessens RT-induced apoptosis and favors survival pathway through the activation of NFκB signaling. In addition, PFC-dependent MMP9 regulates 19 critical molecular determinants that play a pivotal role in tumor evolution. Interestingly, seven of 19 genes possess NFκB-binding sites, demonstrating that MMP9 regulates these molecules by activating NFκB. Collectively, these data suggest that RT-triggered NFκB-dependent MMP9 actuates feedback to NFκB though IKKβ- and ERK1/2-dependent IκBα phosphorylation. This RT-triggered PFC prompts MMP9-dependent survival advantage, tumor growth, and dissemination. Targeting therapy-pressure-driven PFC and/or selective inhibition of MMP9 maintenance could serve as promising therapeutic strategies for treatment of progressive NB.

Anti-GD2 Based Immunotherapy Prevents Late Events in High-Risk Neuroblastoma Patients over 18 Months at Diagnosis.

Simple SummaryHigh-risk neuroblastoma accounts for 4% of newly diagnosed pediatric malignancies, but for 9–10% of pediatric cancer mortality. To reduce the number of (late) recurrences and subsequently improve survival, anti-GD2 monoclonal antibody based immunotherapy has been added to the maintenance phase of treatment. The first randomized study (ANBL0032) was ground breaking, showing a 20% improved event free survival. Subsequently immunotherapy was included in all international high-risk treatment regimens. Randomization will never be repeated. In this article we present additional data from our retrospective cohort to corroborate the ANBL0032 study. Our cohort contains 84 Dutch high-risk neuroblastoma patients. They were treated with GPOH or POG induction, followed by immunotherapy according to original ANBL0032 protocol (immunotherapy group) or single-agent isotretinoin (historical control group). In the complete cohort, 5 year OS was 64 ± 7% and 49 ± 8% for the immunotherapy group and the control group, respectively (p = 0.16). Five year EFS was 57 ± 7% and 41 ± 8%, respectively (p = 0.16). In the subgroup of patients ≥ 18 months, 5-yr OS was 63 ± 8% and 39 ± 9, respectively (p = 0.04) and EFS 54 ± 8% and 29 ± 8%, respectively (p = 0.05). Our five year data suggest a role for the immunotherapy in preventing late events, especially in patients ≥ 18 months old.Background: Anti-GD2 based immunotherapy has improved overall (OS) and event free survival (EFS) for high-risk neuroblastoma (HR-NBL) patients. Here, we evaluate the long-term efficacy of anti-GD2 immunotherapy in combination with isotretinoin, GM-CSF, and IL-2. Methods: Dutch HR-NBL patients treated with immunotherapy according to the COG-ANBL0032 protocol (n = 47) were included and compared to historical controls (n = 37) treated with single-agent isotretinoin maintenance therapy. Survival time was calculated from start of the maintenance therapy. Results: The study and control group were similar concerning baseline characteristics. In the complete cohort, 5 year OS was 64 ± 7% and 49 ± 8% for the immunotherapy group and the control group, respectively (p = 0.16). Five year EFS was 57 ± 7% and 41 ± 8%, respectively (p = 0.16). In the subgroup of patients ≥ 18 months, 5-yr OS was 63 ± 8% and 39 ± 9, respectively (p = 0.04) and EFS 54 ± 8% and 29 ± 8%, respectively (p = 0.05). Landmark analysis for EFS with landmark point at 6 months after start of maintenance suggests a larger effect on the prevention of late than early events. Conclusions: This study is the first to confirm the results of the COG-ANBL0032 study in a cohort treated with a different induction regimen. Anti-GD2 immunotherapy prevents late events, most significantly in patients older than 18 months of age at diagnosis.

MEIS2 Is an Adrenergic Core Regulatory Transcription Factor Involved in Early Initiation of TH-MYCN-Driven Neuroblastoma Formation.

Simple SummaryNeuroblastoma is a pediatric tumor originating from the sympathetic nervous system responsible for 10–15% of all childhood cancer deaths. Half of all neuroblastoma patients present with high-risk disease, of which nearly 50% relapse and die of their disease. In addition, standard therapies cause serious lifelong side effects and increased risk for secondary tumors. Further research is crucial to better understand the molecular basis of neuroblastomas and to identify novel druggable targets. Neuroblastoma tumorigenesis has to this end been modeled in both mice and zebrafish. Here, we present a detailed dissection of the gene expression patterns that underlie tumor formation in the murine TH-MYCN-driven neuroblastoma model. We identified key factors that are putatively important for neuroblastoma tumor initiation versus tumor progression, pinpointed crucial regulators of the observed expression patterns during neuroblastoma development and scrutinized which factors could be innovative and vulnerable nodes for therapeutic intervention.Roughly half of all high-risk neuroblastoma patients present with MYCN amplification. The molecular consequences of MYCN overexpression in this aggressive pediatric tumor have been studied for decades, but thus far, our understanding of the early initiating steps of MYCN-driven tumor formation is still enigmatic. We performed a detailed transcriptome landscaping during murine TH-MYCN-driven neuroblastoma tumor formation at different time points. The neuroblastoma dependency factor MEIS2, together with ASCL1, was identified as a candidate tumor-initiating factor and shown to be a novel core regulatory circuit member in adrenergic neuroblastomas. Of further interest, we found a KEOPS complex member (gm6890), implicated in homologous double-strand break repair and telomere maintenance, to be strongly upregulated during tumor formation, as well as the checkpoint adaptor Claspin (CLSPN) and three chromosome 17q loci CBX2, GJC1 and LIMD2. Finally, cross-species master regulator analysis identified FOXM1, together with additional hubs controlling transcriptome profiles of MYCN-driven neuroblastoma. In conclusion, time-resolved transcriptome analysis of early hyperplastic lesions and full-blown MYCN-driven neuroblastomas yielded novel components implicated in both tumor initiation and maintenance, providing putative novel drug targets for MYCN-driven neuroblastoma.

Unraveling the extracellular matrix-tumor cell interactions to aid better targeted therapies for neuroblastoma

Treatment in children with high-risk neuroblastoma remains largely unsuccessful due to the development of metastases and drug resistance. The biological complexity of these tumors and their microenvironment represent one of the many challenges to face. Matrix glycoproteins such as vitronectin act as bridge elements between extracellular matrix and tumor cells and can promote tumor cell spreading. In this study, we established through a clinical cohort and preclinical models that the interaction of vitronectin and its ligands, such as αv integrins, are related to the stiffness of the extracellular matrix in high-risk neuroblastoma. These marked alterations found in the matrix led us to specifically target tumor cells within these altered matrices by employing nanomedicine and combination therapy. Loading the conventional cytotoxic drug etoposide into nanoparticles significantly increased its efficacy in neuroblastoma cells. We noted high synergy between etoposide and cilengitide, a high-affinity cyclic pentapeptide αv integrin antagonist. The results of this study highlight the need to characterize cell-extracellular matrix interactions, to improve patient care in high-risk neuroblastoma.

The embryonic trunk neural crest microenvironment regulates the plasticity and invasion of human neuroblastoma via TrkB signaling

Mistakes in trunk neural crest (NC) cell migration may lead to birth defects of the sympathetic nervous system (SNS) and neuroblastoma (NB) cancer. Receptor tyrosine kinase B (TrkB) and its ligand BDNF critically regulate NC cell migration during normal SNS development and elevated expression of TrkB is correlated with high-risk NB patients. However, in the absence of a model with in vivo interrogation of human NB cell and gene expression dynamics, the mechanistic role of TrkB in NB disease progression remains unclear. Here, we study the functional relationship between TrkB, cell invasion and plasticity of human NB cells by taking advantage of our validated in vivo chick embryo transplant model. We find that LAN5 (high TrkB) and SHSY5Y (moderate TrkB) human NB cells aggressively invade host embryos and populate typical NC targets, however loss of TrkB function significantly reduces cell invasion. In contrast, NB1643 (low TrkB) cells remain near the transplant site, but over-expression of TrkB leads to significant cell invasion. Invasive NB cells show enhanced expression of genes indicative of the most invasive host NC cells. In contrast, transplanted human NB cells down-regulate known NB tumor initiating and stem cell markers. Human NB cells that remain within the dorsal neural tube transplant also show enhanced expression of cell differentiation genes, resulting in an improved disease outcome as predicted by a computational algorithm. These in vivo data support TrkB as an important biomarker and target to control NB aggressiveness and identify the chick embryonic trunk neural crest microenvironment as a source of signals to drive NB to a less aggressive state, likely acting at the dorsal neural tube.

Dinutuximab (Unituxin)

CADTH reimbursement reviews are comprehensive assessments of the clinical effectiveness and cost-effectiveness, as well as patient and clinician perspectives, of a drug or drug class. The assessments inform non-binding recommendations that help guide the reimbursement decisions of Canada's federal, provincial, and territorial governments, with the exception of Quebec. This review assesses dinutuximab (Unituxin) 17.5 mg/m2 per day, administered as an IV infusion over 10 hours to 20 hours for 4 consecutive days per treatment cycle. Indication: For the treatment of high-risk neuroblastoma patients in their first relapse or determination of refractory disease in combination with irinotecan, temozolomide, and granulocyte-macrophage colony-stimulating factor.

Association of BRAF V600E mutations with vasoactive intestinal peptide syndrome in MYCN-amplified neuroblastoma.

Very rarely, vasoactive intestinal peptide-related diarrhea (VIP-D) is observed in patients with high-risk neuroblastoma (HR-NB) where the associated fluid and electrolyte abnormalities can pose a major clinical challenge for administering the required aggressive multimodality treatment. Two patients with HR-NB developed VIP-D during induction and were found to have a somatic BRAF V600E mutation. Serum VIP levels and diarrhea promptly resolved in both patients after initiating treatment with BRAF and MEK inhibitors. This illustrates an association of VIP-D with BRAF V600E mutations and demonstrates a therapeutic strategy in the specific context of VIP-D and BRAF V600E mutations in HR-NB patients. The addition of BRAF and MEK inhibitors allows continued conventional tumor-directed treatment by decreasing the severity of symptoms caused by this life-threatening complication.

Analysis of SARS-CoV-2 infection associated cell entry proteins ACE2, CD147, PPIA, and PPIB in datasets from non SARS-CoV-2 infected neuroblastoma patients, as potential prognostic and infection biomarkers in neuroblastoma

SARS-CoV-2 viral contagion has given rise to a worldwide pandemic. Although most children experience minor symptoms from SARS-CoV-2 infection, some have severe complications including Multisystem Inflammatory Syndrome in Children. Neuroblastoma patients may be at higher risk of severe infection as treatment requires immunocompromising chemotherapy and SARS-CoV-2 has demonstrated tropism for nervous cells. To date, there is no sufficient epidemiological data on neuroblastoma patients with SARS-CoV-2. Therefore, we evaluated datasets of non-SARS-CoV-2 infected neuroblastoma patients to assess for key genes involved with SARS-CoV-2 infection as possible neuroblastoma prognostic and infection biomarkers. We hypothesized that ACE2, CD147, PPIA and PPIB, which are associated with viral-cell entry, are potential biomarkers for poor prognosis neuroblastoma and SARS-CoV-2 infection.We have analysed three publicly available neuroblastoma gene expression datasets to understand the specific molecular susceptibilities that high-risk neuroblastoma patients have to the virus. Gene Expression Omnibus (GEO) GSE49711 and GEO GSE62564 are the microarray and RNA-Seq data, respectively, from 498 neuroblastoma samples published as part of the Sequencing Quality Control initiative. TARGET, contains microarray data from 249 samples and is part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. ACE2, CD147, PPIA and PPIB were identified through their involvement in both SARS-CoV-2 infection and cancer pathogenesis.In-depth statistical analysis using Kaplan-Meier, differential gene expression, and Cox multivariate regression analysis, demonstrated that overexpression of ACE2, CD147, PPIA and PPIB is significantly associated with poor-prognosis neuroblastoma samples. These results were seen in the presence of amplified MYCN, unfavourable tumour histology and in patients older than 18 months of age. Previously, we have shown that high levels of the nerve growth factor receptor NTRK1 together with low levels of the phosphatase PTPN6 and TP53 are associated with increased relapse-free survival of neuroblastoma patients. Interestingly, low levels of expression of ACE2, CD147, PPIA and PPIB are associated with this NTRK1-PTPN6-TP53 module, suggesting that low expression levels of these genes are associated with good prognosis. These findings have implications for clinical care and therapeutic treatment. The upregulation of ACE2, CD147, PPIA and PPIB in poor-prognosis neuroblastoma samples suggests that these patients may be at higher risk of severe SARS-CoV-2 infection. Importantly, our findings reveal ACE2, CD147, PPIA and PPIB as potential biomarkers and therapeutic targets for neuroblastoma.

Timing of adjuvant chemotherapy after laparotomy for Wilms tumor and neuroblastoma.

To describe the timing of chemotherapy initiation after surgery for Wilms tumor (WT) and neuroblastoma within a dedicated children's cancer center. A single-institution retrospective cohort study identified patients that underwent resection of unilateral WT or high-risk neuroblastoma and received adjuvant chemotherapy treatment. Adjuvant chemotherapy initiation and postoperative complications were recorded. Among 47 WT patients, the median time to chemotherapy initiation was 11days [interquartile range IQR 7-14]. 3 WT patients had post-operative complications, but all preceded chemotherapy. Among 83 patients treated for high-risk neuroblastoma, the median time to chemotherapy was 11days [IQR 9-14]. High-risk neuroblastoma patients with 30-day postoperative complications had a significantly longer time to initiation of adjuvant chemotherapy (odds ratio 1.13; p = 0.008). Many of these complications preceded and delayed the initiation of post-operative chemotherapy. No complications occurred in the group of 12 (25%) WT patients or 16 (19.3%) neuroblastoma patients who started chemotherapy ≤ 7days after surgery. There is no association between early initiation of adjuvant chemotherapy and post-operative complications including wound healing. Early initiation of chemotherapy (≤ 7days) is feasible in unilateral WT or high-risk neuroblastoma patients who are otherwise doing well without resulting in a preponderance of wound healing complications.

Oral Metronomic Maintenance Therapy Can Improve Survival in High-Risk Neuroblastoma Patients Not Treated with ASCT or Anti-GD2 Antibodies.

Simple SummaryLow-dose metronomic chemotherapy has anti-angiogenic activity and inhibits tumor growth. Therefore, we investigated the benefits of low-dose metronomic maintenance therapy (MT) in high-risk neuroblastoma (NB) patients who are unable to undergo autologous stem cell transplantation (ASCT) or anti-GD2 antibody therapy. A total of 217 high-risk NB patients were enrolled. One hundred and eighty-five (85%) had a complete/very good partial remission/partial remission (CR/VGPR/PR) to treatment, of them, 167 patients with stage 4, that did or did not receive oral metronomic MT, 3 years of event-free survival (EFS) were 42.5% versus 29.4%, and overall survival (OS) was 71.1% versus 59.4%, respectively. Totally, 117 high-risk patients with oral metronomic MT had an EFS rate of 42.7%. The results were similar to those of ASCT from other studies. The toxicities of metronomic MT were lower. Our study showed that oral metronomic MT is an optimal option for high-risk NB patients without ASCT or anti-GD2 antibody therapy.Despite aggressive treatment, the prognosis of high-risk NB patients is still poor. This retrospective study investigated the benefits of metronomic maintenance treatment (MT) in high-risk NB patients without ASCT or GD2 antibody therapy. Patients aged ≤ 21 years with newly diagnosed high-risk NB were included. Patients with complete/very good partial remission (CR/VGPR/PR) to conventional treatment received, or not, oral metronomic MT for 1 year. Two hundred and seventeen high-risk NB patients were enrolled. One hundred and eighty-five (85%) had a CR/VGPR/PR to conventional treatment, of the patients with stage 4, 106 receiving and 61 not receiving oral metronomic MT, and the 3-year event-free survival (EFS) rate was 42.5 ± 5.1% and 29.6 ± 6%, respectively (p = 0.017), and overall survival (OS) rate was 71.1 ± 4.7% and 59.4 ± 6.4%, respectively (p = 0.022). A total of 117 high-risk patients with oral metronomic MT had EFS rate of 42.7 ± 4.8%. The toxicity of MT was mild. For high-risk NB patients without ASCT or anti-GD2 antibody therapy, stage 4, MYCN amplication and patients with stage 4 not receiving oral metronomic MT after CR/VGPR/PR were independent adverse prognostic factors. Oral metronomic MT can improve survival in high-risk NB patients in CR/VGPR/PR without ASCT or anti-GD2 antibodies therapy.

Abstract 639: Analysis of DNA minimal residual disease markers in pediatric solid cancers using quantitative real time PCR and droplet digital PCR

Abstract Background: Minimal Residual Disease (MRD) detection is vital for therapy monitoring and relapse prediction in cancers. While RNA-based MRD assays are shown to be effective at diagnostic time-points, serial response monitoring does not provide additional predictive value.DNA-based assays may be more sensitive and allow longitudinal monitoring of the disease trajectory. This study aimed to develop MRD assays based on whole genome sequencing (WGS) data for high-risk neuroblastoma (HRNB) and Ewing sarcoma (ES) patients, to establish their sensitivity in quantitative PCR (qPCR) anddroplet digital PCR (ddPCR) formats, and to directly compare against RNA-based MRD assays. Methodology: We analyzed WGS data to identify patient-specific chromosomal breakpoints in HRNB (N=6) and ES (N=6) patients, then established qPCR and ddPCR assays. Assay performance was validated using patient-derived cells spiked into bone marrow samples and in serially collected clinical samples as available. The DNA-MRD markers were compared against a panel of RNA-MRD markers for HRNB (TH/DCX/PHOX2B) and ES (EWS-FLI1/ERG/ETV1). Results: DNA-MRD markers showed high sensitivity and quantitation ranging from 10-4(0.01%) to 10-5(0.001%) in qPCR assays and ddPCR assays. In clinical samples of HRNB and ES, DNA-MRD markers were detected in diagnosis specimens and predicted disease trajectory. Among RNA-MRD markers, TH & EWS-FLI1 transcripts showed the maximum detection sensitivity (0.01%), however was less sensitive than the patient specific DNA-MRD markers. Conclusion: This study identifies DNA-MRD analysis as a reliable complimentary approach to RNA-MRD assays and highlights its enhanced sensitivity and clinical utility for monitoring treatment response and disease progression in solid pediatric tumors. Citation Format: Vinod Vijay Subhash, Alvin Kamili, Marie-Wong Erasmus, Dan Chen, Libby Huang, Vanessa Tyrell, Caroline Atkinson, Nicola Venn, Mark Cowley, Glenn Marshall, Paul Ekert, Michelle Henderson, Rosemary Sutton, Murray Norris, Michelle Haber, Jamie Fletcher, Toby Trahair. Analysis of DNA minimal residual disease markers in pediatric solid cancers using quantitative real time PCR and droplet digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 639.

Abstract 1346: Direct targeting of MAPK/ERK signaling pathway is a novel therapeutic approach for high-risk neuroblastoma

Abstract Neuroblastoma is a highly heterogenous pediatric tumor that develops during the early embryonic stages, and accounts for almost 15% of pediatric cancer related deaths. Despite advanced and intensive therapeutic approaches that combine surgery, radiation, and chemotherapy, high-risk neuroblastoma (NB) patients' long-term survival is still less than 50%. Tumor metastasis, relapse, drug-resistance, and treatment related toxicities mandate the development of novel therapeutic approaches to treat NB patients. MAPK/ERK pathway plays an important oncogenic role in different types of cancers, including NB. Activation of the MAPK/ERK pathway is known to induce cancer cell proliferation, progression, metabolism, and resistance to drug-induced apoptosis. In the present study, we investigated the effect of a small-molecule MEK inhibitor that targets MEK1 and MEK2 to block MAPK/ERK pathway in NB. Cytotoxicity assays using Cell-Titer One solution in different MYCN-amplified (NGP, LAN-5, CHLA-255-MYCN) and MYCN-non-amplified (SH-SY5Y, SK-N-AS, CHLA-255) NB cell lines show that MEK inhibitor significantly reduce the NB cell proliferation in a dose-dependent manner. We further performed clonogenic assays and 3D spheroidal assays to mimic the NB tumor growth, and our results show that MEK inhibitor significantly inhibit NB colony formation, inhibit 3D spheroidal tumor size by inducing cell death in 3D spheroidal tumors, in comparison to controls. Additionally, MEK inhibitor in a dose-dependent manner induces apoptosis and blocks cell cycle progression in NB cells in comparison to control, as determined by Annexin V apoptosis assays and Click-iT EdU cell proliferation assay respectively. Furthermore, gene expression analysis show that MEK inhibitor significantly reduce the mRNA expression of specific MAPK/ERK pathway targets such as MEK1, MEK2, and ERK2. Western blot assays further confirm the efficacy of MEK inhibitor in blocking MAPK/ERK pathway by significantly inhibiting the phosphorylation of key pathway proteins. Taken together, our results highlight that: a) MAPK/ERK pathway is implicated in NB growth, and b) direct targeting of MAPK/ERK signaling pathway by using a novel small molecule MEK inhibitor inhibit NB proliferation and 3D tumor growth. We will further combine this MEK inhibitor with current chemotherapy drugs to develop effective therapeutic approaches for NB patients. Citation Format: Rameswari Chilamakuri, Bharti Sharma, Danielle C. Rouse, Saurabh Agarwal. Direct targeting of MAPK/ERK signaling pathway is a novel therapeutic approach for high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1346.