High-risk Neuroblastoma Patients Research Articles

Investigation of patient factors associated with the number of transfusions required during chemotherapy for high-risk neuroblastoma.

Blood transfusion is an important supportive care for high-risk neuroblastoma. When the number of transfusions increases, transfusion-associated adverse reactions may be more problematic. However, the factors determining the degree of myelosuppression and the number of transfusions during chemotherapy for high-risk neuroblastoma remain unclear. We investigated patient factors determining the number of required transfusions in 15 high-risk neuroblastoma patients who received five courses of chemotherapy. Clinical data, cytokine profile and colony-forming assay with bone marrow samples at diagnosis were analysed. The required number of transfusions of both platelets and erythrocytes decreased once in the second course and then increased as the course progressed. The variability among cases increased as the chemotherapy course progressed. In cases of low peripheral blood platelet count and lower fibrinogen level at diagnosis, the number of platelet transfusions was higher during chemotherapy. In contrast, there was a negative correlation between the forming ability of granulocyte-macrophage or erythroid colonies and the number of erythrocyte transfusions in the latter period. In the early stages of chemotherapy, bone marrow infiltration in neuroblastoma and/or coagulopathy complication may cause thrombocytopenia and requirement of platelet transfusion; conversely, in the later stages, the number of erythrocyte transfusions may be defined by the patient's inherent hematopoietic ability. These factors may be useful in predicting the required number of transfusions.

Sarcopenia and preserved bone mineral density in paediatric survivors of high-risk neuroblastoma with growth failure.

BackgroundSurvival from paediatric high‐risk neuroblastoma (HR‐NBL) has increased, but cis‐retinoic acid (cis‐RA), the cornerstone of HR‐NBL therapy, can cause osteoporosis and premature physeal closure and is a potential threat to skeletal structure in HR‐NBL survivors. Sarcopenia is associated with increased morbidity in survivors of paediatric malignancies. Low muscle mass may be associated with poor prognosis in HR‐NBL patients but has not been studied in these survivors. The study objective was to assess bone density, body composition and muscle strength in HR‐NBL survivors compared with controls.MethodsThis prospective cross‐sectional study assessed areal bone mineral density (aBMD) of the whole body, lumbar spine, total hip, femoral neck, distal 1/3 and ultradistal radius and body composition (muscle and fat mass) using dual‐energy X‐ray absorptiometry (DXA) and lower leg muscle strength using a dynamometer. Measures expressed as sex‐specific standard deviation scores (Z‐scores) included aBMD (adjusted for height Z‐score), bone mineral apparent density (BMAD), leg lean mass (adjusted for leg length), whole‐body fat mass index (FMI) and ankle dorsiflexion peak torque adjusted for leg length (strength‐Z). Muscle‐specific force was assessed as strength relative to leg lean mass. Outcomes were compared between HR‐NBL survivors and controls using Student's t‐test or Mann–Whitney U test. Linear regression models examined correlations between DXA and dynamometer outcomes.ResultsWe enrolled 20 survivors of HR‐NBL treated with cis‐RA [13 male; mean age: 12.4 ± 1.6 years; median (range) age at therapy initiation: 2.6 (0.3–9.1) years] and 20 age‐, sex‐ and race‐matched controls. Height‐Z was significantly lower in HR‐NBL survivors compared with controls (−1.73 ± 1.38 vs. 0.34 ± 1.12, P < 0.001). Areal BMD‐Z, BMAD‐Z, FMI‐Z, visceral adipose tissue and subcutaneous adipose tissue were not significantly different in HR‐NBL survivors compared with controls. Compared with controls, HR‐NBL survivors had lower leg lean mass‐Z (−1.46 ± 1.35 vs. − 0.17 ± 0.84, P < 0.001) and strength‐Z (−1.13 ± 0.86 vs. − 0.15 ± 0.71, P < 0.001). Muscle‐specific force was lower in HR‐NBL survivors compared with controls (P < 0.05).ConclusionsBone mineral density and adiposity are not severely impacted in HR‐NBL survivors with growth failure, but significant sarcopenia persists years after treatment. Future studies are needed to determine if sarcopenia improves with muscle‐specific interventions in this population of cancer survivors.

Chimeric Antigen Receptor 4SCAR-GD2-Modified T Cells Targeting High-Risk and Recurrent Neuroblastoma: A Phase II Multi-Center Trial in China

Background: Neuroblastoma (NB) is one of the most common childhood malignancies responsible for 15% of pediatric cancer deaths. 70% of children with NB present with high-risk features characterized by severe metastasis and poor long-term survival even if the multimodal intensive combination therapies are used. There is ongoing challenge for developing novel therapeutic strategies for NB. GD2 is a well-characterized tumor antigen in NB. Anti-GD2 antibodies and T cells genetically modified to express GD2-specific chimeric antigen receptor (GD2-CART) have demonstrated anti-cancer activity. Here we report the safety and efficacy of a new generation of GD2-CAR (4SCAR-GD2) targeting high-risk NB with a 1-year follow-up (NCI registrations: NCT02992210 and NCT02765243).Patients and Methods: Total 34 high-risk NB patients, after confirmation for GD2 expression, are enrolled from 5 hospitals in China. These patients had undergone a series of standard therapies, including surgery, chemo- and radio-therapy before CART treatment. CD3 positive cells were selected from apheresis lymphocytes and activated before lentiviral infection. The cells were transduced with a caspase 9-inducible, safety-engineered lentivector CAR containing multiple intracellular signaling domains: GD2-scFv/CD28/CD27/CD3ζ-iCasp9. Before CART infusion, patients received cyclophosphamide and fludarabine conditioning. The CART infusion dose was at average 1.1x10^6/kg. Toxicity was determined based on NCI common toxicity criteria. The International Neuroblastoma Response Criteria were used to define response after CART treatment. The total observation time for all patients is 1 year after infusion.Results: GD2 expression was scored 0 to 4 by immunohistochemical staining of tumor sections, and total 142 specimens were stained. The results showed that 9% of specimens were negative and more than 71% scored more than 2. Total 34 patients were infused with 4SCAR-GD2 T cells: 24% of them experienced no adverse reactions (per NCI-CTC v.4.0), 50% had grade 1 and 26% had grade 2 toxicity responses including fever, skin rash and peripheral nerve pain. No grade 3 or 4 toxicity was observed. CART expansion kinetics were monitored by quantitative PCR of CAR in peripheral blood. The 4SCAR-GD2 illustrated long term persistence with the highest level of copy number detected at 30% on day 180 after CART infusion. Marked tumor regression was observed in 2 patients. At 28 days after infusion, by ultrasound the mediastinal and peritoneal tumors in one patient were reduced by 94% and 99.5%, respectively. The other patient had progressive tumor located in retroperitoneum and PET-CT showed shrinking tumor two months after CART treatment; retroperitoneal SUV decreased from 2.5 to 1.5. At the one-year observation point, 38% of patients were in stable disease, 15% had partial response and 47% were in disease progression. The 1-year overall survival rate is 74%.Conclusion: This multicenter trial demonstrates that the 4SCAR-GD2 T cells are safe and effective for treating high-risk stage IV NB children. The correlations of GD2 expression in tumor, CART kinetics in blood and treatment responses could not be statistically established, suggesting a vast disease heterogeneity. Important therapeutic correlates require a large patient cohort and long-term follow-ups to elucidate. DisclosuresYu:H3 Biomedicine Inc.: Employment.

The Olive Leaves Extract Has Anti-Tumor Effects against Neuroblastoma through Inhibition of Cell Proliferation and Induction of Apoptosis.

Neuroblastoma (NB) is the most common extra-cranial solid tumor of pediatric age. The prognosis for high-risk NB patients remains poor, and new treatment strategies are desirable. The olive leaf extract (OLE) is constituted by phenolic compounds, whose health beneficial effects were reported. Here, the anti-tumor effects of OLE were investigated in vitro on a panel of NB cell lines in terms of (i) reduction of cell viability; (ii) inhibition of cell proliferation through cell cycle arrest; (iii) induction of apoptosis; and (iv) inhibition of cell migration. Furthermore, cytotoxicity experiments, by combining OLE with the chemotherapeutic topotecan, were also performed. OLE reduced the cell viability of NB cells in a time- and dose-dependent manner in 2D and 3D models. NB cells exposed to OLE underwent inhibition of cell proliferation, which was characterized by an arrest of the cell cycle progression in G0/G1 phase and by the accumulation of cells in the sub-G0 phase, which is peculiar of apoptotic death. This was confirmed by a dose-dependent increase of Annexin V+ cells (peculiar of apoptosis) and upregulation of caspases 3 and 7 protein levels. Moreover, OLE inhibited the migration of NB cells. Finally, the anti-tumor efficacy of the chemotherapeutic topotecan, in terms of cell viability reduction, was greatly enhanced by its combination with OLE. In conclusion, OLE has anti-tumor activity against NB by inhibiting cell proliferation and migration and by inducing apoptosis.

Naxitamab combined with granulocyte-macrophage colony-stimulating factor as consolidation for high-risk neuroblastoma patients in complete remission.

Naxitamab is a humanized anti-disialoganglioside (GD2) monoclonal antibody approved for treatment of bone/bone marrow refractory high-risk neuroblastoma (HR-NB). Compassionate use (CU) expanded access program at Hospital Sant Joan de Deu permitted treatment of patients in complete remission (CR). We here report the survival, toxicity, and relapse pattern of patients in first or second CR treated with naxitamab and sargramostim (GM-CSF). Seventy-three consecutive patients with HR-NB (stage M at age >18months or MYCN-amplified stages L1/L2 at any age) were treated in first or second CR. Treatment comprised five cycles of subcutaneous (SC) GM-CSF for 5days at 250μg/m2 /day (days -4 to 0), followed by naxitamab + SC GM-CSF for 5days at 500μg/m2 /day (days 1-5). Naxitamab was infused over 30minutes at 3mg/kg/day, days 1, 3, and 5, outpatient. Fifty-five patients were in first CR and 18 in second CR. Seventeen patients had MYCN-amplified NB and 11 detectable minimal residual disease in the bone marrow. Fifty-eight (79.5%) patients completed therapy. Four (5%) experienced grade 4 toxicities and 10 (14%) early relapse. Three-year event-free survival (EFS) 58.4%, 95% CI=(43.5%, 78.4%) and overall survival (OS) 82.4%, 95% CI=(66.8%, 100%). First CR patients 3-year EFS 74.3%, 95% CI=(62.7%, 88.1%), and OS 91.6%, 95% CI=(82.4%, 100%). EFS is significantly different between first and second CR (p=.0029). The pattern of relapse is predominantly (75%) of an isolated organ, mainly bone (54%). Univariate Cox models show prior history of relapse as the only statistically significant predictor of EFS but not OS. Consolidation with naxitamab and GM-CSF resulted in excellent survival rates for HR-NB patients in CR.

Efficacy of naxitamab in patients with refractory/relapse (R/R) high-risk neuroblastoma (HR-NB) by bone/bone marrow (BM) evaluation, potential sites of residual disease.

10022 Background: NB is the most common extracranial solid tumor in children and half of patients present with high-risk disease. Bone and BM are frequent sites of metastatic disease and can serve as a reservoir for residual disease driving relapse. Naxitamab, a GD2-binding monoclonal antibody, was recently approved in the United States in combination with GM-CSF for the treatment of pediatric patients ≥1 year of age and adult patients with R/R HR-NB in the bone/BM who have demonstrated a partial response (PR), minor response (MR), or stable disease (SD) to prior therapy. Here we describe outcomes from the registrational Trial 201 (NCT03363373) detailed by bone/BM involvement. Methods: HR-NB patients with primary refractory disease or incomplete response to salvage treatment following relapse or progressive disease (PD) (in both cases including SD, MR and PR) with disease limited to bone and/or BM were eligible. Naxitamab was administered over ≥30 min in the outpatient setting on Days 1, 3 and 5 at 3 mg/kg/infusion (9 mg/kg/cycle) in combination with GM-CSF at 250 µg/m2/day on Days -4 to 0 and at 500 µg/m2/day on days 1 to 5. Treatment cycles were repeated every 4 weeks. Response was assessed after Cycle 2 and then every 2-3 months by revised International Neuroblastoma Response Criteria (INRC) using BM biopsies/aspirates and 123I-MIBG scintigraphy or FDG-PET. Effectiveness was concluded if the lower limit of the Clopper-Pearson exact 95% confidence interval (CI) of overall response rate (ORR) was &gt;20%. We report efficacy data on 22 patients and safety data on the first 25 patients enrolled. Results: 13 (59%) patients had NB in bone, 2 (9%) had NB in BM, and 7 (32%) had NB in both bone and BM. Summary of overall response and response by compartment. Conclusions: Naxitamab provided clinically meaningful activity in both bone and BM with ORR of 68% and had a manageable AE profile. Clinical trial information: NCT03363373. [Table: see text]

Naxitamab and GM-CSF for consolidation of high-risk neuroblastoma (HR-NB) patients in complete remission.

10021 Background: Naxitamab was recently approved in the US in combination with GM-CSF for the treatment of patients (pts) with relapse/refractory HR-NB in the bone/bone marrow who have demonstrated a partial response (PR), minor response (MR), or stable disease (SD) to prior therapy. Here we describe the use of 5 cycles of naxitamab and GM-CSF through compassionate use for consolidation of HR-NB pts in first or subsequent complete remission (CR). Methods: HR-NB pts in CR (first or subsequent) after initial multimodal induction treatment were eligible. Disease status was assessed at study entry by histology of BM biopsies/aspirates obtained from bilateral posterior and bilateral anterior iliac crests, I-MIBG SPECT scan, and whole body MRI. FDG-PET was used for MIBG non-avid cases at diagnosis. Quantitative reverse transcription-polymerase chain reaction was used to assess MRD in pooled heparinized BM aspirates. Disease response was defined according to the revised INRC. Four BM aspirates and 123I-MIBG SPECT scan or FDG-PET scans were performed after cycles 2 and 5 and every 3 months thereafter for one year in all pts to assess response. Naxitamab was administered over ≥30 min in the outpatient setting on Days 1, 3 and 5 at 3.0 mg/kg/infusion (9.0 mg/kg/cycle) in combination with GM-CSF at 250 ug/m2/day on Days -4 to 0 and at 500 ug/m2/day on days 1 to 5. Treatment cycles were repeated every 4 weeks. Survival curves were built from the time of naxitamab treatment initiation by Kaplan-Meier methods and compared using the log-rank test. Results: From June 2017 to November 30 2020, 73 pts were treated: 55 (75%) in first CR and 18 (25%) in second or more CR. Majority of pts were MYCN non-amplified (n=56, 77%), all stage 4, median age at treatment initiation 4.5 years. 61 (84%) pts had received &gt;5 cycles of induction chemotherapy; 22 (30%) high-dose chemotherapy and autologous stem cell transplant (ASCT); and 36 (49%) radiotherapy before receiving naxitamab. 58 (79.5%) pts completed naxitamab therapy, 53 (73%) in continued CR. 10 (14%) pts relapsed during treatment and 5 (7%) had grade 4 toxicities: 2 apnea related to naxitamab; and 2 non-related: 1 opioid related chest rigidity syndrome and 1 stroke. 3-y event-free-survival (EFS) for all pts is 58% [95% CI, 43.5; 78.4], 74% for first CR and 19% for second or more CR (p=0.0029). 3-y OS for the whole group is 82% [95% CI, 66.8; 100.0], 92% for first CR and 66% for second or more CR pts (p=0.18). Univariate Cox models for CR group, MYCN status, number of chemotherapies, ASCT, radiotherapy, MRD, and age showed significant p value only for prior relapse as predictor of EFS (p=0.047). Conclusions: Naxitamab for HR-NB pts in CR provided excellent 3-y OS rates regardless of previous management. The only predictor for relapse is prior history of recurrence.

Predicting response to chemotherapy in neuroblastoma using deep learning: A report from the International Neuroblastoma Risk Group.

10039 Background: Metaiodobenzylguanidine (MIBG) scans are a radionucleotide imaging modality used to evaluate neuroblastoma stage at diagnosis and also determine disease response following therapy. Curie scoring is used to semi-quantitatively assess disease burden from an MIBG scan on a scale from none (0) to widespread throughout the body (30). While a Curie score ≤2 after six cycles of induction chemotherapy has been shown to be prognostic of outcome, there is no established correlation between diagnostic Curie score and outcome. Deep learning models, such as convolutional neural networks (CNN), have been shown to learn generalizable patterns within images for successful classification of metastases and detection of multiple adult cancers. We hypothesized a CNN could be developed to predict response to induction chemotherapy, a proxy for outcome, using diagnostic MIBG scans. Methods: DICOM MIBG scans and associated clinical data from a Children’s Oncology Group (COG) pilot study for children diagnosed with high-risk neuroblastoma (ANBL12P1; NCT1798004) were deidentified and linked to clinical data by the Pediatric Cancer Data Commons and obtained from the International Neuroblastoma Risk Group Data Commons. Patients were defined as having a poor response to induction chemotherapy if their Curie score after four cycles of induction chemotherapy was ≥2. An independent external validation cohort was comprised of 29 images from 26 high-risk patients treated at the University of Chicago with clinically-annotated diagnostic and post-cycle six induction DICOM MIBG scans. The CNN was trained using 2D whole body MIBG scans obtained at diagnosis. We developed the CNN using a transfer learning approach using the Xception architecture as the base layer. Hyperparameter optimization was performed using an 80%-20% train-validation strategy. Model performance was evaluated using area under the receiver operating characteristic curve (AUROC). Results: Among 146 patients with high-risk neuroblastoma enrolled on ANBL12P1, 104 had available diagnostic and end-induction MIBG scans. There were no differences in clinical or biological characteristics between included and excluded patients. The base model CNN was able to predict which patients had a poor response to induction chemotherapy with an AUROC of 0.72 in the validation set from the ANBL12P1 cohort. Additionally, the CNN was able to predict patient response to therapy with an AUROC of 0.64 in an independent external dataset from University of Chicago. Conclusions: Our study suggests it is feasible to apply machine learning of diagnostic MIBG scans to predict response to chemotherapy for high-risk neuroblastoma patients. Given these promising results, further work to improve AUROC and performance within larger datasets is ongoing.

Limited correlation between tumor markers and minimal residual disease detected by seven neuroblastoma-associated mRNAs in high-risk neuroblastoma patients.

Vanillylmandelic acid (VMA), homovanillic acid (HVA), neuron-specific enolase (NSE) and lactate dehydrogenase (LDH) are classical tumor markers and are used as standard clinical evaluations for patients with neuroblastoma (NB). Minimal residual disease (MRD) can be monitored by quantifying several sets of NB-associated mRNAs in the bone marrow (BM) and peripheral blood (PB) of patients with NB. Although MRD in BM and PB has been revealed to be a strong prognostic factor that is independent of standard clinical evaluations, its interrelation with tumor markers remains uncharacterized. The present study determined the levels of tumor markers (VMA, HVA, NSE and LDH) and MRD (BM-MRD and PB-MRD) in 133 pairs of concurrently collected BM, PB and urine samples from 19 patients with high-risk NB. The patients were evaluated during the entire course of treatment, which included 10 diagnoses, 32 treatments, 36 post-treatment, 9 relapses and 46 post-relapse sample pairs. The level of BM-MRD and PB-MRD was determined by quantifying 7 NB-mRNAs (collapsin response mediator protein 1, dopamine beta-hydroxylase, dopa decarboxylase, growth-associated protein 43, ISL LIM homeobox 1, pairedlike homeobox 2b and tyrosine hydroxylase) using droplet digital PCR. In overall sample pairs, tumor markers (VMA, HVA, NSE and LDH) demonstrated weak but significant correlations (P<0.011) with BM-MRD and PB-MRD. In subgroups according to each patient evaluation, the degree of correlation between tumor markers and MRD became stronger in patients with adrenal gland tumors, BM metastasis at diagnosis and relapse/regrowth compared with overall sample pairs. In contrast, tumor markers demonstrated variable correlations with MRD in subgroups according to each sample evaluation (BM infiltration at sampling, collection time point and disease status). The results suggested that tumor markers may demonstrate limited correlation with MRD in patients with high-risk NB.

Minimal residual disease in high-risk neuroblastoma shows a dynamic and disease burden-dependent correlation between bone marrow and peripheral blood

Neuroblastoma (NB) is the most common extracranial solid tumor in children and originates from sympathoadrenal or Schwann cell precursors derived from neural crest. These neural crest derivatives also constitute the hematopoietic and mesenchymal stem cells in bone marrow (BM) that is the most frequent site of NB metastasis and relapse. In NB patients, NB cells have been pathologically detected in BM and peripheral blood (PB), and minimal residual disease (MRD) in BM and PB (BM-MRD and PB-MRD) can be monitored by quantitating several sets of NB-associated mRNAs (NB-mRNAs). Although previous studies have shown varying degrees of correlation between BM-MRD and PB-MRD, the underlying factors and/or mechanisms remains unknown. In the present study, we determined the levels of BM-MRD and PB-MRD by quantitating seven NB-mRNAs in 133 pairs of concurrently collected BM and PB samples from 19 high-risk NB patients with clinical disease evaluation, and examined their correlation in overall and subgroups of sample pairs. The levels of BM-MRD and PB-MRD were moderately (r=0.418, p<0.001) correlated with each other in overall sample pairs. The correlation became strong (r=0.725, p<0.001), weak (r=0.284, p=0.008), and insignificant (p=0.194) in progression, stable, and remission subgroups of sample pairs, respectively. It also became stronger in subgroups of sample pairs with poor treatment responses and poor prognostic factors. Present study suggests that MRD in high-risk NB shows a dynamic and disease burden-dependent correlation between BM and PB.

Analysis of Monoamine and Organic Cation Transporter Expression in Neuroblastoma and Relationship with Overall Survival

Neuroblastoma (NB) is a neuroendocrine cancer with great clinical heterogeneity and most prevalent in children. Despite recent improvements in therapy, long term survival of high-risk NB patients is still below 50%. Although amplification of the proto-oncogene MYCN is a well-established risk factor in NB, little is known about prognosis markers for high-risk NB patients without MYCN amplification. On account of being a cancer originated from early sympathetic nerve cells, the majority of NB tumors express the norepinephrine transporter (NET). NET is a solute carrier (SLC) monoamine transporter known to play an important role in targeting radiopharmaceuticals (e.g. 123I- or 131I-mIBG) for NB diagnosis and treatment. However, little is currently known about the expression of other functionally related SLC transporters in NB. Here, we analyzed the expression of MYCN, NET and other monoamine and organic cation transporters in NB patient samples and explored their association with overall survival of those patients. In order to perform this analysis, we mined the data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program database. The TARGET database contains gene expression data of tumor samples from multiple NB clinical trials consisting of predominantly high-risk or relapsed NB patients with predetermined MYCN amplification status. To compare transporter expression in NB, we analyzed mRNAseq data of NET, the dopamine and serotonin transporters (DAT and SERT), the plasma membrane monoamine transporter (PMAT), vesicular monoamine transporters (VMAT1/2), organic cation transporters (OCT1-3), and multidrug and toxin extrusion proteins (MATE1/2-K) in NB tumor samples and normalized their expression to GAPDH. The association between transporter expression and the overall survival of patients was then explored using Gene Expression Array data from 243 NB patient samples matched to clinical outcomes. Patient samples were divided into high and low expression for each transporter and survival analysis was performed using Kaplan-Meier curves. Our data revealed that NET, PMAT and VMAT1/2 are highly expressed in NB tumors especially in MYCN non-amplified samples. In contrast, DAT, SERT, OCT1-3, and MATE1/2-K are minimally or not expressed in NB tumor samples. Interestingly, survival analysis using Kaplan-Meier curves and Log-rank test indicates a significant association between low expression of PMAT and VMAT1/2 and poor overall survival, especially in patients without MYCN amplification. Our study suggests that besides NET, PMAT and VMAT1/2 are highly expressed in NB tumor samples and can be explored for targeting therapeutics in NB tumor cells. The apparent association of PMAT and VMAT1/2 with overall survival suggests that these transporters could be further explored as biomarkers for disease progression and risk prediction for NB patients without MYCN amplification.

Immune Monitoring during Therapy Reveals Activitory and Regulatory Immune Responses in High-Risk Neuroblastoma.

Simple SummaryNeuroblastoma is a type of childhood cancer accounting for approximately 15% of childhood cancer deaths. Despite intensive treatment, including immunotherapy, prognosis of high-risk neuroblastoma is poor. Increasing amounts of research show that the fighting capacity of the immune system is very important for the outcome of neuroblastoma patients. Therefore, we investigated the fighting capacity of immune cells in blood at diagnosis and during the different phases of therapy. In this study, we observed both processes that stimulate and processes that decrease fighting capacity of immune cells in neuroblastoma patients during therapy. Despite this, we show that overall fighting capacity of the immune system of neuroblastoma patients is impaired at diagnosis as well as during therapy. In addition, we observed a lot of variation between patients, which might explain differences in therapy efficacy between patients. This study provides insight for improvement of therapy timing as well as new therapy strategies enhancing immune cell fighting capacity.Despite intensive treatment, including consolidation immunotherapy (IT), prognosis of high-risk neuroblastoma (HR-NBL) is poor. Immune status of patients over the course of treatment, and thus immunological features potentially explaining therapy efficacy, are largely unknown. In this study, the dynamics of immune cell subsets and their function were explored in 25 HR-NBL patients at diagnosis, during induction chemotherapy, before high-dose chemotherapy, and during IT. The dynamics of immune cells varied largely between patients. IL-2- and GM-CSF-containing IT cycles resulted in significant expansion of effector cells (NK-cells in IL-2 cycles, neutrophils and monocytes in GM-CSF cycles). Nonetheless, the cytotoxic phenotype of NK-cells was majorly disturbed at the start of IT, and both IL-2 and GM-CSF IT cycles induced preferential expansion of suppressive regulatory T-cells. Interestingly, proliferative capacity of purified patient T-cells was impaired at diagnosis as well as during therapy. This study indicates the presence of both immune-enhancing as well as regulatory responses in HR-NBL patients during (immuno)therapy. Especially the double-edged effects observed in IL-2-containing IT cycles are interesting, as this potentially explains the absence of clinical benefit of IL-2 addition to IT cycles. This suggests that there is a need to combine anti-GD2 with more specific immune-enhancing strategies to improve IT outcome in HR-NBL.

Non-inferiority of fragmented care for high-risk pediatric neuroblastoma patients: a single institution analysis

Pediatric neuroblastoma (NB) patients receive multi-modal therapy and may experience care fragmented among multiple institutions with a significant travel burden, which has been associated with poor outcomes for some adult cancers. We hypothesized that fragmented care for pediatric NB patients is associated with inferior outcomes compared to treatment consolidated at one location. We reviewed paper and electronic records for pediatric NB patients who received ≥1 hematopoietic stem cell transplant (HSCT) at Duke from 1990-2017. Fragmented care was defined by treatment at >1 institution and grouped by 2 institutions vs. 3+ institutions. Distances were calculated using Google Maps. To compare all care groups, we used Fisher’s Exact and Kruskal-Wallis tests for demographic and treatment characteristics, Kaplan-Meier for unadjusted overall survival (OS), and Cox proportional hazards for factors associated with OS. Of 127 eligible patients, 102 (80.3%) patients experienced fragmented care, with 17 treated at 3+ facilities. Kaplan-Meier analysis did not associate fragmented care with increased mortality (log-rank p = 0.13). With multivariate analysis, only earlier diagnostic decade and greater distance to HSCT remained significantly associated with worsened OS. In this single institutional study, we found fragmented care did not impact overall survival. Worsened overall survival was associated with increased travel distance for HSCT and further research should aim to improve supportive processes for patients undergoing HSCT for high-risk neuroblastoma.

Unraveling Gene Fusions for Drug Repositioning in High-Risk Neuroblastoma.

High-risk neuroblastoma (NB) remains a significant therapeutic challenge facing current pediatric oncology patients. Structural variants such as gene fusions have shown an initial promise in enhancing mechanistic understanding of NB and improving survival rates. In this study, we performed a comprehensive in silico investigation on the translational ability of gene fusions for patient stratification and treatment development for high-risk NB patients. Specifically, three state-of-the-art gene fusion detection algorithms, including ChimeraScan, SOAPfuse, and TopHat-Fusion, were employed to identify the fusion transcripts in a RNA-seq data set of 498 neuroblastoma patients. Then, the 176 high-risk patients were further stratified into four different subgroups based on gene fusion profiles. Furthermore, Kaplan-Meier survival analysis was performed, and differentially expressed genes (DEGs) for the redefined high-risk group were extracted and functionally analyzed. Finally, repositioning candidates were enriched in each patient subgroup with drug transcriptomic profiles from the LINCS L1000 Connectivity Map. We found the number of identified gene fusions was increased from clinical the low-risk stage to the high-risk stage. Although the technical concordance of fusion detection algorithms was suboptimal, they have a similar biological relevance concerning perturbed pathways and regulated DEGs. The gene fusion profiles could be utilized to redefine high-risk patient subgroups with significant onset age of NB, which yielded the improved survival curves (Log-rank p value ≤ 0.05). Out of 48 enriched repositioning candidates, 45 (93.8%) have antitumor potency, and 24 (50%) were confirmed with either on-going clinical trials or literature reports. The gene fusion profiles have a discrimination power for redefining patient subgroups in high-risk NB and facilitate precision medicine-based drug repositioning implementation.

Clinical Relevance of CD4 Cytotoxic T Cells in High-Risk Neuroblastoma.

Neuroblastoma is the most common extracranial childhood solid tumor. The majority of high-risk neuroblastoma is resistant/refractory to the current high intensity therapy, and the survival of these patients remains poor for the last three decades. To effectively treat these extremely unfavorable neuroblastomas, innovative immunotherapy approaches would be the most promising. In this article, we discuss the identity of tumor-infiltrating effector cells and immunosuppressive cells in high-risk neuroblastoma. Neuroblastoma is unique in that it expresses little or no classical HLA Class I and II. In contrast, high-risk neuroblastomas express the stress-responsive non-classical Class I, HLA-E molecule. HLA-E is the ligand of activating receptors NKG2C/E that are expressed on memory NK cells, CD8+T cells and CD4 CTLs. By examining a comprehensive RNA-seq gene expression dataset, we detected relatively high levels of CD4 expression in high-risk neuroblastoma tissues. The majority of CD4+ cells were CD3+, and thus they were likely tumor-associated CD4+T cells. In addition, high-level of both CD4 and NKG2C/E expression was associated with prolonged survival of the high-risk neuroblastoma patients, but CD8 levels were not, further suggesting that the CD4+ NKG2C/E+ T cells or CD4 CTL conferred cytotoxicity against the neuroblastoma cells. However, this T cell mediated- “protective effect” declined over time, in part due to the progressive formation of immunosuppressive tumor microenvironment. These observations suggest that to improve survival of high-risk neuroblastoma patients, it is essential to gain insights into how to enhance CD4 CTL cytotoxicity and control the immunosuppressive tumor microenvironment during the course of the disease.

Combined Effects of Myeloid Cells in the Neuroblastoma Tumor Microenvironment.

Simple SummaryNeuroblastoma is a type of childhood solid cancer often characterized by resistance to treatment and with poor prognosis. The tumors contain a mixture of cell types as well as cancer cells, and one of the most common is a type of blood cells called myeloid cells. In this review article we present a summary of what is known about the function of myeloid cells within neuroblastoma tumors.Despite multimodal treatment, survival chances for high-risk neuroblastoma patients remain poor. Immunotherapeutic approaches focusing on the activation and/or modification of host immunity for eliminating tumor cells, such as chimeric antigen receptor (CAR) T cells, are currently in development, however clinical trials have failed to reproduce the preclinical results. The tumor microenvironment is emerging as a major contributor to immune suppression and tumor evasion in solid cancers and thus has to be overcome for therapies relying on a functional immune response. Among the cellular components of the neuroblastoma tumor microenvironment, suppressive myeloid cells have been described as key players in inhibition of antitumor immune responses and have been shown to positively correlate with more aggressive disease, resistance to treatments, and overall poor prognosis. This review article summarizes how neuroblastoma-driven inflammation induces suppressive myeloid cells in the tumor microenvironment and how they in turn sustain the tumor niche through suppressor functions, such as nutrient depletion and generation of oxidative stress. Numerous preclinical studies have suggested a range of drug and cellular therapy approaches to overcome myeloid-derived suppression in neuroblastoma that warrant evaluation in future clinical studies.

Thiotepa-melphalan myeloablative therapy for high-risk neuroblastoma.

Appropriate high-dose chemotherapy (HDC) for high-risk neuroblastoma has not yet been established. In Japan, a unique HDC regimen that comprises two cycles of a total of 800mg/m2 of thiotepa and a total of 280mg/m2 of melphalan is widely utilized. To evaluate the safety and efficacy of this thiotepa-melphalan high-dose therapy for high-risk neuroblastoma, we reviewed the medical records of 41 patients with high-risk neuroblastoma who underwent this regimen followed by autologous peripheral blood stem cell rescue between 2002 and 2012. MYCN-amplified high-risk neuroblastomas were observed in 23 patients. All patients underwent intensive multidrug induction chemotherapy, but none underwent anti-GD2 antibody immunotherapy. The primary tumor was resected at the adequate time point. The median follow-up duration for living patients was 9.2years (range 5.5-14.0years). The 5-year event-free survival (EFS) and overall survival from treatment initiation were 41.5±7.7% and 56.1±7.8%, respectively. The 5-year EFS of MYCN-amplified high-risk neuroblastoma patients was 60.9±10.2%, which was significantly superior compared with those with MYCN-nonamplified high-risk neuroblastoma (16.7±8.8%; p<.001). MYCN amplification was the most favorable prognostic factor for EFS (hazard ratio=0.29; 95% confidence interval=0.12-0.66). Of the 41 patients, three died because of regimen-related toxicity (infection, n=2; microangiopathy, n=1). The thiotepa-melphalan high-dose therapy with thiotepa and melphalan may be effective for high-risk neuroblastoma. However, this regimen is toxic and warrants special attention in clinical practice.

Temporal Quantitative Proteomics Analysis of Neuroblastoma Cells Treated with Bovine Milk-Derived Extracellular Vesicles Highlights the Anti-Proliferative Properties of Milk-Derived Extracellular Vesicles.

Neuroblastoma (NBL) is a pediatric cancer that accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc occurs in 20% of NBL patients and is considered high risk as it correlates with aggressiveness, treatment resistance and poor prognosis. Even though the treatment strategies have improved in the recent years, the survival rate of high-risk NBL patients remain poor. Hence, it is crucial to explore new therapeutic avenues to sensitise NBL. Recently, bovine milk-derived extracellular vesicles (MEVs) have been proposed to contain anti-cancer properties. However, the impact of MEVs on NBL cells is not understood. In this study, we characterised MEVs using Western blotting, NTA and TEM. Importantly, treatment of NBL cells with MEVs decreased the proliferation and increased the sensitivity of NBL cells to doxorubicin. Temporal label-free quantitative proteomics of NBL cells highlighted the depletion of proteins involved in cell metabolism, cell growth and Wnt signalling upon treatment with MEVs. Furthermore, proteins implicated in cellular senescence and apoptosis were enriched in NBL cells treated with MEVs. For the first time, this study highlights the temporal proteomic profile that occurs in cancer cells upon MEVs treatment.

Pediatric high risk neuroblastoma with autologous stem cell transplant - 20 years of experience.

Background and ObjectiveNeuroblastoma is the most common extracranial solid tumor found in pediatric patients. High-risk neuroblastoma (HR-NBL) can be characterized by metastasis, age, and other tumor characteristics that result in an adverse outlook for this patient cohort. The standard of care includes induction chemotherapy, surgery, followed by stem cell autologous transplant (ASCT), and later, antidisialoganglioside (anti-GD2) antibodies. In this study, we provide the survival and toxicity data of our HR-NBL patients treated with a single ASCT.MethodsWe retrospectively analyzed pediatric HR-NBL patients treated with single ASCT after a carboplatin, etoposide, and melphalan (CEM) regimen in our institution between January 1993 and December 2014.ResultsThere were 99 evaluable patients with male predominance. The median age at diagnosis was 3 years. Most of our HR-NBL patients were stage 4 (88%). All patients received ASCT. Peripheral blood was the graft source in 58% of the patients. Time for hematological count recovery with bone marrow as a graft source was prolonged but not statistically significant when compared with PBSCs. Of all the patients, 58% received radiation therapy to residual disease. Overt secondary leukemia was not seen in any of these patients. Three-year overall survival (OS) was 68.5% ± 5.2% and the 3-year event-free survival (EFS) was (48.3% ± 5.2%).ConclusionOur HR-NBL patients tolerated high-dose chemotherapy well followed by single autologous stem cell transplant. Tandem transplant is a feasible option in our patient cohort. Apart from secondary solid tumors, there were no long-term complications seen.

Treatment-Related Toxicities During Anti-GD2 Immunotherapy in High-Risk Neuroblastoma Patients.

The introduction of immunotherapy using an anti-GD2 antibody (dinutuximab, ch14.18) has significantly improved survival rates for high-risk neuroblastoma patients. However, this improvement in survival is accompanied by a substantial immunotherapy-related toxicity burden. The primary objective of this study was to describe treatment-related toxicities during immunotherapy with dinutuximab, IL-2, GM-CSF, and isotretinoin. A retrospective, single center analysis of immunotherapy-related toxicities was performed in twenty-six consecutive high-risk neuroblastoma patients who received immunotherapy as maintenance therapy in the Princess Máxima Center (Utrecht, Netherlands). Toxicities were recorded and graded according to the CTCAE. Particular attention was drawn to pain and fever management and toxicities leading to dose modifications of dinutuximab and IL-2. Twenty-three patients (88%) completed all six courses of immunotherapy. Disease progression, isotretinoin-associated liver toxicity, and catheter-related infection in combination with peripheral neuropathy were reasons for immunotherapy discontinuation. The most common grade ≥3 toxicities for courses 1–5, respectively, were pain, catheter-related infections, and fever. In total, 310 grade ≥3 toxicities were recorded in 124 courses. Thirty-three grade 4 toxicities in 19/26 patients and no grade 5 toxicities (death) were seen. Fifty-nine percent of grade ≥3 toxicities were recorded in the two courses with IL-2. Catheter-related bloodstream infections were identified in 81% of patients. Four of these episodes led to intensive care admission followed by full recovery (grade 4).