Enhanced anti-tumor activity mediated by combination chimeric antigen receptor T cells targeting GD2 and GPC2 in high-risk neuroblastoma.

BACKGROUND AIMS: Chimeric antigen receptor T (CAR-T) cells targeting single antigens show limited activity against solid tumors due to poor T cell persistence, low efficiency infiltration, and exhaustion together with heterogeneous tumor-associated antigen (TAA) expression. This is also true in high-risk neuroblastoma (HRNB), a lethal pediatric extracranial malignancy. To overcome these obstacles, a combinational strategy using GD2-specific and GPC2-specific CAR-T cells was developed to improve immunotherapeutic efficacy. METHODS: We individually developed GD2-specific and GPC2-specific CARs containing a selective domain (sCAR) which was a peptide of 10 amino acids derived from human nuclear autoantigen La/SS-B. These constructs allowed us to generate two different HRNB antigen-specific CAR-T cells with enhanced biological activity through stimulating sCAR-engrafted T cells via a selective domain-specific monoclonal antibody (SmAb). Binding affinity and stimulation of GD2- and GPC2-specific sCARs by SmAb were measured, and transient and persistent anti-tumor cytotoxicity of GD2sCAR-T and GPC2sCAR-T cells were quantified in neuroblastoma cell lines expressing different TAA levels. The anti-tumor pharmaceutical effects and cellular mechanisms mediated by single or combinational sCAR-T cells were evaluated in vitro and in vivo. RESULTS: GD2- and GPC2-specific sCARs had antigen-specific binding affinity similar to their parental counterparts and were recognized by SmAb. SmAb-mediated stimulation selectively activated sCAR-T proliferation and increased central memory T cells in the final products. SmAb-stimulated sCAR-T cells had enhanced transient cytolytic activity, and combination therapy extended long-term anti-tumor activity in vitro through TNF-α and IL-15 release. Stimulated sCAR-T cells overcame heterogeneous antigen expression in HRNB, and the multi-TAA-targeting strategy was especially efficacious in vivo, inducing apoptosis through the caspase-3/PARP pathway and inhibiting the release of several tumor-promoting cytokines. CONCLUSIONS: These data suggest that combined targeting of multiple TAAs is a promising strategy to overcome heterogenous antigen expression in solid tumors and extend CAR-T cell persistence for HRNB immunotherapy.

Analysis of the incidence, characteristics, and risk factors of complications during induction chemotherapy in children with high-risk neuroblastoma.

Patients with high-risk neuroblastoma (HR-NB) exhibit suboptimal 5-year survival rates, leading to a widespread international preference for high-intensity chemotherapeutic regimens in these children. We analyzed the incidence and risk factors for complications during induction chemotherapy in children with HR-NB and tried to assist clinicians in predicting such complications and optimizing therapeutic strategy. The clinical data of children with HR-NB admitted to our hospital from January 2007 to December 2019 were retrospectively analyzed. The incidence, characteristics, and risk factors of complications (infection, hemorrhage, and chemotherapy-related adverse reactions (CRAR)) requiring hospitalization during induction chemotherapy in these children were explored. (1) A total of 108 patients with HR-NB were included in the final analysis. The overall infection rate was 92.6% (100/108), with the highest incidence of 71.3% observed during the first cycle. FN, bacterial infection, as well as fungal infection were common infectious complications in children with HR-NB during induction chemotherapy. (2) The overall hemorrhage rate was 24.1% (26/108), with the highest incidence of 14.8% also observed in the first cycle. Among the children with hemorrhage, there were 72% with bone marrow involved, while 65.0% of them had a high vanillylmandelic acid (VMA) value. And children with hemorrhage also exhibited neuron-specific enolase (NSE) ≥ 200 µg/L in 88.5% of cases and lactic dehydrogenase (LDH) ≥ 1000U/L in 73.1% of cases. (3) The incidence of CRAR rate was 100%, and 99.1% (107/108) patients experienced myelosuppression. The incidence of myelosuppression peaked in the third cycle, reaching up to 85.2%. Most children suffered severe myelosuppression existed with bone marrow metastases (76.3%), abnormal VMA (67.5%), and LDH ≥ 1000 U/L (60%). (4) Non-myelosuppressive adverse effects were observed in 75.9% children (82/108), with the highest incidence occurring in the third cycle at 42.6%. (5) Patients who experienced three types of complications had a lower median survival time (MST) of 54.4 months, a 3-year event-free survival (EFS) rate of (44.2 ± 10.7)%, and a 3-year overall survival (OS) rate of (75.8 ± 8.6)%, in comparison to those with only one or two complications, who had a higher MST of 59.5 months, a 3-year EFS rate of (73.5 ± 5.2)% (X2 = 10.457, P = 0.001), and a 3-year OS rate of (84.8 ± 4.1)% (X2 = 10.511, P = 0.001). CONCLUSION: The presence of bone marrow involved and increased VMA were high-risk factors for infection, while NSE ≥ 200 µg/L and LDH ≥ 1000 U/L were high-risk factors for hemorrhage. For those children who had experienced severe myelosuppression, the presence of bone marrow metastases, increased VMA, and LDH ≥ 1000 U/L were their risk factors. The presence of bone involvement was a high-risk factor for children to have non-myelosuppressive adverse effects. Complications that arise during induction chemotherapy could negatively impact the children's prognosis and overall quality of life. WHAT IS KNOWN: • The high-risk neuroblastoma (HR-NB) had a worse prognosis; there was a general international preference for high-intensity chemotherapeutic regimens in the induction phase to these children. WHAT IS NEW: • We analyzed the incidence and risk factors of complications during induction chemotherapy in children with HR-NB and tried to help clinicians predict such complications and adopt optimized therapeutic strategy.

High-dose carboplatin-irinotecan-temozolomide is an effective salvage chemotherapy for relapsed or refractory neuroblastoma.

There is no clear consensus on the most effective treatment for relapsed/refractory high-risk neuroblastoma (NB). We retrospectively assessed seven NB patients with relapsed/refractory disease who received high-dose carboplatin-irinotecan-temozolomide (HD-CIT). Five of seven patients showed favorable therapeutic response (complete remission or partial remission). Regarding toxicity, the cytopenia period tended to prolong when more than three cycles were repeated, but nonhematological toxicities were controllable with general supportive care. Due to its antitumor efficacy and well-tolerated nonhematologic toxicity, HD-CIT is a promising salvage chemotherapy for relapsed/refractory NB. However, it is important to pay attention to the exacerbation of hematological toxicity when repeating the regimen.

Prognostic value of xenograft engraftment in patients with metastatic high-risk neuroblastoma.

BACKGROUND: Successful engraftment of human cancer biopsies in immunodeficient mice correlates with the poor prognosis of patients. This was reported 30 years ago for children with neuroblastoma, but the standard of care treatment evolved significantly during the last 15 years, leading to improved survival of these patients. Here, we evaluated the association of patient-derived xenograft (PDX) engraftment and prognosis in patients receiving up-to-date treatments for cancers classified as metastatic (stage M) high-risk neuroblastoma (HR-NB) by the International Neuroblastoma Risk Group Staging System (INRGSS). METHODS: We obtained biopsies from patients with stage M HR-NB. We inoculated biopsy fragments subcutaneously in mice. We studied the association of PDX engraftment with event-free survival (EFS) and overall survival (OS) of patients. RESULTS: Since 2009, we established 17 PDX from 97 samples of 66 patients with stage M HR-NB, with a follow-up of at least two years. Factors associated with higher probability of engraftment were the death as outcome (p = .0006) and the amplification of the gene MYCN in tumors (p = .0271). Patients whose biopsies established a PDX had significantly shorter EFS and OS (p = .0039 and .0002, respectively) than patients whose samples did not engraft. The association of PDX engraftment and OS was significant in patients without MYCN amplification (p = .0041), but not in patients with MYCN amplification (p = .2707). CONCLUSION: Positive PDX engraftment is a factor related to poor prognosis and fatal outcome in patients with stage M HR-NB treated with up-to-date therapies.

Prognostic significance of NT5E/CD73 in neuroblastoma and its function in CSC stemness maintenance.

Cluster of differentiation 73 (CD73), a cell surface enzyme that catalyzes adenosine monophosphate (AMP) breakdown to adenosine, is differentially expressed in cancers and has prognostic significance. We investigated its expression profile in neuroblastoma (NB), its association with NB clinical outcomes, and its influence in the regulation of cancer stem cells' (CSCs) stemness maintenance. RNA-Seq data mining (22 independent study cohorts, total n = 3836) indicated that high CD73 can predict good NB prognosis. CD73 expression (immunohistochemistry) gauged in an NB patient cohort (n = 87) showed a positive correlation with longer overall survival (OS, P = 0.0239) and relapse-free survival (RFS, P = 0.0242). Similarly, high CD73 correlated with longer OS and RFS in advanced disease stages, MYCN non-amplified (MYCN-na), and Stage-4-MYCN-na subsets. Despite no definite association in children < 2 years old (2Y), high CD73 correlated with longer OS (P = 0.0294) and RFS (P = 0.0315) in children > 2Y. Consistently, high CD73 was associated with better OS in MYCN-na, high-risk, and stage-4 subsets of children > 2Y. Multivariate analysis identified CD73 as an independent (P = 0.001) prognostic factor for NB. Silencing CD73 in patient-derived (stage 4, progressive disease) CHLA-171 and CHLA-172 cells revealed cell-line-independent activation of 58 CSC stemness maintenance molecules (QPCR profiling). Overexpressing CD73 in CHLA-20 and CHLA-90 cells with low CD73 and silencing in CHLA-171 and CHLA-172 cells with high CD73 showed that CD73 regulates epithelial to mesenchymal transition (E-Cadherin, N-Cadherin, Vimentin), stemness maintenance (Sox2, Nanog, Oct3/4), self-renewal capacity (Notch), and differentiation inhibition (leukemia inhibitory factor, LIF) proteins (confocal-immunofluorescence). These results demonstrate that high CD73 can predict good prognosis in NB, and further suggest that CD73 regulates stemness maintenance in cells that defy clinical therapy.

Development and validation of a nomogram based on common biochemical indicators for survival prediction of children with high-risk neuroblastoma: A valuable tool for resource-limited hospitals.

BACKGROUND: Despite multiple attempts have been made to develop risk stratification within high-risk neuroblastoma (NB) patients (age of diagnosis ≥ 18 month-old with metastatic NB), the definition of "ultra high-risk NB" is still lack of consensus, and indicators for identifying this subgroup are still unclear. This study aimed to develop a nomogram based on easy-to-obtain blood-derived biofactors for identifying ultra high-risk NB patients with highest risk of death within 3 or 5 years. METHODS: One hundred sixty-seven NB patients who treated at Sun Yat-sen University Cancer Center between 2015 and 2023 were recruited and clustered randomly into training and validation cohorts (116 and 51 cases, respectively). Univariate and multivariate Cox analysis were performed in training set to screen independent prognostic indicators for constructing nomogram model of predicting 1-, 3- and 5-year overall survival (OS). The discrimination power of the nomogram in training and validation sets were assessed by concordance index (C-index) and calibration plot. Based on the risk score obtained from nomogram model, the prognostic accuracy of 1-, 3- and 5-year OS rates in training and validation cohorts were further evaluated using the area under receiver operating characteristic (ROC) curves (AUC). RESULTS: Through univariate and multivariate Cox analysis, independent prognostic indicators, including serum lactate dehydrogenase (LDH) and albumin (ALB), were identified in training set, and used to establish a nomogram model. The model showed good discrimination power with C-index in training cohort being 0.706 (95%CI: 0.633-0.788). According to the cut-point calculated based on the established nomogram, patients with a nomogram score > 34 points could be stratified to ultra high-risk NB subgroup, and this subgroup had poorer OS than those in non-ultra one (p < 0.001). AUC values of ROC curves for 3- and 5-year OS rates in the training set were 0.758 and 0.756, respectively. Moreover, based on the cut-point score (34 points) developed in training set, The model also showed good discrimination power with C-index of 0.773 (95%CI: 0.664-0.897) and powerful prognostic accuracy of AUC for 3- and 5-year OS rates being 0.825 and 0.826, respectively, in validation cohort. CONCLUSIONS: We developed a simple-to-use nomogram based on common laboratory indicators to identify the subgroup of ultra high-risk NB before treatment, providing these children even from developing countries or regions access to intensified multimodal treatments earlier and thus improving their long-term outcome.

Biological Insight and Recent Advancement in the Treatment of Neuroblastoma.

One of the most frequent solid tumors in children is neuroblastoma, which has a variety of clinical behaviors that are mostly influenced by the biology of the tumor. Unique characteristics of neuroblastoma includes its early age of onset, its propensity for spontaneous tumor regression in newborns, and its high prevalence of metastatic disease at diagnosis in individuals older than 1 year of age. Immunotherapeutic techniques have been added to the previously enlisted chemotherapeutic treatments as therapeutic choices. A groundbreaking new treatment for hematological malignancies is adoptive cell therapy, specifically chimeric antigen receptor (CAR) T cell therapy. However, due to the immunosuppressive nature of the tumor microenvironment (TME) of neuroblastoma tumor, this treatment approach faces difficulties. Numerous tumor-associated genes and antigens, including the MYCN proto-oncogene (MYCN) and disialoganglioside (GD2) surface antigen, have been found by the molecular analysis of neuroblastoma cells. The MYCN gene and GD2 are two of the most useful immunotherapy findings for neuroblastoma. The tumor cells devise numerous methods to evade immune identification or modify the activity of immune cells. In addition to addressing the difficulties and potential advancements of immunotherapies for neuroblastoma, this review attempts to identify important immunological actors and biological pathways involved in the dynamic interaction between the TME and immune system.

Phase I/II clinical trial of high-dose [131I] meta-iodobenzylguanidine therapy for high-risk neuroblastoma preceding single myeloablative chemotherapy and haematopoietic stem cell transplantation.

PURPOSE: Paediatric high-risk neuroblastoma has poor prognosis despite modern multimodality therapy. This phase I/II study aimed to determine the safety, dose-limiting toxicity (DLT), and efficacy of high-dose 131I-meta-iodobenzylguanidine (131I-mIBG) therapy combined with single high-dose chemotherapy (HDC) and haematopoietic stem cell transplantation (HSCT) in high-risk neuroblastoma in Japan. METHODS: Patients received 666 MBq/kg of 131I-mIBG and single HDC and HSCT from autologous or allogeneic stem cell sources. The primary endpoint was DLT defined as adverse events associated with 131I-mIBG treatment posing a significant obstacle to subsequent HDC. The secondary endpoints were adverse events/reactions, haematopoietic stem cell engraftment and responses according to the Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST 1.1) and 123I-mIBG scintigraphy. Response was evaluated after engraftment. RESULTS: We enrolled eight patients with high-risk neuroblastoma (six females; six newly diagnosed and two relapsed high-risk neuroblastoma; median age, 4 years; range, 1-10 years). Although all patients had adverse events/reactions after high-dose 131I-mIBG therapy, we found no DLT. Adverse events and reactions were observed in 100% and 25% patients during single HDC and 100% and 12.5% patients during HSCT, respectively. No Grade 4 complications except myelosuppression occurred during single HDC and HSCT. The response rate according to RECIST 1.1 was observed in 87.5% (7/8) in stable disease and 12.5% (1/8) were not evaluated. Scintigraphic response occurred in 62.5% (5/8) and 37.5% (3/8) patients in complete response and stable disease, respectively. CONCLUSION: 131I-mIBG therapy with 666 MBq/kg followed by single HDC and autologous or allogeneic SCT is safe and efficacious in patients with high-risk neuroblastoma and has no DLT. TRIAL REGISTRATION NUMBER: jRCTs041180030. NAME OF REGISTRY: Feasibility of high-dose iodine-131-meta-iodobenzylguanidine therapy for high-risk neuroblastoma preceding myeloablative chemotherapy and haematopoietic stem cell transplantation (High-dose iodine-131-meta-iodobenzylguanidine therapy for high-risk neuroblastoma). URL OF REGISTRY: https://jrct.niph.go.jp/en-latest-detail/jRCTs041180030 . DATE OF ENROLMENT OF THE FIRST PARTICIPANT TO THE TRIAL: 12/01/2018.

Clinical impact of primary tumour 123ImIBG response to induction chemotherapy in children with high-risk neuroblastoma.

BACKGROUND: More than 50% children with high-risk neuroblastoma (HR-NBL) experience disease progression, which we hypothesise is due to non-response of primary tumour to treatment. Current imaging techniques are unable to characterise response in primary tumour (necrotic versus viable tissue) at diagnosis or follow-up. OBJECTIVES: Compare clinico-histological characteristics between primary 123ImIBG-avid tumours that became entirely 123ImIBG-non-avid (responders) after induction chemotherapy (IC) versus primary 123ImIBG-avid tumour that remained 123ImIBG-avid (non-responders). METHODS: Retrospective review of clinico-radiological data of children diagnosed with 123ImIBG-avid HR-NBL at our centre (2005-2016). Patients received Rapid COJEC IC and two additional courses of TVD if metastatic response was inadequate. Primary tumour 123ImIBG response was assessed qualitatively as positive, negative or intermediate at diagnosis and after IC. Post-surgical histopathology slices were marked considering percentage of viable tissue. RESULTS: Sixteen of 61 patients showed complete primary tumour 123ImIBG response, 20 partial response, while 25 no response. There was no statistically significant difference between clinical demographics of complete responders and group of non- or partial responders. Mean percentage of viable tumour cells was higher in non-responders than in complete responders (44.6% vs 20.6%; p = 0.05). Five-year EFS was significantly higher in complete responders than non-responders (43 ± 15% vs 7 ± 6%; p < 0.005). CONCLUSIONS: 123ImIBG response in primary HR-NBL correlates with amount of necrotic tissue, skeletal metastatic 123ImIBG response and outcome. An entirely 123ImIBG non-avid tumour can still harbour viable tumour cells. Therefore, our findings do not support utility of primary tumour 123ImIBG response in decision making regarding residual tumour surgery. Combining both, primary and metastatic 123ImIBG response will improve interpretability of clinical trial results.

The expression of PHOX2B in bone marrow and peripheral blood predicts adverse clinical outcome in non-high-risk neuroblastoma.

Paired-like homeobox 2B (PHOX2B) is a highly sensitive and specific biomarker for diagnosing neuroblastoma, as well as detecting minimal residual disease in neuroblastoma. The clinical significance of PHOX2B expression in bone marrow (BM) and peripheral blood (PB) samples of newly diagnosed patients with very low-, low- and intermediate-risk neuroblastoma remains unknown, to the best of our knowledge. The expression level of PHOX2B in paired BM and PB samples of patients with newly diagnosed neuroblastoma was validated using reverse transcription-quantitative polymerase chain reaction (RTqPCR). Among the 132 patients, 26 exhibited a positive PHOX2B expression BM (19.7%) and 11 in PB (8.3%) samples. PHOX2B was highly expressed in BM and PB samples from patients aged <18 months, with International Neuroblastoma Risk Group Staging System stages M and MS, 1p loss of heterozygosity, and high levels of lactate dehydrogenase, serum ferritin and neuron-specific enolase (p < 0.05). In all eligible patients, the 2-year event-free survival (EFS) and overall survival (OS) rates were 94.7 ± 2.0% and 97.7 ± 1.3%, respectively. However, the 2-year EFS rates were significantly decreased to 76.9 ± 8.3% and 63.6 ± 14.5% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). Similarly, the 2-year OS rates were also decreased to 88.5 ± 6.3% and 81.8 ± 11.6% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). In conclusion, a positive PHOX2B expression in BM and PB samples at diagnosis had a strong adverse prognostic effect on patients with non-high-risk neuroblastoma.

Comparison Between Diffusion-Weighted MRI and 123 I-mIBG Uptake in Primary High-Risk Neuroblastoma.

BACKGROUND: High-risk neuroblastoma (HR-NB) has a variable response to preoperative chemotherapy. It is not possible to differentiate viable vs. nonviable residual tumor before surgery. PURPOSE: To explore the association between apparent diffusion coefficient (ADC) values from diffusion-weighted magnetic resonance imaging (DW-MRI), 123 I-meta-iodobenzyl-guanidine (123 I-mIBG) uptake, and histology before and after chemotherapy. STUDY TYPE: Retrospective. SUBJECTS: Forty patients with HR-NB. FIELD STRENGTH/SEQUENCE: 1.5T axial DW-MRI (b = 0,1000 s/mm2 ) and T2 -weighted sequences. 123 I-mIBG scintigraphy planar imaging (all patients), with additional 123 I-mIBG single-photon emission computed tomography / computerized tomography (SPECT/CT) imaging (15 patients). ASSESSMENT: ADC maps and 123 I-mIBG SPECT/CT images were coregistered to the T2 -weighted images. 123 I-mIBG uptake was normalized with a tumor-to-liver count ratio (TLCR). Regions of interest (ROIs) for primary tumor volume and different intratumor subregions were drawn. The lower quartile ADC value (ADC25prc ) was used over the entire tumor volume and the overall level of 123 I-mIBG uptake was graded into avidity groups. STATISTICAL TESTS: Analysis of variance (ANOVA) and linear regression were used to compare ADC and MIBG values before and after treatment. Threshold values to classify tumors as viable/necrotic were obtained using ROC analysis of ADC and TLCR values. RESULTS: No significant difference in whole-tumor ADC25prc values were found between different 123 I-mIBG avidity groups pre- (P = 0.31) or postchemotherapy (P = 0.35). In the "intratumor" analysis, 5/15 patients (prechemotherapy) and 0/14 patients (postchemotherapy) showed a significant correlation between ADC and TLCR values (P < 0.05). Increased tumor shrinkage was associated with lower pretreatment tumor ADC25prc values (P < 0.001); no association was found with pretreatment 123 I-mIBG avidity (P = 0.17). Completely nonviable tumors had significantly lower postchemotherapy ADC25prc values than tumors with >10% viable tumor (P < 0.05). Both pre- and posttreatment TLCR values were significantly higher in patients with >50% viable tumor than those with 10-50% viable tumor (P < 0.05). DATA CONCLUSION: 123 I-mIBG avidity and ADC values are complementary noninvasive biomarkers of therapeutic response in HR-NB. LEVEL OF EVIDENCE: 4. TECHNICAL EFFICACY STAGE: 3.

The reliability of bone marrow cytology as response criterion in metastatic neuroblastoma.

BACKGROUND: The quantitative assessment of neuroblastoma cell content in bone marrow aspirates for response evaluation has been introduced recently. Data on the concordance of interobserver reports are lacking so far. METHODS: Investigators of seven European countries representing national reference or large oncological centers convened in 2016. They agreed to quantitatively assess routine bone marrow smears of the participating institutions and to discuss the discrepant results in joint meetings. RESULTS: From 2017 through 2019, three cytology rounds with 24, 28, and 28 bone marrow samples were run evaluating the representativity of the smears (yes/[restricted]/no) and the presence of tumor cells (yes/no and %). The comparison of the reports using κ (Fleiss) and α (Krippendorff) statistics demonstrated no robust reliabilities. The agreement on the representativity was moderate to poor, on the presence of tumor cells moderate to good, and on the percentage of tumor cells slight to moderate. Though the value of cytology is unquestioned to detect even tiny metastatic cells in bone marrow, the investigators unanimously agreed that a reliable quantification of the tumor cell content in bone marrow smears is unrealistic. For the key issue of representativity, a new practical definition was developed. CONCLUSION: For any work with bone marrow aspirates, the representativity of the material is of paramount importance. A practical definition is proposed. A reliable quantitative cytological assessment of tumor cell content in bone marrow aspirates is not feasible in metastatic neuroblastoma. Therefore, its use as response criterion should be reconsidered.

A nomogram of clinical and biologic factors to predict survival in children newly diagnosed with high-risk neuroblastoma: An International Neuroblastoma Risk Group project.

BACKGROUND: Long-term outcome remains poor for children with high-risk neuroblastoma (five-year overall survival [OS] ∼50%). Our objectives were to (a) identify prognostic biomarkers and apply them in a nomogram to identify the subgroup of ultra-high-risk patients at highest risk of disease progression/death, for whom novel frontline therapy is urgently needed; and (b) validate the nomogram in an independent cohort. METHODS: A total of 1820 high-risk patients (≥18 months old with metastatic neuroblastoma), diagnosed 1998-2015, from the International Neuroblastoma Risk Groups (INRG) Data Commons were analyzed in a retrospective cohort study. Using multivariable Cox regression of OS from diagnosis, a nomogram was created from prognostic biomarkers to predict three-year OS. External validation was performed using the SIOPEN HR-NBL1 trial cohort (n = 521), evidenced by receiver operating characteristic curves. RESULTS: The nomogram, including MYCN status (P < 0.0001), lactate dehydrogenase (LDH) (P = 0.0007), and presence of bone marrow metastases (P = 0.004), had robust performance and was validated. Applying the nomogram at diagnosis (a) gives prognosis of an individual patient and (b) identifies patients predicted to have poor outcome (three-year OS was 30% ± 5% for patients with a nomogram score of > 82 points; 58% ± 1% for those ≤82 points). Median follow-up time was 5.5 years (range, 0-14.1). CONCLUSIONS: In high-risk neuroblastoma, a novel, publicly available nomogram using prognostic biomarkers (MYCN status, LDH, presence of bone marrow metastases; https://neuroblastoma.shinyapps.io/High-Risk-Neuroblastoma-Nomogram/) has the flexibility to apply a clinically suitable and context-specific cutoff to identify patients at highest risk of death. This will facilitate testing urgently needed new frontline treatment options to improve outcome for these children.

131 I-Meta-iodobenzylguanidine followed by busulfan and melphalan and autologous stem cell rescue in high-risk neuroblastoma.

INTRODUCTION: Despite the progress in current treatments, the event-free survival of high-risk neuroblastoma (HR-NB) patients does not exceed 40%-50%, and the prognosis of refractory or relapsed patients is poor, still representing a challenge for pediatric oncologist. Therapeutic Iodine-131 meta-iodobenzylguanidine (Th-131 I-MIBG) is a recognized safe and potentially effective treatment for NB. MATERIALS: This retrospective study reports the outcomes of 28 MIBG-avid NB patients with advanced disease either refractory or relapsed, which was undertaken from 1996 to 2014. Th-131 I-MIBG was administered shortly before (median: 17 days) high-dose chemotherapy with busulfan and melphalan (HD-BuMel) and autologous stem cell rescue (ASCR) at the Gaslini Institute in Genoa, with the aim of analyzing the feasibility, safety, and efficacy of this approach. RESULTS: Engraftment occurred in all patients after a median of 14 (11-29) and 30 days (13-80) from ASCR for neutrophils and platelets, respectively. No treatment-related deaths were observed. The main high-grade (3-4) toxicity observed was oral and gastrointestinal mucositis in 78.6% and 7.1% of patients, respectively, whereas high-grade hepatic toxicity was observed in 10.7%. Two patients developed veno-occlusive-disease (7.1%), completely responsive to defibrotide. Hypothyroidism was the main late complication that occurred in nine patients (31.1%). After Th-131 MIBG and HD-BuMel, 19 patients (67.8%) showed an improvement in disease status. Over a median follow-up of 15.9 years, the three-year and five-year overall survival (OS) probabilities were 53% (CI 0.33-0.69) and 41% (CI 0.22-0.59), and the three-year and five-year rates of cumulative risk of progression/relapse were 64% (CI 0.47-0.81) and 73% (CI 0.55-0.88), respectively. MYCN amplification emerged as the only risk factor significantly associated with OS (HR, 3.58;P = 0.041). CONCLUSION: Th-131 I-MIBG administered shortly before HD-BuMel is a safe and effective regimen for patients with advanced MIBG-avid NB. These patients should be managed in centers with proven expertise.

Heterogeneities in Cell Cycle Checkpoint Activation Following Doxorubicin Treatment Reveal Targetable Vulnerabilities in TP53 Mutated Ultra High-Risk Neuroblastoma Cell Lines.

Most chemotherapeutics target DNA integrity and thereby trigger tumour cell death through activation of DNA damage responses that are tightly coupled to the cell cycle. Disturbances in cell cycle regulation can therefore lead to treatment resistance. Here, a comprehensive analysis of cell cycle checkpoint activation following doxorubicin (doxo) treatment was performed using flow cytometry, immunofluorescence and live-cell imaging in a panel of TP53 mutated ultra high-risk neuroblastoma (NB) cell lines, SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C. Following treatment, a dose-dependent accumulation in either S- and/or G2/M-phase was observed. This coincided with a heterogeneous increase of cell cycle checkpoint proteins, i.e., phos-ATM, phos-CHK1, phos-CHK2, Wee1, p21Cip1/Waf1, and p27Kip among the cell lines. Combination treatment with doxo and a small-molecule inhibitor of ATM showed a delay in regrowth in SK-N-DZ, of CHK1 in BE(2)-C, of Wee1 in SK-N-FI and BE(2)-C, and of p21 in Kelly and BE(2)-C. Further investigation revealed, in all tested cell lines, a subset of cells arrested in mitosis, indicating independence on the intra-S- and/or G2/M-checkpoints. Taken together, we mapped distinct cell cycle checkpoints in ultra high-risk NB cell lines and identified checkpoint dependent and independent druggable targets.

Kinome multigenic panel identified novel druggable EPHB4-V871I somatic variant in high-risk neuroblastoma.

Neuroblastoma (NB) is the most common extracranial neoplasm in children. The overall outcome for high-risk NB patients is still unacceptable, therefore, it is critical to deeply understand molecular mechanisms associated with NB, which in turn can be utilized for developing drugs towards the treatment of NB. Protein kinases (TKs) play an essential role in the regulation of cell survival and proliferation. Different kinases, such as anaplastic lymphoma kinase (ALK), Aurora kinase, RET receptor tyrosine kinase, are potential therapeutic targets in various cancers, including NB. We analysed a cohort of 45 high-risk NB patients and 9 NB cell lines by a targeted-(t)NGS custom gene panel (genes codifying for the kinase domains of 90 TKs). We identified somatic variants in four TK genes (ALK, EPHB4, LMTK3 and EPHB6) in NB patients and we functionally characterized an interesting somatic variant, V871I, in EPHB4 gene. EPHB4 plays a crucial role in cardiovascular development and regulates vascularization in cancer-promoting angiogenesis, tumour growth and metastasis. Several EPHB4 mutations have previously been identified in solid and haematological tumour specimens but EPHB4 mutations were not described until now in NB. Interestingly, a re-analysis of public CGH-array showed that the EPHB4 gain is associated with advanced diseases in NB. We further demonstrated that higher EPHB4 expression is correlated to stage 4 of NB and with poor overall survival. Additionally, we also revealed that the EPHB4-V871I accounts for increased proliferation, migration and invasion properties in two NB cell lines by acting on VEGF, c-RAF and CDK4 target genes and by increasing the phosphorylation of ERK1-2 pathway. The use of two EPHB4 inhibitors, JI-101 and NVP-BHG712, was able to rescue the phenotype driven by the variant. Our study suggested that EPHB4 is a promising therapeutic target in high-risk NB.

A subset analysis of a phase II trial evaluating the use of DFMO as maintenance therapy for high-risk neuroblastoma.

Neuroblastoma is a sympathetic nervous system tumor, primarily presenting in children under 6 years of age. The long-term prognosis for patients with high-risk neuroblastoma (HRNB) remains poor despite aggressive multimodal therapy. This report provides an update to a phase II trial evaluating DFMO as maintenance therapy in HRNB. Event-free survival (EFS) and overall survival (OS) of 81 subjects with HRNB treated with standard COG induction, consolidation and immunotherapy followed by 2 years of DFMO on the NMTRC003/003b Phase II trial were compared to a historical cohort of 76 HRNB patients treated at Beat Childhood Cancer Research Consortium (BCC) hospitals who were disease-free after completion of standard upfront therapy and did not receive DFMO. The 2- and 5-year EFS were 86.4% [95% confidence interval (CI) 79.3%-94.2%] and 85.2% [77.8%-93.3%] for the NMTRC003/003b subset vs 78.3% [69.5%-88.3%] and 65.6% [55.5%-77.5%] for the historical control group. The 2- and 5-year OS were 98.8% [96.4-100%] and 95.1% [90.5%-99.9%] vs 94.4% [89.3%-99.9%] and 81.6% [73.0%-91.2%], respectively. DFMO maintenance for HRNB after completion of standard of care therapy was associated with improved EFS and OS relative to historical controls treated at the same institutions. These results support additional investigations into the potential role of DFMO in preventing relapse in HRNB.

Extended induction chemotherapy does not improve the outcome for high-risk neuroblastoma patients: results of the randomized open-label GPOH trial NB2004-HR.

BACKGROUND: Long-term survival of high-risk neuroblastoma patients is still below 50% despite intensive multimodal treatment. This trial aimed to address whether the addition of two topotecan-containing chemotherapy courses compared to standard induction therapy improves event-free survival (EFS) of these patients. PATIENTS AND METHODS: An open-label, multicenter, prospective randomized controlled trial was carried out at 58 hospitals in Germany and Switzerland. Patients aged 1-21 years with stage 4 neuroblastoma and patients aged 6 months to 21 years with MYCN-amplified tumors were eligible. The primary endpoint was EFS. Patients were randomly assigned to standard induction therapy with six chemotherapy courses or to experimental induction chemotherapy starting with two additional courses of topotecan, cyclophosphamide, and etoposide followed by standard induction chemotherapy (eight courses in total). After induction chemotherapy, all patients received high-dose chemotherapy with autologous hematopoietic stem cell rescue and isotretinoin for consolidation. Radiotherapy was applied to patients with active tumors at the end of induction chemotherapy. RESULTS: Of 536 patients enrolled in the trial, 422 were randomly assigned to the control arm (n = 211) and the experimental arm (n = 211); the median follow-up time was 3.32 years (interquartile range 1.65-5.92). At data lock, the 3-year EFS of experimental and control patients was 34% and 32% [95% confidence Interval (CI) 28% to 40% and 26% to 38%; P = 0.258], respectively. Similarly, the 3-year overall survival of the patients did not differ [54% and 48% (95% CI 46% to 62% and 40% to 56%), respectively; P = 0.558]. The response to induction chemotherapy was not different between the arms. The median number of non-fatal toxicities per patient was higher in the experimental group while the median number of toxicities per chemotherapy course was not different. CONCLUSION: While the burden for the patients was increased by prolonging the induction chemotherapy and the toxicity, the addition of two topotecan-containing chemotherapy courses did not improve the EFS of high-risk neuroblastoma patients and thus cannot be recommended. CLINICAL TRIALS. GOV NUMBER: NCT number 03042429.

Long-term renal prognosis and risk for hypertension after myeloablative therapies in survivors of childhood high-risk neuroblastoma: A nationwide study.

BACKGROUND: Patients with high-risk neuroblastoma (HR NBL) treated with myeloablative regimens are reported to be at risk for cardiovascular morbidity, and this risk may be increased by impaired renal function. PROCEDURE: Long-term renal function was assessed in a national cohort of 18 (age 22.4 ± 4.9 years) HR NBL survivors by plasma creatinine (P-Cr), urea, and cystatin C (P-Cys C) concentrations, urine albumin/creatinine ratio (ACR), and estimated glomerular filtration rate (eGFR). Ambulatory blood pressure was monitored, and common carotid intima-media thickness (CIMT) and left ventricular mass index (LVMI) were evaluated. RESULTS: No significant difference in P-Cr, P-Cys C, or eGFR was found between the NBL survivors and the age- and sex-matched 20 controls. P-Cys C-based eGFR (eGFRcysc) was significantly lower than the P-Cr-based eGFRcr (97 ± 17 mL/min/1.73 m2 vs 111 ± 19 mL/min/1.73 m2 , P < 0.001) among the NBL survivors. The eGFRcysc was below normal in 28%, and ACR was above normal in 22% of the NBL survivors. Abnormal blood pressure was found in 56% of the survivors, and an additional 17% were normotensive at daytime but had significant nocturnal hypertension. Both ACR and P-Cys C were associated with nighttime diastolic hypertension. CONCLUSIONS: Long-term survivors of childhood HR NBL showed signs of only mild renal dysfunction associated with diastolic hypertension. Elevated ACR and P-Cys C were the most sensitive indicators of glomerular renal dysfunction and hypertension in this patient cohort.

Is high-risk neuroblastoma induction chemotherapy possible without G-CSF? A pilot study of safety and treatment delays in the absence of primary prophylactic hematopoietic growth factors.

BACKGROUND/OBJECTIVES: Standard supportive care during induction therapy for high-risk neuroblastoma (HR-NBL) includes primary prophylactic granulocyte colony-stimulating factor (G-CSF) aimed at limiting duration of neutropenia, reducing infection risk, and minimizing treatment delays. Preclinical models suggest that G-CSF promotes maintenance of neuroblastoma cancer stem cells and may reduce the efficacy of chemotherapy. This study's objective was to determine the safety and feasibility of administering induction chemotherapy without routine use of prophylactic G-CSF. DESIGN/METHODS: Children with newly diagnosed HR-NBL received six-cycle induction chemotherapy regimen without prophylactic G-CSF in four cycles. G-CSF was administered for stem cell mobilization after cycle 3 and granulocyte-monocyte colony-stimulating factor after cycle 5 prior to surgical resection of primary disease. The primary outcome measure was the incidence of grade 3 or higher infection. We hypothesized that the per patient infection rate would be comparable to our institutional baseline rate of 58% in patients with HR-NBL receiving induction chemotherapy with prophylactic growth factor support. The trial used an A'Hern single-stage design. RESULTS: Twelve patients with HR-NBL received 58 cycles of chemotherapy on study. Three patients completed the entire six-cycle regimen with no infections. Nine patients experienced grade 3 infections (bacteremia four, urinary tract infection two, skin/soft tissue infection three). No patients experienced grade 4 infections or required intensive care treatment for infection. CONCLUSION: A greater than expected number of serious bacterial infections were observed during administration of induction chemotherapy for HR-NBL without primary prophylactic G-CSF. These results support continued prophylactic administration growth factor during induction chemotherapy.