Patterns of recurrence after radiotherapy for high-risk neuroblastoma: Implications for radiation dose and field.

BACKGROUND: Prognosis for patients with high-risk neuroblastoma (HR-NBL) is guarded despite aggressive therapy, and few studies have characterized outcomes after radiotherapy in relation to radiation treatment fields. METHODS: Multi-institutional retrospective cohort of 293 patients with HR-NBL who received autologous stem cell transplant (ASCT) and EBRT between 1997-2021. LRR was defined as recurrence at the primary site or within one nodal echelon beyond disease present at diagnosis. Follow-up was defined from the end of EBRT. Event-free survival (EFS) and OS were analyzed by Kaplan-Meier method. Cumulative incidence of locoregional progression (CILP) was analyzed using competing risks of distant-only relapse and death with Gray's test. RESULTS: Median follow-up was 7.0 years (range: 0.01-22.4). Five-year CILP, EFS, and OS were 11.9 %, 65.2 %, and 77.5 %, respectively. Of the 31 patients with LRR and imaging review, 15 (48.4 %) had in-field recurrences (>12 Gy), 6 (19.4 %) had marginal failures (≤12 Gy), and 10 (32.3 %) had both in-field and marginal recurrences. No patients receiving total body irradiation (12 Gy) experienced marginal-only failures (p = 0.069). On multivariable analyses, MYCN amplification had higher risk of LRR (HR: 2.42, 95 % CI: 1.06-5.50, p = 0.035) and post-consolidation isotretinoin and anti-GD2 antibody therapy (HR: 0.42, 95 % CI: 0.19-0.94, p = 0.035) had lower risk of LRR. CONCLUSIONS: Despite EBRT, LRR remains a contributor to treatment failure in HR-NBL with approximately half of LRRs including a component of marginal failure. Future prospective studies are needed to explore whether radiation fields and doses should be defined based on molecular features such as MYCN amplification, and/or response to chemotherapy.

The MYCN 5' UTR as a therapeutic target in neuroblastoma.

Tumor MYCN amplification is seen in high-risk neuroblastoma, yet direct targeting of this oncogenic transcription factor has been challenging. Here, we take advantage of the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis to inhibit the activity of eukaryotic translation initiation factor 4A1 (eIF4A1) using an amidino-rocaglate, CMLD012824. Consistent with the role of this RNA helicase in resolving structural barriers in 5' untranslated regions (UTRs), CMLD012824 increased eIF4A1 affinity for polypurine-rich 5' UTRs, including that of the MYCN and associated transcripts with critical roles in cell proliferation. CMLD012824-mediated clamping of eIF4A1 spanned the full lengths of mRNAs, while translational inhibition was mediated through 5' UTR binding in a cap-dependent and -independent manner. Finally, CMLD012824 led to growth inhibition in MYCN-amplified neuroblastoma models without generalized toxicity. Our studies highlight the key role of eIF4A1 in MYCN-amplified neuroblastoma and demonstrate the therapeutic potential of disrupting its function.

MYCN immunohistochemistry as surrogate marker for MYCN-amplified spinal ependymomas.

MYCN (master regulator of cell cycle entry and proliferative metabolism) gene amplification defines a molecular subgroup of spinal cord ependymomas that show high-grade morphology and aggressive behavior. Demonstration of MYCN amplification by DNA methylation or fluorescence-in situ hybridization (FISH) is required for diagnosis. We aimed to (i) assess prevalence and clinicopathological features of MYCN-amplified spinal ependymomas and (ii) evaluate utility of immunohistochemistry (IHC) for MYCN protein as a surrogate for molecular testing. A combined retrospective-prospective study spanning 8 years was designed during which all spinal cord ependymomas with adequate tissue were subjected to MYCN FISH and MYCN IHC. Among 77 spinal cord ependymomas included, MYCN amplification was identified in 4 samples from 3 patients (3/74, 4%) including two (1st and 2nd recurrences) from the same patient. All patients were adults (median age at diagnosis of 32 years) including two females and one male. The index tumors were located in thoracic (n = 2) and lumbar (n = 1) spinal cord. One of the female patients had neurofibromatosis type 2 (NF2). All four tumors showed anaplastic histology. Diffuse expression of MYCN protein was seen in all four MYCN-amplified samples but in none of the non-amplified cases, thus showing 100% concordance with FISH results. On follow-up, the NF2 patient developed widespread spinal dissemination while another developed recurrence proximal to the site of previous excision. To conclude, MYCN-amplified spinal ependymomas are rare tumors, accounting for ~ 4% of spinal cord ependymomas. Within the limitation of small sample size, MYCN IHC showed excellent concordance with MYCN gene amplification.

Ceftriaxone exerts antitumor effects in MYCN-driven retinoblastoma and neuroblastoma by targeting DDX3X for translation repression.

MYCN proto-oncogene, bHLH transcription factor (MYCN) amplification is associated with aggressive retinoblastoma (RB) and neuroblastoma (NB) cancer recurrence that is resistant to chemotherapies. Therefore, there is an urgent need to identify new therapeutic tools. This study aimed to evaluate the potential repurposing of ceftriaxone for the treatment of MYCN-amplified RB and NB, based on the clinical observations that the drug was serendipitously found to decrease the volume of the MYCN-driven RB subtype. Using patient-derived tumor organoids and tumor cell lines, we demonstrated that ceftriaxone is a potent and selective growth inhibitor targeting MYCN-driven RB and NB cells. Profiling of drug-induced transcriptomic changes, cell-cycle progression, and apoptotic death indicated cell-cycle arrest and death of drug-treated MYCN-amplified tumor cells. Drug target identification, using an affinity-based proteomic and molecular docking approach, and functional studies of the target proteins revealed that ceftriaxone targeted DEAD-box helicase 3 X-linked (DDX3X), thereby inhibiting translation in MYCN-amplified tumors but not in MYCN-nonamplified cells. The data suggest the feasibility of repurposing ceftriaxone as an anticancer drug and provide insights into the mechanism of drug action, highlighting DDX3X as a potential target for treating MYCN-driven tumors.

Predictive value of 2-deoxy-2-fluorine-18-fluoro-D-glucose positron emission tomography/computed tomography parameters for MYCN amplification in high-risk neuroblastoma.

OBJECTIVES: To investigate the predictive value of 2-deoxy-2-fluorine-18-fluoro-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) parameters for MYCN amplification in high-risk neuroblastoma (HR-NB). MATERIALS AND METHODS: A retrospective analysis was performed by reviewing 68 HR-NB patients who underwent MYCN testing and 18F-FDG PET/CT imaging at our hospital between January 2018 and December 2019. Based on the results of MYCN testing, patients were categorized into either the MYCN-amplified (MNA) or MYCN non-amplified (MYCN-NA) group. The 18F-FDG PET/CT parameters, including maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), peak standardized uptake value (SUVpeak), tumor metabolic volume (MTV), total lesion glycolysis (TLG), coefficient of variation (COV), and areas under the curve of cumulative SUV-volume histogram index (AUC-CSH index) were evaluated. Independent predictors were identified through univariate and multivariate logistic regression analyses, and their diagnostic performance was evaluated using the receiver-operating characteristic (ROC) curve. RESULTS: Univariate logistic regression analysis revealed that SUVpeak was significantly associated with MYCN amplification. Multivariate logistic regression analysis showed that SUVpeak was an independent predictor of MYCN amplification in HR-NB [Odds ratio (OR) = 0.673, 95 % confidence interval (95 % CI): 0.494-0.917, P = 0.012]. ROC curve analysis demonstrated that the predictive model including SUVpeak had higher diagnostic performance [area under the curve (AUC): 0.790, 95 % CI: 0.677-0.881, sensitivity: 0.861, specificity: 0.591, positive predictive value (PPV): 0.820, negative predictive value (NPV): 0.722] compared to using SUVpeak alone (AUC: 0.640, 95 % CI: 0.514-0.752, sensitivity: 0.630, specificity: 0.682, PPV: 0.806, NPV: 0.469). CONCLUSION: SUVpeak can predict the MYCN amplification in HR-NB patients. The predictive model constructed by combining SUVpeak and age can distinguish MYCN status in HR-NB non-invasively with superior efficacy compared to using SUVpeak alone.

Chromosome 1p36 candidate gene ZNF436 predicts the prognosis of neuroblastoma: a bioinformatic analysis.

BACKGROUND: Genetic 1p deletion is reported in 30% of all neuroblastoma and is associated with the unfavorable prognosis of neuroblastoma. The expressions and prognosis of 1p candidate genes in neuroblastoma are unclear. METHODS: Public neuroblastoma cohorts were obtained for secondary analysis. The prognosis of 1p candidate genes in neuroblastoma was determined using Kaplan-Meier and cox regression analysis. The prediction of the nomogram model was determined using timeROC. RESULTS: First, we confirmed the bad prognosis of 1p deletion in neuroblastoma. Moreover, zinc finger protein 436 (ZNF436) located at 1p36 region was down-regulated in 1p deleted neuroblastoma and higher ZNF436 expression was associated with the longer event free survival and overall survival of neuroblastoma. The expression levels of ZNF436 were lower in neuroblastoma patients with MYCN amplification or age at diagnosis ≥ 18months, or with stage 4 neuroblastoma. ZNF436 had robust predictive values of MYCN amplification and overall survival of neuroblastoma. Furthermore, the prognostic significance of ZNF436 in neuroblastoma was independent of MYCN amplification and age of diagnosis. Combinations of ZNF436 with MYCN amplification or age of diagnosis achieved better prognosis. At last, we constructed a nomogram risk model based on age, MYCN amplification and ZNF436. The nomogram model could predict the overall survival of neuroblastoma with high specificity and sensitivity. CONCLUSIONS: Chromosome 1p36 candidate gene ZNF436 was a prognostic maker of neuroblastoma.

MYCN amplification, TERT rearrangements and ATRX mutations in neuroblastoma: clinicopathological correlates- an Indian perspective.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumour in childhood with a diverse clinical presentation and course. The early age of onset, high frequency of metastatic disease at diagnosis and tendency for spontaneous regression in infancy sets it apart from other childhood tumors. This heterogeneity is largely attributed to underlying genetic aberrations which are distinct in low-risk and high-risk NB. To this end, we sought to analyse our NB cases for the molecular alterations and find its correlation with clinical behaviour. METHODS: NB cases (n = 50) diagnosed over last 7 years were retrospectively analysed for MYCN amplification (fluorescent-in-situ hybridization), TERT rearrangements (qRT-PCR), ATRX mutations (immunohistochemistry). These findings were correlated with demographic profiles, histologic features and clinical outcome. RESULTS: Age ranged from 1 month to 30 years (mean 2.8 years) with male preponderance. Poorly differentiated subtype constituted the majority (64%), followed by differentiating (28%) and undifferentiated subtype (8%) which were equally distributed across all age groups. MYCN amplification, TERT-mRNA upregulation and ATRX mutations was observed in 30%, 42% and 24%, respectively. Cases with TERT-mRNA upregulation were distributed equally across all histological subtypes while those with ATRX mutations and MYCN amplification were frequent in poorly differentiated NB. ATRX mutation was mutually exclusive of TERT-mRNA upregulation and MYCN amplification. Kaplan-Meier analysis revealed significantly shorter overall and progression-free survival for tumors harboring MYCN amplification and TERT-mRNA upregulation, while that for ATRX mutant tumors was not significant. CONCLUSIONS: Our results provide data indicating poor clinical outcome in NB carrying MYCN amplification and TERT-mRNA upregulation.

CCNB1IP1 prevents ubiquitination-mediated destabilization of MYCN and potentiates tumourigenesis of MYCN-amplificated neuroblastoma.

BACKGROUND: MYCN amplification as a common genetic alteration that correlates with a poor prognosis for neuroblastoma (NB) patients. However, given the challenge of directly targeting MYCN, indirect strategies to modulate MYCN by interfering with its cofactors are attractive in NB treatment. Although cyclin B1 interacting protein 1 (CCNB1IP1) has been found to be upregulated in MYCN-driven mouse NB tissues, its regulation with MYCN and collaboration in driving the biological behaviour of NB remains unknown. METHODS: To evaluate the expression and clinical significance of CCNB1IP1 in NB patients, public datasets, clinical NB samples and cell lines were explored. MTT, EdU incorporation, colony and tumour sphere formation assays, and a mouse xenograft tumour model were utilized to examine the biological function of CCNB1IP1. The reciprocal manipulation of CCNB1IP1 and MYCN and the underlying mechanisms involved were investigated by gain- and loss-of-function approaches, dual-luciferase assay, chromatin immunoprecipitation (CHIP) and co-immunoprecipitation (Co-IP) experiments. RESULTS: CCNB1IP1 was upregulated in MYCN-amplified (MYCN-AM) NB cell lines and patients-derived tumour tissues, which was associated with poor prognosis. Phenotypic studies revealed that CCNB1IP1 facilitated the proliferation and tumourigenicity of NB cells in cooperation with MYCN in vitro and in vivo. Mechanistically, MYCN directly mediates the transcription of CCNB1IP1, which in turn attenuated the ubiquitination and degradation of MYCN protein, thus enhancing CCNB1IP1-MYCN cooperativity. Moreover, CCNB1IP1 competed with F box/WD-40 domain protein 7 (FBXW7) for MYCN binding and enabled MYCN-mediated tumourigenesis in a C-terminal domain-dependent manner. CONCLUSIONS: Our study revealed a previously uncharacterized mechanism of CCNB1IP1-mediated MYCN protein stability and will provide new prospects for precise treatment of MYCN-AM NB based on MYCN-CCNB1IP1 interaction.

Ganglioneuroblastoma intermixed: Clinicopathological implications of diagnosis at presentation and genomic correlations.

BACKGROUND: Ganglioneuroblastoma intermixed (GNBI) is classified as "favorable" histology by International Neuroblastoma Pathology Classification system. However, the International Neuroblastoma Risk Group (INRG) stratifies patients using wider clinicopathological and cytogenetic/molecular parameters. While the diagnosis of GNBI is typically made on resected tumor, it may sometimes be rendered on initial biopsy. We studied GNBI noted at diagnosis to evaluate its correlation with INRG staging and other clinicopathological and molecular features. METHODS: In this retrospective study, clinical, radiological, pathological, cytogenetic, and molecular information from patients with GNBI at diagnosis seen between 1995 and 2021 was analyzed. INRG staging was performed. RESULTS: Of the 15,827 neuroblastoma specimens, GNBI was noted in 237 patients. Of these, 53 had the initial pathological diagnosis of GNBI; median follow-up 3.5 (range: 0.2-14) years. Disease was locoregional in 41 (77%, 16 stage L1 and 25 L2); none relapsed. Twelve (23%) had metastatic disease at presentation; six (50%) relapsed, and two died of disease. MYCN was amplified in two metastatic tumors. Six of 31 (19%) tumors tested had recurrent cytogenetic abnormalities and nonrecurrent somatic gene mutations in 10/23 (43%). The presence of any adverse molecular/cytogenetic findings was associated with metastatic disease (p < .05). For patients with localized GNBI undergoing both biopsy and resection, GNBI was diagnosed in both in 17/19 (90%). CONCLUSIONS: Localized GNBI at diagnosis has excellent long-term clinical outcome even without cytotoxic therapy. For localized GNBI, a biopsy sample is adequate to make the diagnosis. When associated with metastasis at diagnosis, prognosis is poorer, possibly due to associated adverse biological features.

MYCN Amplification Is Associated with Reduced Expression of Genes Encoding γ-Secretase Complex and NOTCH Signaling Components in Neuroblastoma.

Amplification of the MYCN oncogene is found in ~20% of neuroblastoma (NB) cases and correlates with high-risk disease and poor prognosis. Despite the plethora of studies describing the role of MYCN in NB, the exact molecular mechanisms underlying MYCN's contribution to high-risk disease are not completely understood. Herein, we implemented an integrative approach combining publicly available RNA-Seq and MYCN ChIP-Seq datasets derived from human NB cell lines to define biological processes directly regulated by MYCN in NB. Our approach revealed that MYCN-amplified NB cell lines, when compared to non-MYCN-amplified cell lines, are characterized by reduced expression of genes involved in NOTCH receptor processing, axoneme assembly, and membrane protein proteolysis. More specifically, we found genes encoding members of the γ-secretase complex, which is known for its ability to liberate several intracellular signaling molecules from membrane-bound proteins such as NOTCH receptors, to be down-regulated in MYCN-amplified NB cell lines. Analysis of MYCN ChIP-Seq data revealed an enrichment of MYCN binding at the transcription start sites of genes encoding γ-secretase complex subunits. Notably, using publicly available gene expression data from NB primary tumors, we revealed that the expression of γ-secretase subunits encoding genes and other components of the NOTCH signaling pathway was also reduced in MYCN-amplified tumors and correlated with worse overall survival in NB patients. Genetic or pharmacological depletion of MYCN in NB cell lines induced the expression of γ-secretase genes and NOTCH-target genes. Chemical inhibition of γ-secretase activity dampened the expression of NOTCH-target genes upon MYCN depletion in NB cells. In conclusion, this study defines a set of MYCN-regulated pathways that are specific to MYCN-amplified NB tumors, and it suggests a novel role for MYCN in the suppression of genes of the γ-secretase complex, with an impact on the NOTCH-target gene expression in MYCN-amplified NB.

Macrophage infiltration promotes regrowth in MYCN-amplified neuroblastoma after chemotherapy.

Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA+) tumors compared to PRE MNA+ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA+ tumor cells were more immunogenic compared to PRE MNA+ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA+ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA+ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA+ neuroblastoma by targeting the immune microenvironment.

ALK Gene Mutation and ALK Protein Expression in Advanced Neuroblastoma and the Potential Value in Risk Stratification in Fine-Needle Aspiration Biopsy Samples.

OBJECTIVES: The protein ALK is targeted for therapy in neuroblastoma, and ALK mutation confers a poor prognosis. We evaluated ALK in a cohort of patients with advanced neuroblastoma diagnosed by fine-needle aspiration biopsy (FNAB). METHODS: Fifty-four cases of neuroblastoma were evaluated for ALK protein expression by immunocytochemistry and ALK gene mutation by next-generation sequencing. MYCN amplification by fluorescence in situ hybridization, International Neuroblastoma Risk Group (INRG) staging, and risk assignment was performed, and patients were managed accordingly. All parameters were correlated with overall survival (OS). RESULTS: ALK protein showed cytoplasmic expression in 65% cases and did not correlate with MYCN amplification (P = .35), INRG groups (P = .52), and OS (P = .2); however, ALK-positive, poorly differentiated neuroblastoma showed better prognosis (P = .02). ALK negativity was associated with poor outcome by Cox proportional hazard model (hazard ratio, 2.36). Two patients showed ALK gene F1174L mutation with 8% and 54% allele frequency and high ALK protein expression; they died of disease 1 and 17 months following diagnosis, respectively. A novel IDH1 exon 4 mutation was also detected. CONCLUSIONS: ALK expression is a promising prognostic and predictive marker in advanced neuroblastoma that can be evaluated in cell blocks from FNAB samples along with traditional prognostic parameters. ALK gene mutation confers a poor prognosis for patients with this disease.

Paediatric-type diffuse high-grade gliomas in the 5th CNS WHO Classification.

As a relevant element of novelty, the fifth CNS WHO Classification highlights the distinctive pathobiology underlying gliomas arising primarily in children by recognizing for the first time the families of paediatric-type diffuse gliomas, both high-grade and low-grade. This review will focus on the family of paediatric-type diffuse high-grade gliomas, which includes four tumour types: 1) Diffuse midline glioma H3 K27-altered; 2) Diffuse hemispheric glioma H3 G34-mutant; 3) Diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype; and 4) Infant-type hemispheric glioma. The essential and desirable diagnostic criteria as well as the entities entering in the differential will be discussed for each tumour type. A special focus will be given on the issues encountered in the daily practice, especially regarding the diagnosis of the diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype. The advantages and the limits of the multiple molecular tests which may be utilised to define the entities of this tumour family will be evaluated in each diagnostic context.

A signature based on five immune-related genes to predict the survival and immune characteristics of neuroblastoma.

BACKGROUND: MYCN amplification (MNA) has been proved to be related to poor prognosis in neuroblastoma (NBL), but the MYCN-related immune signatures and genes remain unclear. METHODS: Enrichment analysis was used to identify the significant enrichment pathways of differentially expressed immune-related genes (DEIRGs). Weight gene coexpression network analysis (WGCNA) was applied to reveal the correlation between these DEIRGs and MYCN status. Univariate and multivariate Cox analyses were used to construct risk model. The relevant fractions of immune cells were evaluated by CIBERSORT and single-sample gene set enrichment analysis (ssGSEA). RESULTS: Five genes, including CHGA, PTGER1, SHC3, PLXNC1, and TRIM55 were enrolled into the risk model. Kaplan-Meier survival analysis and receiver operating characteristic (ROC) curve showed that our model performed well in predicting the outcomes of NBL (3-years AUC = 0.720, 5-year AUC = 0.775, 10-years AUC = 0.782), which has been validated in the GSE49711 dataset and the E-MTAB-8248 dataset. By comparing with the tumor immune dysfunction and exclusion (TIDE) and tumor inflammation signature (TIS), we further proved that our model is reliable. Univariate and multivariate Cox regression analyses indicated that the risk score, age, and MYCN can serve as independent prognostic factors in the E-MATB-8248. Functional enrichment analysis showed the DEIRGs were enriched in leukocyte adhesion-related signaling pathways. Gene set enrichment analysis (GSEA) revealed the significantly enriched pathways of the five MYCN-related DEIRGs. The risk score was negatively correlated with the immune checkpoint CD274 (PD-L1) but no significant difference with the TMB. We also confirmed the prognostic value of our model in predicting immunotherapeutics. CONCLUSION: We constructed and verified a signature based on DEIRG that related to MNA and predicted the survival of NBL based on relevant immune signatures. These findings could provide help for predicting prognosis and developing immunotherapy in NBL.

Chromosome 11q loss and MYCN amplification demonstrate synthetic lethality with checkpoint kinase 1 inhibition in neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor found in children and despite intense multi-modal therapeutic approaches, low overall survival rates of high-risk patients persist. Tumors with heterozygous loss of chromosome 11q and MYCN amplification are two genetically distinct subsets of neuroblastoma that are associated with poor patient outcome. Using an isogenic 11q deleted model system and high-throughput drug screening, we identify checkpoint kinase 1 (CHK1) as a potential therapeutic target for 11q deleted neuroblastoma. Further investigation reveals MYCN amplification as a possible additional biomarker for CHK1 inhibition, independent of 11q loss. Overall, our study highlights the potential power of studying chromosomal aberrations to guide preclinical development of novel drug targets and combinations. Additionally, our study builds on the growing evidence that DNA damage repair and replication stress response pathways offer therapeutic vulnerabilities for the treatment of neuroblastoma.

Targeting EP2 receptor with multifaceted mechanisms for high-risk neuroblastoma.

Prostaglandin E2 (PGE2) promotes tumor cell proliferation, migration, and invasion, fostering an inflammation-enriched microenvironment that facilitates angiogenesis and immune evasion. However, the PGE2 receptor subtype (EP1-EP4) involved in neuroblastoma (NB) growth remains elusive. Herein, we show that the EP2 receptor highly correlates with NB aggressiveness and acts as a predominant Gαs-coupled receptor mediating PGE2-initiated cyclic AMP (cAMP) signaling in NB cells with high-risk factors, including 11q deletion and MYCN amplification. Knockout of EP2 in NB cells blocks the development of xenografts, and its conditional knockdown prevents established tumors from progressing. Pharmacological inhibition of EP2 by our recently developed antagonist TG6-129 suppresses the growth of NB xenografts in nude mice and syngeneic allografts in immunocompetent hosts, accompanied by anti-inflammatory, antiangiogenic, and apoptotic effects. This proof-of-concept study suggests that the PGE2/EP2 signaling pathway contributes to NB malignancy and that EP2 inhibition by our drug-like compounds provides a promising strategy to treat this deadly pediatric cancer.

Prognostic analysis of E2F transcription factors E2F1 and E2F3 in four independent pediatric neuroblastoma cohorts.

BACKGROUND: Previously, we had analyzed the prognosis of E2F transcription factors across adult tumor types. However, the expressions and prognosis of E2F transcription factors in pediatric neuroblastoma have not yet been fully studied. METHODS: The prognosis of E2F transcription factors was determined in four independent pediatric neuroblastoma cohorts from Therapeutically Applicable Research to Generate Effective Treatments (TARGET), Gene Expression Omnibus (GEO) and European ArrayExpres datasets using Kaplan-Meier and cox regression analysis. RESULTS: E2F regulated gene set was associated with the event free survival and the overall survival of neuroblastoma. E2F1 and E2F3 were prognostic factors in all four independent pediatric neuroblastoma cohorts. Over-expressions of E2F1 or E2F3 were correlated with the shorted event free survival and overall survival of neuroblastoma. Expression levels of E2F1 and E2F3 were higher in neuroblastoma patients with MYCN amplification or age at diagnosis ≥ 18 months. Moreover, the prognostic significance of E2F1 or E2F3 in neuroblastoma was independent of MYCN amplification and age of diagnosis. Combinations of E2F1, E2F3 with MYCN amplification or age of diagnosis achieved better prognosis of neuroblastoma. Identification of 234 genes were associated with E2F1 and E2F3 expressions in neuroblastoma and those genes were significantly enriched in cell cycle signaling pathway. Also, higher scores of cell cycle signaling pathway were correlated with the adverse prognosis of neuroblastoma. CONCLUSIONS: E2F transcription factors E2F1 and E2F3 were prognostic makers of neuroblastoma.

Circulating Cell-Free DNA Assessment in Biofluids from Children with Neuroblastoma Demonstrates Feasibility and Potential for Minimally Invasive Molecular Diagnostics.

Liquid biopsy strategies in pediatric patients are challenging due to low body weight. This study investigated cfDNA size distribution and concentration in blood, bone marrow, cerebrospinal fluid, and urine from 84 patients with neuroblastoma classified as low (n = 28), intermediate (n = 6), or high risk (n = 50) to provide key data for liquid biopsy biobanking strategies. The average volume of blood and bone marrow plasma provided ranged between 1 and 2 mL. Analysis of 637 DNA electropherograms obtained by Agilent TapeStation measurement revealed five different major profiles and characteristic DNA size distribution patterns for each of the biofluids. The proportion of samples containing primarily cfDNA was, at 85.5%, the highest for blood plasma. The median cfDNA concentration amounted to 6.28 ng/mL (blood plasma), 58.2 ng/mL (bone marrow plasma), 0.08 ng/mL (cerebrospinal fluid), and 0.49 ng/mL (urine) in samples. Meta-analysis of the dataset demonstrated that multiple cfDNA-based assays employing the same biofluid sample optimally require sampling volumes of 1 mL for blood and bone marrow plasma, 2 mL for cerebrospinal fluid, and as large as possible for urine samples. A favorable response to treatment was associated with a rapid decrease in blood-based cfDNA concentration in patients with high-risk neuroblastoma. Blood-based cfDNA concentration was not sufficient as a single parameter to indicate high-risk disease recurrence. We provide proof of concept that monitoring neuroblastoma-specific markers in very small blood volumes from infants is feasible.

Prediction of MYCN Amplification, 1p and 11q Aberrations in Pediatric Neuroblastoma via Pre-therapy 18F-FDG PET/CT Radiomics.

Purpose: This study aimed to assess the predictive ability of 18F-FDG PET/CT radiomic features for MYCN, 1p and 11q abnormalities in NB. Method: One hundred and twenty-two pediatric patients (median age 3. 2 years, range, 0.2-9.8 years) with NB were retrospectively enrolled. Significant features by multivariable logistic regression were retained to establish a clinical model (C_model), which included clinical characteristics. 18F-FDG PET/CT radiomic features were extracted by Computational Environment for Radiological Research. The least absolute shrinkage and selection operator (LASSO) regression was used to select radiomic features and build models (R-model). The predictive performance of models constructed by clinical characteristic (C_model), radiomic signature (R_model), and their combinations (CR_model) were compared using receiver operating curves (ROCs). Nomograms based on the radiomic score (rad-score) and clinical parameters were developed. Results: The patients were classified into a training set (n = 86) and a test set (n = 36). Accordingly, 6, 8, and 7 radiomic features were selected to establish R_models for predicting MYCN, 1p and 11q status. The R_models showed a strong power for identifying these aberrations, with area under ROC curves (AUCs) of 0.96, 0.89, and 0.89 in the training set and 0.92, 0.85, and 0.84 in the test set. When combining clinical characteristics and radiomic signature, the AUCs increased to 0.98, 0.91, and 0.93 in the training set and 0.96, 0.88, and 0.89 in the test set. The CR_models had the greatest performance for MYCN, 1p and 11q predictions (P < 0.05). Conclusions: The pre-therapy 18F-FDG PET/CT radiomics is able to predict MYCN amplification and 1p and 11 aberrations in pediatric NB, thus aiding tumor stage, risk stratification and disease management in the clinical practice.

The Thermal Dose of Photothermal Therapy Generates Differential Immunogenicity in Human Neuroblastoma Cells.

Photothermal therapy (PTT) is an effective method for tumor eradication and has been successfully combined with immunotherapy. However, besides its cytotoxic effects, little is known about the effect of the PTT thermal dose on the immunogenicity of treated tumor cells. Therefore, we administered a range of thermal doses using Prussian blue nanoparticle-based photothermal therapy (PBNP-PTT) and assessed their effects on tumor cell death and concomitant immunogenicity correlates in two human neuroblastoma cell lines: SH-SY5Y (MYCN-non-amplified) and LAN-1 (MYCN-amplified). PBNP-PTT generated thermal dose-dependent tumor cell killing and immunogenic cell death (ICD) in both tumor lines in vitro. However, the effect of the thermal dose on ICD and the expression of costimulatory molecules, immune checkpoint molecules, major histocompatibility complexes, an NK cell-activating ligand, and a neuroblastoma-associated antigen were significantly more pronounced in SH-SY5Y cells compared with LAN-1 cells, consistent with the high-risk phenotype of LAN-1 cells. In functional co-culture studies in vitro, T cells exhibited significantly higher cytotoxicity toward SH-SY5Y cells relative to LAN-1 cells at equivalent thermal doses. This preliminary report suggests the importance of moving past the traditional focus of using PTT solely for tumor eradication to one that considers the immunogenic effects of PTT thermal dose to facilitate its success in cancer immunotherapy.