Survival Of High-risk Neuroblastoma Patients Research Articles

S-100 and MATH-1 Protein Expressions Can Be Useful for the Prediction of Clinical Outcome in Neuroblastoma Patients.

Neuroblastoma (NB) is the most frequent extracranial solid tumor of childhood, remarkable for its broad spectrum of clinical behavior. This diversity in behavior correlates closely with defined clinical and biological features and combinations of prognostic variables are used for risk-group assignment. S-100 proteins have roles in differentiation and were shown to be frequently dysregulated in NB. MATH-1 protein plays role in neuronal cell differentiation through development. However, up to date, there are no studies evaluating the relationship between MATH-1 and NB. Grb2-associated binding (Gab) proteins have roles in the regulation of cell growth and differentiation. Gab1 was reported to be related to poor survival of high-risk NB patients. The aim of this study was to investigate the relationship between differentiation-related S-100, MATH-1, and Gab1 proteins and risk group and/or stages of NB. A significant relation was found between S-100 and early stages of NB. This study also revealed a significant association between MATH-1 and low-risk groups. S-100 and MATH-1 were also shown to provide survival advantages among stages and risk groups. The findings of this study support the assumption that S-100 and MATH-1 can be potential prognostic biomarkers for staging and risk-group assignment of NB patients. These proteins can be useful tools for clinicians to guide through treatment options, especially for the evaluation of tumor differentiation.

The Plasma Membrane Monoamine Transporter is Highly Expressed in Neuroblastoma and Functions as an mIBG Transporter.

Neuroblastoma (NB) is a pediatric cancer with low survival rates in high-risk patients. 131I-mIBG has emerged as a promising therapy for high-risk NB and kills tumor cells by radiation. Consequently, 131I-mIBG tumor uptake and retention are major determinants for its therapeutic efficacy. mIBG enters NB cells through the norepinephrine transporter (NET), and accumulates in mitochondria through unknown mechanisms. Here we evaluated the expression of monoamine and organic cation transporters in high-risk NB tumors and explored their relationship with MYCN amplification and patient survival. We found that NB mainly expresses NET, the plasma membrane monoamine transporter (PMAT), and the vesicular membrane monoamine transporter 1/2 (VMAT1/2), and that the expression of these transporters is significantly reduced in MYCN-amplified tumor samples. PMAT expression is the highest and correlates with overall survival in high-risk NB patients without MYCN amplification. Immunostaining showed that PMAT resides intracellularly in NB cells and co-localizes with mitochondria. Using cells expressing PMAT, mIBG was identified as a PMAT substrate. In mitochondria isolated from NB cell lines, mIBG uptake was reduced by ∼50% by a PMAT inhibitor. Together, our data suggest that PMAT is a previously unrecognized transporter highly expressed in NB and could impact intracellular transport and therapeutic response to 131I-mIBG. SIGNIFICANCE STATEMENT: This study identified that plasma membrane monoamine transporter (PMAT) is a novel transporter highly expressed in neuroblastoma and its expression level is associated with overall survival rate in high-risk patients without MYCN amplification. PMAT is expressed intracellularly in neuroblastoma cells, transports meta-iodobenzylguanidine (mIBG) and thus could impact tumor retention and response to 131I-mIBG therapy. These findings have important clinical implications as PMAT could represent a novel molecular marker to help inform disease prognosis and predict response to 131I-mIBG therapy.

Axial Skeleton Radiomics of 18F-FDG PET/CT: Impact on Event-Free Survival Prediction in High-Risk Pediatric Neuroblastoma

To construct and validate a combined model based on axial skeleton radiomics of 18F-FDG PET/CT for predicting event-free survival in high-risk pediatric neuroblastoma patients. Eighty-seven high-risk neuroblastoma patients were retrospectively enrolled in this study and randomized in a 7:3 ratio to the training and validation cohorts. The radiomics model was constructed using radiomics features that were extracted from the axial skeleton. A univariate Cox regression analysis was then performed to screen clinical risk factors associated with event-free survival for building clinical model. Radiomics features and clinical risk factors were incorporated to construct the combined model for predicting the event-free survival in high-risk neuroblastoma patients. The performance of the models was evaluated by the C-index. Eighteen radiomics features were selected to build the radiomics model. The radiomics model achieved better event-free survival prediction than the clinical model in the training cohort (C-index: 0.846 vs. 0.612) and validation cohort (C-index: 0.754 vs. 0.579). The combined model achieved the best prognostic prediction performance with a C-index of 0.863 and 0.799 in the training and validation cohorts, respectively. The combined model integrating radiomics features and clinical risk factors showed more accurate predictive performance for event-free survival in high-risk pediatric neuroblastoma patients, which helps to design individualized treatment strategies and regular follow-ups.

Inducing mismatch repair deficiency sensitizes immune-cold neuroblastoma to anti-CTLA4 and generates broad anti-tumor immune memory

Immune checkpoint blockade can induce potent and durable responses in patients with highly immunogenic mismatch repair-deficient tumors; however, these drugs are ineffective against immune-cold neuroblastoma tumors. To establish a role for a Tcell-based therapy against neuroblastoma, we show that Tcell and memory Tcell-dependent gene expression are associated with improved survival in high-risk neuroblastoma patients. To stimulate anti-tumor immunity and reproduce this immune phenotype in neuroblastoma tumors, we used CRISPR-Cas9 to knockout MLH1-a crucial molecule in the DNA mismatch repair pathway-to induce mismatch repair deficiency in a poorly immunogenic murine neuroblastoma model. Induced mismatch repair deficiency increased the expression of proinflammatory genes and stimulated Tcell infiltration into neuroblastoma tumors. In contrast to adult cancers with induced mismatch repair deficiency, neuroblastoma tumors remained unresponsive to anti-PD1 treatment. However, anti-CTLA4 therapy was highly effective against these tumors. Anti-CTLA4 therapy promoted immune memory and Tcell epitope spreading in cured animals. Mechanistically, the effect of anti-CTLA4 therapy against neuroblastoma tumors with induced mismatch repair deficiency is CD4+ Tcell dependent, as depletion of these cells abolished the effect. Therefore, a therapeutic strategy involving mismatch repair deficiency-based Tcell infiltration of neuroblastoma tumors combined with anti-CTLA4 can serve as a novel Tcell-based treatment strategy for neuroblastoma.

Asphericity of tumor [123 I]mIBG uptake as a prognostic factor in high-risk neuroblastoma.

In recent years, many research groups have attempted to identify a subgroup of "ultra-high risk" patients within the high-risk neuroblastoma (NB) category. The aim of our study was to evaluate the prognostic significance of parameters derived from pretherapeutic 123 I-meta-iodobenzylguanidine ([123 I]mIBG) integrated single photon emission computed tomography and computed tomography in high-risk patients with NB. The established parameters metabolic tumor volume (MTV), maximal standardized uptake value (SUVmax ) and the novel parameter tumor asphericity as well as clinical (age, stage) and genetic factors (1p/11q deletions and MYCN amplification) were analyzed in this single-center retrospective study of high-risk patients with newly diagnosed NB. Univariate/multivariable Cox regression and propensity score matching were performed for clinical and radiological parameters. Twenty-eight high-risk patients with NB were included (14 males, median age 28.8 (11.3-41.0), range 3-74months). Multivariable analysis of "full" cohort identified high asphericity (≥65%, adjusted hazard ratio [HR] 5.32, 95% confidence interval [CI]: 1.18-24.07, p=.03) and MTV (≥50ml, adjusted HR 4.31, 95% CI: 1.18-15.80, p=.027) as the only factors associated with worse event-free survival. In matched cohort, tumor asphericity was a significant predictor of relapse/progression (HR 3.83, 95% CI: 1.03-14.26, p=.046). In this exploratory study, imaging parameters related to tumor metabolic activity, tumor asphericity and MTV, provided prognostic value for event-free survival in high-risk NB patients. Asphericity ≥65% and MTV ≥50ml may serve as additional prognostic factors to those already used.

Matched external control analysis of event-free survival (EFS) in patients with high-risk neuroblastoma (HRNB) receiving eflornithine (DFMO) maintenance.

10010 Background: Long term survival in HRNB patients remains a challenge with relapse as the primary cause of mortality. DFMO has been evaluated as a chemopreventative therapy in a single arm study designed to compare EFS outcomes with published rates for the Phase 3 Children’s Oncology Group ch14.18 immunotherapy trial, ANBL0032. In order to address the limitations associated with comparison to a historical control group, we used ANBL0032 patient-level data and propensity-score matching (PSM) to simulate a randomized study comparing EFS of patients treated with DFMO after ch14.18 (treated) to ANBL0032 patients who did not subsequently receive DFMO (control). Methods: A phase 2 trial enrolled a total of 140 HRNB patients in remission at the completion of disease treatment from 2012 to 2016. Patients received 2 years of continuous treatment with DFMO 750 ±250 mg/m2 BID and were followed for up to 7 years. ANBL0032 enrolled a total of 1328 HRNB patients from 2001 to 2015 who were assigned to treatment with ch14.18 immunotherapy and followed for up to 10 years. With FDA input, we defined selection rules to identify like groups of treated and control patients eligible for matching, covariates of potential prognostic importance, and matching algorithm details. PSM was used to balance cohorts on their baseline demographic and characteristics, matching each treated patient with the 3 most closely scored control patients with an exact match on MYCN. The Kaplan-Meier method and Cox regression analyses were used to compare EFS (primary) and overall survival (OS) (key secondary) endpoints. Multiple sensitivity analyses were performed to further investigate the primary comparison. Results: A total of 92 treated patients and 852 control patients met selection criteria, with 91 and 516, respectively, having complete covariate data required for the analysis. Eighty-seven (94.6%) of the treated group had verified participation in ANBL0032 immediately prior to enrollment in the DFMO trial. EFS from end of immunotherapy was significantly improved in the matched DFMO group (n=90) vs. control group (n=270), with a hazard ratio of 0.48 (95% CI: 0.27, 0.85) and a p-value of 0.0114. Four-year EFS was 84.4% (95% CI 75.2, 90.5) in the DFMO group versus 72.8% (95% CI 67.0, 77.7) in the control group. OS rates were also higher for DFMO group in the matched population, with a hazard ratio of 0.36 (95% CI: 0.16, 0.79) and a p-value of 0.0105. EFS sensitivity analyses demonstrated consistent results, including those challenging selection criteria for the control population. Conclusions: Patients in remission after standard upfront therapy treated with DFMO had approximately half the risk of relapse compared to matched control patients. The PSM comparisons represent the most statistically robust findings to date supporting the benefit of DFMO as a maintenance treatment for HRNB. Clinical trial information: NCT02395666, NCT00026312.

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.

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.

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.

Identification of novel prognostic markers of survival time in high-risk neuroblastoma using gene expression profiles.

Neuroblastoma is the most common extracranial solid tumor in childhood. Patients in high-risk group often have poor outcomes with low survival rates despite several treatment options. This study aimed to identify a genetic signature from gene expression profiles that can serve as prognostic indicators of survival time in patients of high-risk neuroblastoma, and that could be potential therapeutic targets. RNA-seq count data was downloaded from UCSC Xena browser and samples grouped into Short Survival (SS) and Long Survival (LS) groups. Differential gene expression (DGE) analysis, enrichment analyses, regulatory network analysis and machine learning (ML) prediction of survival group were performed. Forty differentially expressed genes (DEGs) were identified including genes involved in molecular function activities essential for tumor proliferation. DEGs used as features for prediction of survival groups included EVX2, NHLH2, PRSS12, POU6F2, HOXD10, MAPK15, RTL1, LGR5, CYP17A1, OR10AB1P, MYH14, LRRTM3, GRIN3A, HS3ST5, CRYAB and NXPH3. An accuracy score of 82% was obtained by the ML classification models. SMIM28 was revealed to possibly have a role in tumor proliferation and aggressiveness. Our results indicate that these DEGs can serve as prognostic indicators of survival in high-risk neuroblastoma patients and will assist clinicians in making better therapeutic and patient management decisions.

Anti-GD2 induced allodynia in rats can be reduced by pretreatment with DFMO.

Anti-GD2 therapy with dinutuximab is effective in improving the survival of high-risk neuroblastoma patients in remission and after relapse. However, allodynia is the major dose-limiting side effect, hindering its use for neuroblastoma patients at higher doses and for other GD2-expressing malignancies. As polyamines can enhance neuronal sensitization, including development of allodynia and other forms of pathological pain, we hypothesized that polyamine depletion might prove an effective strategy for relief of anti-GD2 induced allodynia. Sprague-Dawley rats were allowed to drink water containing various concentrations of difluoromethylornithine (DFMO) for several days prior to behavioral testing. Anti-GD2 (14G2a) was injected into the tail vein of lightly sedated animals and basal mechanical hindpaw withdrawal threshold assessed by von Frey filaments. Endpoint serum DFMO and polyamines, assessed 24h after 14G2a injection, were measured by HPLC and mass spectrometry. An i.v. injection of 14G2a causes increased paw sensitivity to light touch in this model, a response that closely mimics patient allodynia. Animals allowed to drink water containing 1% DFMO exhibited a significant reduction of 14G2a-induced pain sensitivity (allodynia). Increasing the dosage of the immunotherapeutic increased the magnitude (intensity and duration) of the pain behavior. Administration of DFMO attenuated the enhanced sensitivity. Consistent with the known actions of DFMO on ornithine decarboxylase (ODC), serum putrescene and spermidine levels were significantly reduced by DFMO, though the decrease in endpoint polyamine levels did not directly correlate with the behavioral changes. Our results demonstrate that DFMO is an effective agent for reducing anti-GD2 -induced allodynia. Using DFMO in conjunction with dinutuximab may allow for dose escalation in neuroblastoma patients. The reduction in pain may be sufficient to allow new patient populations to utilize this therapy given the more acceptable side effect profile. Thus, DFMO may be an important adjunct to anti-GD2 immunotherapy in addition to a role as a potential anti-cancer therapeutic.

Monitoring Immune Responses in Neuroblastoma Patients during Therapy.

Neuroblastoma (NBL) is the most common extracranial solid tumor in childhood. Despite intense treatment, children with this high-risk disease have a poor prognosis. Immunotherapy showed a significant improvement in event-free survival in high-risk NBL patients receiving chimeric anti-GD2 in combination with cytokines and isotretinoin after myeloablative consolidation therapy. However, response to immunotherapy varies widely, and often therapy is stopped due to severe toxicities. Objective markers that help to predict which patients will respond or develop toxicity to a certain treatment are lacking. Immunotherapy guided via immune monitoring protocols will help to identify responders as early as possible, to decipher the immune response at play, and to adjust or develop new treatment strategies. In this review, we summarize recent studies investigating frequency and phenotype of immune cells in NBL patients prior and during current treatment protocols and highlight how these findings are related to clinical outcome. In addition, we discuss potential targets to improve immunogenicity and strategies that may help to improve therapy efficacy. We conclude that immune monitoring during therapy of NBL patients is essential to identify predictive biomarkers to guide patients towards effective treatment, with limited toxicities and optimal quality of life.

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

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. 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. 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. 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. NCT number03042429.

Identification of a minimal region of loss on chromosome 6q27 associated with poor survival of high-risk neuroblastoma patients

ABSTRACTPatients with high-risk neuroblastoma (HR-NB) often initially respond to therapy, but afterward they become resistant and disease recurred. Unfortunately, it does not exist one or more specific chromosome defects associated with relapse or refractory NB. Recently, genomic evidence from primary tumors indicated that the distal region of chromosome 6q is loss in HR-NB patients with fatal outcome. We identified a minimal common region of loss of chromosome 6q27 spanning an area of 2.09 Mb by high-resolution DNA copy number data of a small cohort of HR-NB samples carrying 6q loss. This region of loss harbored five genes T, SFT2D1, RPS6KA2, FGFR1OP, and UNC93A. We found that low SFT2D1, RPS6KA2, and FGFR1OP gene expression predicted poor outcome in HR-NB patients using public R2 Platform. Further functional studies will be essential to confirm the presumed tumor suppressor gene(s) located within 6q27 region. These results suggest that SFT2D1, RPS6KA2, and FGFR1OP genes may be responsible for poor prognosis of HR-NB tumors with 6q27 loss, and their haploinsufficiency may be crucial in accelerating tumor progression.

Anti-GD2 antibodies in treatment of high-risk Neuroblastoma: present and perspectives

Neuroblastoma is the most frequent extra-cranial pediatric solid tumor, occurring in young children, 90% being less than 5 years at diagnosis. It remains a therapeutic challenge since survival of high-risk neuroblastoma patients that represent around 50% of the patients is around 50% in spite of extensive combined treatments. Immunotherapy based on the use of antibodies directed to GD2, a ganglioside strongly expressed by almost all neuroblastoma cells, has been developed during the last decade. In SIOPEN studies have shown that dinatuximab beta (Qarziba®) is effective on refractory/relapsed patients and improves survival when administered in the first line maintenance treatment. Other strategies are currently explored using combination with chemotherapy at relapse and evaluating the benefits of an earlier administration during the induction treatment. In addition, more selective antibodies are also developed to decrease toxicity, especially neuropathic pain that is one of the major toxic effect.

Activated Natural Killer Cells in Combination with Anti-GD2 Antibody Dinutuximab Improve Survival of Mice after Surgical Resection of Primary Neuroblastoma.

Immunotherapy of neuroblastoma that remains after myeloablative chemotherapy with anti-GD2 antibody dinutuximab has increased the two-year event-free and overall survival of high-risk neuroblastoma patients; however, 40% of patients develop recurrent disease during or after this treatment. To determine the potential of such antibody-based immunotherapy earlier in treatment, a mouse model was developed in which surgical resection of the primary tumor was followed by therapy of residual disease with dinutuximab combined with ex vivo-activated human natural killer (aNK) cells. The effect of combining dinutuximab with human aNK cells was determined in vitro with cellular cytotoxicity and Matrigel invasion assays. The in vivo efficacy of dinutuximab and aNK cells against neuroblastoma was assessed following resection of primary tumors formed by two cell lines or a patient-derived xenograft (PDX) in immunodeficient NOD-scid gamma mice. In vitro, the combination of aNK cells and dinutuximab caused cytotoxicity and decreased invasiveness of three human neuroblastoma cell lines. Treatment of mice with dinutuximab combined with aNK cells after surgical resection of primary intrarenal tumors formed by two cell lines or a PDX decreased tumor cells in liver and bone marrow as evaluated by histopathology and bioluminescence imaging. Survival of mice after resection of these tumors was most significantly increased by treatment with dinutuximab combined with aNK cells compared with that of untreated mice. The combination of dinutuximab and adoptively transferred human aNK cells following surgical resection of primary neuroblastomas significantly improves survival of immunodeficient mice.

Abstract LB-300: Natural antibodies to non-human glycans Neu5Gc and alpha-gal correlate with outcome of high-risk neuroblastoma patients treated with dinutuximab on COG ANBL0032 and ANBL0931

Abstract Immunotherapy with ch14.18 (dinutuximab) has significantly improved the survival of high-risk neuroblastoma patients, though late relapses and allergic reaction remain concerning. N-glycolylneuraminic acid (Neu5Gc) and galactose alpha-1,3-galactose (α-gal) are glycans present in most mammals except human. Humans have circulating antibodies against these glycans due to their presence in dairy products and red meats. We investigated whether anti-glycan antibodies influence efficacy or allergic reactions associated with dinutuximab. Using ELISA, we measured anti-Neu5Gc and anti-α-gal IgG and IgE levels in plasma collected from courses 1 (days -1 & 6), 4 (days 80 & 90) & 5 (days 111 & 118) of patients on two immunotherapy trials of neuroblastoma (ANBL0032 n= 219; ANBL0931 n=100). Levels of IgG and IgE antibodies against both glycans were highest at pretreatment, decreasing significantly over the entire course of therapy as well as within each course of therapy (p<0.001, Wilcoxon signed rank). For patients on ANBL0032 immunotherapy, anti-α-gal was 924 ± 208 ng/ml (n=118) at pretreatment, decreasing to 102 ± 573 ng/ml (n=82) at day 118. Similar decreases in anti-glycan levels were also noted in patients treated with isotretinoin alone. No association between anti-glycan levels and the occurrence of anaphylaxis, urticaria or other allergic reaction during immunotherapy was observed. Patients were next dichotomized by median value into low vs. high anti-glycan subgroups and associations with EFS and OS assessed using log-rank test. While there was no correlation of anti-glycan levels at pretreatment with outcome, patients treated with immunotherapy on ANBL0032 with lower levels of anti-α-gal and anti-Neu5Gc IgG at later assessment times were significantly associated with better EFS. For example, patients with less than median levels of anti-α-gal on day 90 (n=93, p=0.05) and day 118 (n=86, p=0.05), and anti-Neu5Gc on day 90 (p=0.009) and day 118 (p=0.05), had better EFS. These results remained valid when the ANBL0032 and ANBL0931 cohorts were combined. In contrast, there was no significant correlation of anti-glycan levels and OS with immunotherapy, or EFS or OS in the non-immunotherapy cohort at any treatment timepoint. Our results suggest that lower levels of circulating antibodies against non-human glycans in neuroblastoma patients treated with dinutuximab are associated with better EFS, presumably due to less interaction of glycans on dinutuximab. Citation Format: Mitchell B. Diccianni, Jenna Mielke, Richard Williams, Karen Messer, Fevzi Ozkaynak, Andrew L. Gilman, Arlene Naranjo, Wendy London, Paul M. Sondel, Julie Park, Alice L. Yu, Childrens Oncology Group. Natural antibodies to non-human glycans Neu5Gc and alpha-gal correlate with outcome of high-risk neuroblastoma patients treated with dinutuximab on COG ANBL0032 and ANBL0931 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-300.

Genomic Amplifications and Distal 6q Loss: Novel Markers for Poor Survival in High-risk Neuroblastoma Patients.

BackgroundNeuroblastoma is characterized by substantial clinical heterogeneity. Despite intensive treatment, the survival rates of high-risk neuroblastoma patients are still disappointingly low. Somatic chromosomal copy number aberrations have been shown to be associated with patient outcome, particularly in low- and intermediate-risk neuroblastoma patients. To improve outcome prediction in high-risk neuroblastoma, we aimed to design a prognostic classification method based on copy number aberrations.MethodsIn an international collaboration, normalized high-resolution DNA copy number data (arrayCGH and SNP arrays) from 556 high-risk neuroblastomas obtained at diagnosis were collected from nine collaborative groups and segmented using the same method. We applied logistic and Cox proportional hazard regression to identify genomic aberrations associated with poor outcome.ResultsIn this study, we identified two types of copy number aberrations that are associated with extremely poor outcome. Distal 6q losses were detected in 5.9% of patients and were associated with a 10-year survival probability of only 3.4% (95% confidence interval [CI] = 0.5% to 23.3%, two-sided P = .002). Amplifications of regions not encompassing the MYCN locus were detected in 18.1% of patients and were associated with a 10-year survival probability of only 5.8% (95% CI = 1.5% to 22.2%, two-sided P < .001).ConclusionsUsing a unique large copy number data set of high-risk neuroblastoma cases, we identified a small subset of high-risk neuroblastoma patients with extremely low survival probability that might be eligible for inclusion in clinical trials of new therapeutics. The amplicons may also nominate alternative treatments that target the amplified genes.

Accumulation of potential driver genes with genomic alterations predicts survival of high-risk neuroblastoma patients

BackgroundNeuroblastoma is the most common pediatric malignancy with heterogeneous clinical behaviors, ranging from spontaneous regression to aggressive progression. Many studies have identified aberrations related to the pathogenesis and prognosis, broadly classifying neuroblastoma patients into high- and low-risk groups, but predicting tumor progression and clinical management of high-risk patients remains a big challenge.ResultsWe integrate gene-level expression, array-based comparative genomic hybridization and functional gene-interaction network of 145 neuroblastoma patients to detect potential driver genes. The drivers are summarized into a driver-gene score (DGscore) for each patient, and we then validate its clinical relevance in terms of association with patient survival. Focusing on a subset of 48 clinically defined high-risk patients, we identify 193 recurrent regions of copy number alterations (CNAs), resulting in 274 altered genes whose copy-number gain or loss have parallel impact on the gene expression. Using a network enrichment analysis, we detect four common driver genes, ERCC6, HECTD2, KIAA1279, EMX2, and 66 patient-specific driver genes. Patients with high DGscore, thus carrying more copy-number-altered genes with correspondingly up- or down-regulated expression and functional implications, have worse survival than those with low DGscore (P = 0.006). Furthermore, Cox proportional-hazards regression analysis shows that, adjusted for age, tumor stage and MYCN amplification, DGscore is the only significant prognostic factor for high-risk neuroblastoma patients (P = 0.008).ConclusionsIntegration of genomic copy number alteration, expression and functional interaction-network data reveals clinically relevant and prognostic putative driver genes in high-risk neuroblastoma patients. The identified putative drivers are potential drug targets for individualized therapy.ReviewersThis article was reviewed by Armand Valsesia, Susmita Datta and Aleksandra Gruca.

Zebrafish as a model to study neuroblastoma development.

Neuroblastoma is a pediatric solid tumor arising from embryonic neural crest progenitor cells that normally generate the peripheral sympathetic nervous system. As such, the location of neuroblastoma tumors is correlated with the distribution of major post-ganglionic clusters throughout the sympathetic chain, with the highest incidence in the adrenal medulla or lumbar sympathetic ganglia (~65%). Neuroblastoma is an enigmatic tumor that can spontaneously regress with minimal treatment or become highly metastatic and develop resistance to aggressive treatments, including radiation and high-dose chemotherapy. Age of diagnosis, stage of disease and cellular and genetic features often predict whether the tumor will regress or advance to metastatic disease. Recent efforts using molecular and genomic technologies have allowed more accurate stratification of patients into low-, intermediate- and high-risk categories, thereby allowing for minimal intervention in low-risk patients and providing potential new therapeutic targets, such as the ALK receptor tyrosine kinase, for high-risk or relapsed patients. Despite these advances, the overall survival of high-risk neuroblastoma patients is still less than 50%. Furthermore, next-generation sequencing has revealed that almost two-thirds of neuroblastoma tumors do not contain obvious pathogenic mutations, suggesting that epigenetic mechanisms and/or a perturbed cellular microenvironment may heavily influence neuroblastoma development. Understanding the mechanisms that drive neuroblastoma, therefore, will likely require a combination of genomic, developmental and cancer biology approaches in whole animal systems. In this review, we discuss the contributions of zebrafish research to our understanding of neuroblastoma pathogenesis as well as the potential for this model system to accelerate the identification of more effective therapies for high-risk neuroblastoma patients in the future.