MYCN Amplification Status Research Articles

Current and Emerging Biomarkers: Impact on Risk Stratification for Neuroblastoma.

Neuroblastoma has heterogenous clinical presentations that are reflected by several well-defined clinical factors and biomarkers. Combinations of these clinical and biologic prognostic factors have been used for decades to generate classifiers to stratify patients into risk groups (low, intermediate, and high), which in turn are used to inform and tailor treatment as reported in the new NCCN Clinical Practice Guidelines in Oncology for Neuroblastoma. Risk classification uses clinical features, such as age and tumor stage, along with the most significant prognostic tumor biomarkers, including histologic features (differentiation and mitosis-karyorrhexis index), MYCN amplification status, chromosomal copy number alterations (segmental or numerical), and ploidy (DNA content). Recent next-generation sequencing approaches have identified additional tumor-specific genetic factors that have potential roles as prognostic and predictive biomarkers. These emerging biomarkers include telomerase maintenance mechanisms, such as telomerase reverse transcription (TERT) expression and alternative lengthening of telomeres (ALT) status. Somatic alterations of genes, including mutations in the anaplastic lymphoma kinase gene ALK, detected in >10% of patients with newly diagnosed disease, have both prognostic and predictive roles in determining eligibility for targeted therapies (eg, ALK tyrosine kinase inhibitors). In addition to diagnostic tumor-derived biomarkers, significant effort is being directed toward identification of markers to predict response to chemotherapy and immunotherapies. With the increasing use of GD2-containing immunotherapy regimens, efforts are aimed at identifying host or tumor microenvironment immune correlatives that can serve as predictive biomarkers. Understanding the potential role of liquid biopsies as biomarkers during and following treatment, including sequential circulating tumor DNA or tumor-specific mRNA transcripts, is expected to enhance the ability to predict recurrences and also inform understanding of tumor evolution and therapy resistance. These and other emerging biomarkers will lead to refinement and optimization of future neuroblastoma risk classification systems.

Development of red blood cell-derived extracellular particles as a biocompatible nanocarrier of microRNA-204 (REP-204) to harness anti-neuroblastoma effect

Neuroblastoma (NB) is the most common extracranial solid tumor in the pediatric population with a high degree of heterogeneity in clinical outcomes. Upregulation of the tumor suppressor miR-204 in neuroblastoma is associated with good prognosis. Although miR-204 has been recognized as a potential therapeutic candidate, its delivery is unavailable. We hypothesized that REP-204, the red blood cell-derived extracellular particles (REP) with miR-204 loading, can suppress neuroblastoma cells in vitro. After miR-204 loading by electroporation, REP-204, but not REP carriers, inhibited the viability, migration, and 3D spheroid growth of neuroblastoma cells regardless of MYCN amplification status. SWATH-proteomics revealed that REP-204 treatment may trigger a negative regulation of mRNA splicing by the spliceosome, suppression of amino acid metabolism and protein production, and prevent SLIT/ROBO signaling-mediated cell migration, to halt neuroblastoma tumor growth and metastasis. The therapeutic efficacy of REP-204 should be further investigated in preclinical models and clinical studies.

Artificial intelligence-based morphologic classification and molecular characterization of neuroblastic tumors from digital histopathology.

A deep learning model using attention-based multiple instance learning (aMIL) and self-supervised learning (SSL) was developed to perform pathologic classification of neuroblastic tumors and assess MYCN-amplification status using H&E-stained whole slide digital images. The model demonstrated strong performance in identifying diagnostic category, grade, mitosis-karyorrhexis index (MKI), and MYCN-amplification on an external test dataset. This AI-based approach establishes a valuable tool for automating diagnosis and precise classification of neuroblastoma tumors.

MRI compared to 123I-MIBG scan for detection of central nervous system relapse in neuroblastoma.

10040 Background: Neuroblastoma (NB) relapsing in the central nervous system (CNS), though uncommon, is historically incurable. However, a multimodality approach has improved long term survival (1). Timely detection of CNS relapse may reduce or prevent morbidity and mortality. In most centers, surveillance for NB includes whole-body MIBG scans but does not require dedicated anatomical brain imaging. At Memorial Sloan Kettering Cancer Center (MSK), head MRI at regular intervals is standard. The objective of this retrospective study was to determine the optimal imaging modality for detecting CNS relapse in NB. Methods: After MSK IRB approval, records of patients with CNS NB seen at MSK from 2004-2023 were evaluated. In most cases, relapse was diagnosed at other institutions before referral to MSK. Analyzed data included symptoms and findings on brain CT/MRI and MIBG scans. Results: Of 206 patients with MIBG-avid CNS NB, 7 were excluded because they had CNS disease at diagnosis. For the remaining 199 patients, median time to CNS relapse from diagnosis was 18 months. 132 (66%) patients had CNS relapse at a median of 9.8 months after achieving complete remission. In 67 patients with prior systemic progression, median time to CNS relapse was 10.9 months from the last relapse. Relapse was isolated to CNS in 130/199 (65%) patients. 118 (59%) patients had neurological symptoms at time of CNS disease; 81(41%) were asymptomatic, relapse being detected on surveillance scans. Multiple (>1) parenchymal lesions were noted in 74 (37%), diffuse leptomeningeal disease without parenchymal involvement in 15 (7%) and solitary lesions in 110 (55%) patients. Median diameter of the largest lesion was 2.7 (range <0.5-6.8) cm. Anatomical imaging was performed with MRI (65%), CT (14%) or both (34%). Of those undergoing both scans, CNS relapse was missed on CT in 4/67 (6%) patients. 137 patients had MIBG scans before resection of CNS relapse. All sites of CNS disease noted on MRI/CT were positive by MIBG in 46 (33%) patients. However, MIBG scan was either totally or partly negative in 69 (50%) and 22 (16%) patients, respectively. Even for lesions ≥2cm in diameter, MIBG was completely negative in 27/57 (47%). MIBG positivity did not correlate with age, size >2cm, MYCN amplification or ALK mutation status (p>0.05 for each). Lesions <1cm and infratentorially located were more likely to be negative on MIBG scan (p<0.05). Lesions in symptomatic patients, dural lesions and hemorrhagic lesions were more likely to be positive on MIBG scan (p< 0.05). Conclusions: CNS relapse is isolated to the brain in most patients. MIBG scan has poor sensitivity for the detection of CNS NB, regardless of size or location. Although CT or MRI are both effective in detecting CNS relapse, the former can miss some lesions. We recommend brain MRI for surveillance of high-risk NB for ≥2 years after initial diagnosis or last systemic relapse. 1. J. Neurooncology 97:409, 2010.

Image-defined risk factors associated with MYCN oncogene amplification in neuroblastoma and their association with overall survival.

The MYCN oncogene is a critical factor in the development and progression of neuroblastoma, and image-defined risk factors (IDRFs) are radiological findings used for the preoperative staging of neuroblastoma. This study aimed to investigate the specific categories of IDRFs associated with MYCN amplification in neuroblastoma and their association with overall survival. A retrospective analysis was conducted on a cohort of 280 pediatric patients diagnosed with neuroblastoma, utilizing a combination of clinical and radiological data. MYCN amplification status was ascertained through molecular testing, and the assessment of IDRFs was conducted using either contrast-enhanced computed tomography or magnetic resonance imaging. The specific categories of IDRFs associated with MYCN amplification and their association with overall survival were analyzed. MYCN amplification was identified in 19.6% (55/280) of patients, with the majority of primary lesions located in the abdomen (53/55, 96.4%). Lesions accompanied by MYCN amplification exhibited significantly larger tumor volume and a greater number of IDRFs compared with those without MYCN amplification (P < 0.001). Both univariate and multivariate analyses revealed that coeliac axis/superior mesenteric artery encasement and infiltration of adjacent organs/structures were independently associated with MYCN amplification in abdominal neuroblastoma (P < 0.05). Patients presenting with more than four IDRFs experienced a worse prognosis (P = 0.017), and infiltration of adjacent organs/structures independently correlated with overall survival in abdominal neuroblastoma (P = 0.009). The IDRFs are closely correlated with the MYCN amplification status and overall survival in neuroblastoma.

A novel clinical tool and risk stratification system for predicting the event-free survival of neuroblastoma patients: A TARGET-based study.

Neuroblastoma (NB), considered the most common non-intracranial solid tumor in children, accounts for nearly 8% of pediatric malignancies. This study aimed to develop a simple and practical nomogram to predict event-free survival (EFS) in NB patients and establish a new risk stratification system. In this study, 763 patients primarily diagnosed with NB in the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database were included and randomly assigned to a training set (70%) and a validation set (30%) in a 7:3 ratio. First, the independent prognostic factors of EFS for NB patients were identified through univariate and multivariate Cox regression analyses. Second, a nomogram was created based on these factors and was validated for calibration capability, discriminative, and clinical significance by C-curves, receiver operating characteristic (ROC) curves, and decision curve analysis. Finally, a new risk stratification system was established for NB patients based on the nomogram. The univariate Cox analysis demonstrated that NB patients with age at diagnosis >318 days, International Neuroblastoma Staging System (INSS) stage 4, DNA diploidy, MYCN amplification status, and children oncology group (COG) high-risk group had a relatively poor prognosis. However, according to the multivariate Cox regression analysis, only age, INSS stage, and DNA ploidy were independent predictive factors in NB patients regarding EFS, and a nomogram was created based on these factors. The area under the curve (AUC) values of the ROC curves for the 3-, 5-, and 10-year EFS of this nomogram were 0.681, 0.706, and 0.720, respectively. Additionally, the AUC values of individual independent prognostic factors of EFS were lower than those of the nomogram, suggesting that the developed nomogram had a higher predictive reliability for prognosis. In addition, a new risk stratification system was developed to better stratify NB patients and provide clinical practitioners with a better reference for clinical decision-making. NB patients' EFS could be predicted more accurately and easily through the constructed nomogram and event-occurrence risk stratification system, allowing clinicians to better differentiate NB patients and establish individualized treatment plans to maximize patient benefits.

Correlations between contrast-enhanced CT-measured extracellular volume fraction, histopathological features, and MYCN amplification status in abdominal neuroblastoma: a retrospective study.

To retrospectively investigate the correlations between contrast-enhanced CT (CECT)-measured extracellular volume fraction (fECV) and histopathological features, as well as MYCN amplification status, in abdominal neuroblastoma. One hundred and forty-one patients with abdominal neuroblastoma who underwent CECT scanning were retrospectively enrolled. Calculation of fECV involved the measurement of CT values within regions of interest located within the neuroblastoma and aorta on both non-contrast-enhanced CT and equilibrium CECT. The correlations between fECV and various factors, including pathological subtype, mitosis karyorrhexis index (MKI), Shimada classification, MYCN amplification status, International Neuroblastoma Risk Group (INRG) stage, and risk group were analyzed using either the Mann-Whitney U test or Kruskal-Wallis test. Neuroblastoma and ganglioneuroblastoma exhibited fECV values of 0.349 (0.252, 0.424) and 0.438 (0.327, 0.508), respectively, indicating a statistically significant difference (Z = 2.200, P = 0.028). Additionally, the fECV decreased significantly in neuroblastoma with high MKI (H = 8.314, P = 0.016) or unfavorable histology (Z = 3.880, P < 0.001), as well as in those with MYCN amplification (Z = 5.486, P < 0.001). Notably, a significant variation in fECV was observed among different INRG stages (H = 16.881, P <0.001) and risk groups (H = 29.014, P < 0.001). CECT-derived fECV is associated with histopathological features, MYCN amplification status, INRG stage, and risk stratification of abdominal neuroblastoma, reflecting a potential correlation between the extracellular matrix and the biological behavior of neuroblastoma.

Abstract 6157: USP7 is a promising thersapeutic target for neuroblastoma

Abstract Background: The ubiquitin-proteasome system (UPS) plays an essential role in post-translational modification of proteins to maintain proteostasis. Ubiquitination of target protein is coordinated by different enzymes, which add and link ubiquitin to each other in specific ways that determine the fate of the protein for to be degraded or to be included into protein complex to induce signaling pathways. Ubiquitination is further regulated by deubiquitinases, which remove ubiquitin and rescue target protein from being degraded or diverted from it signaling role. UPS dysregulation has been linked to many human diseases, including cancer. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in immune response, tumor suppression and DNA repair. USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including in neuroblastoma (NB). Therefore, USP7 is a potential therapeutic target for NB. Some new specific USP7 inhibitors are highly potent and selective and have demonstrated significant antitumor activity in preclinical models of adult cancer. We hypothesize that inhibition of USP7 alone or in combination with epigenetic drugs like HDAC inhibitors will be more effective against NB tumor growth. To evaluate the efficacy of USP7 inhibition with specific USP7 inhibitors alone or with combination with epigenetic drugs like HDAC inhibitors (Saha) or chemotherapy against NB tumor growth, NB cell lines were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and cell viability using AlamarBlue assay. Apoptosis was measured by caspase cleavage using Caspase 3/7 apoptosis assay as well as by PARP cleavage by Western Blotting (WB). Changes in various proteins were measured by WB and co-immunoprecipitation were performed to analyze the ubiquitin status of specific protein targets regulated by USP7. Pharmacologic inhibition of USP7 resulted in significant decreases in both cell proliferation and cell viability in vitro only in p53 wild-type NB cell lines. There was no correlation seen between MYCN amplification status and treatment response. USP7 inhibition induced apoptosis as well as necroptosis with dose-dependent increase. Treatment with USP7 inhibitors led to decrease p53 ubiquitination leading increased of p53 protein expression in all sensitive cell lines tested and decreased MYCN protein level in MYCN-amplified cell lines. EZH2, which is a predominant target of USP7 is significantly downregulated with increasing dose of USP7 inhibitors. In addition, the combination of USP7 inhibitors with chemotherapy commonly used in NB or with combination with HDAC inhibitors showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, Karen Lee, Peter Zage. USP7 is a promising thersapeutic target for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6157.

Abstract 4517: Novel prognostic and predictive biomarker for neuroblastoma

Abstract Recently we showed that Retinal degeneration protein 3 (RD3) is constitutively expressed in healthy adult and fetal tissues beyond retina and, its gradient expression from high to low in ganglioneuroma, ganglioneuroblastoma and neuroblastoma (NB). Crucially, our sequential in vitro and in vivo studies identified that RD3 regulates NB progression and metastasis, and its loss prompts the evolution of therapy defying progressive disease (PD). Here we investigated the prognostic significance of RD3 in NB, its predictive benefit and, its association to clinical outcomes. RNA-Seq data mining (4 independent study cohorts, total n = 1013) indicated that high RD3 can predict good NB prognosis. RD3 expression (immunohistochemistry) gauged in our NB patient cohort (n = 100) showed a positive correlation with longer overall survival (OS, P = 1.0E-04) and relapse-free survival (RFS, P = 2.1E-02). Similarly, high RD3 correlated with longer OS (P = 1.2E-03) and RFS (P = 2.1-02) in children presented with high-risk NB (HR-NB), irrespective of MYCN amplification status (MYCN amplified, P = 1.4E-02; non-amplified, P = 5.5E-03) and patient age (&amp;lt;2Y age, P = 2.8E-02; &amp;gt;2Y age, P = 1.0E-04) groups. Significantly, we observed a gradient loss of RD3 with NB progression, from stage 1 through stage 4 disease. Consistently, RD3 loss was substantial in metastatic disease when compared to the primary neuroblastomas, and this loss associated with poor OS in patients presented with metastatic disease (P = 4.0E-02). More importantly, RD3 is significantly lost in therapy resistant progressive NB and corroborated significantly (P = 9.5E-03) with the poor progression free survival. Multivariate analysis identified RD3 as an independent (P = 4.4E-02) prognostic factor for NB. These results demonstrate that RD3-loss can forecast poor prognosis in NB, and further imply that assessing RD3 loss could serve as the good predictor for therapy response and/or PD evolution. Together, these data identify a novel prognostic and predictive factor for neuroblastoma. Funding: This work was partially or in full, funded by Oklahoma Center for the Advancement of Science and Technology, OCAST-HR19-045; National Institutes of Health, NIH-P20GM103639 and Department of Defense DoD-CA210339 Citation Format: Dinesh Babu Somasundaram, Zhongxin Yu, Ashley Baker, Natarajan Aravindan. Novel prognostic and predictive biomarker for neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4517.

Mitochondria-associated gene expression perturbation predicts clinical outcomes and shows potential for targeted therapy in neuroblastoma.

Mitochondria have long been considered a potential target in cancer therapy because malignant cells are known for their altered energy production. However, there is a lack of comprehensive research on the involvement of mitochondria-associated proteins (MAPs) in neuroblastoma (NB), and their potential as therapeutic targets is yet to be fully explored. MAP genes were defined based on the protein-coding genes with mitochondrial localization. The mRNA expression patterns and dynamics of MAP genes associated with NB were investigated by integrating publicly available transcriptional profiles at the cellular and tissue levels. Multivariate Cox regression analysis was conducted to reveal the association of MAP genes with the overall survival (OS) and clinical subgroups of NB patients. The single-cell RNA-seq dataset and gene dependency screening datasets were analyzed to reveal the therapeutic potential of targeting MAP genes. We compiled a total of 1,712 MAP genes. We found the global and cell type-specific mRNA expression changes of the MAP genes associated with NB status and survival. Our analyses revealed a group of MAP gene signatures independent of MYCN-amplification status associated with NB outcome. We provided computational evidence with selected MAP genes showing good performance in predicting long-term prognosis. By analyzing gene dependency of the MAP genes in NB cell lines and ex vivo human primary T cells, we demonstrated the therapeutic potential of targeting several MAP genes in NB tumors. Collectively, our study provides evidence for the MAP genes as extended candidates in NB tumor stratification and staging, prognostic prediction, and targeted drug development.

ALK immunopositivity in neuroblastomas: Correlation with MYCN amplification status and clinicopathological features

ALK immunopositivity in neuroblastomas: Correlation with MYCN amplification status and clinicopathological features

Association of Image-Defined Risk Factors with Clinical, Biological Features and Outcome in Neuroblastoma.

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor and the most common cancer encountered in children younger than 12 months of age. Localized tumors have a good prognosis, but some cases undergo treatment failure and recurrence. The aim of the study was to analyze the link between the neuroblastoma risk factors and the prognosis for patients diagnosed with NB. All patients admitted to the department of Pediatric Surgery, "Grigore Alexandrescu" Clinical Emergency Hospital for Children, between 1 January 2010 and 1 July 2022 were included in this analysis when diagnosed with neuroblastoma. Thirty-one patients with NB were admitted to the surgical department, 20 boys and 11 girls. We observed an association between large tumors and positive imaging-defined risk factor (IDRF) status; The Fisher test showed an association between the tumor's diameter when bigger than 8 cm and a positive IDRF status, with p &lt; 0.001. We supposed that positive IDRF status at diagnosis may be linked to other prognostic factors. We discovered that an NSE value over 300 was associated with IDRF status (p &lt; 0.001, phi = 0.692) and death. This study confirms the impact of IDRF status at diagnosis as it can be clearly correlated with other risk factors, such as a high level of NSE, MYCN amplification status, large tumor size, incomplete tumor resection, and an unfavorable outcome.

MYCN protein stability is a better prognostic indicator in neuroblastoma

ObjectiveMYCN oncogene amplification is associated with treatment failure and poor prognosis in neuroblastoma. To date, most detection methods of MYCN focus on DNA copy numbers instead of protein expression, which is the real one performing biological function, for poor antibodies. The current investigation was to explore a fast and reliable way to detect MYCN protein expression and evaluate its performance in predicting prognosis.MethodsSeveral MYCN antibodies were used to detect MYCN protein expression by immunohistochemistry (IHC), and one was chosen for further study. We correlated the IHC results of MYCN from 53 patients with MYCN fluorescence in situ hybridization (FISH) and identified the sensitivity and specificity of IHC. The relationship between patient prognosis and MYCN protein expression was detected from this foundation.ResultsMYCN amplification status detected by FISH was most valuable for INSS stage 3 patients. In the cohort of 53 samples, IHC test demonstrated 80.0–85.7% concordance with FISH results. Further analyzing those cases with inconsistent results, we found that patients with MYCN amplification but low protein expression tumors always had a favorable prognosis. In contrast, if patients with MYCN non-amplified tumors were positive for MYCN protein, they had a poor prognosis.ConclusionMYCN protein level is better than MYCN amplification status in predicting the prognosis of neuroblastoma patients. Joint of FISH and IHC could confirm MYCN protein stability and achieve better prediction effect than the singular method.

Patterns of relapse after immunotherapy in patients with high-risk neuroblastoma.

10043 Background: While the addition of anti-GD2 immunotherapy led to improvement in outcomes in patients on the Children’s Oncology Group (COG) ANBL0032 study, relapse remains a concern. Prior studies demonstrated the prognostic importance of time to first relapse, however, the effect of immunotherapy on timing and patterns of relapse in neuroblastoma (NBL) have yet to be evaluated. The purpose of this exploratory analysis was to describe the impact of immunotherapy on patterns of relapse in patients with high-risk NBL, including a descriptive comparison of sites of relapse based on post-consolidation treatment received [dinutuximab with cytokines and isotretinoin (DIN) vs isotretinoin alone (ISO)]. Methods: A retrospective, descriptive analysis of patients on ANBL0032 was performed, including patients randomized to DIN or ISO and those non-randomly assigned to DIN after ISO arm closure. Pt characteristics including age, stage, MYCN amplification status, tumor grade, mitosis-karyorrhexis index (MKI) and ploidy were summarized descriptively and relapse sites were tabulated. For DIN patients who subsequently relapsed, overall survival (OS) was calculated starting from the time of first relapse after enrollment on ANBL0032 (“post-relapse OS”). Kaplan-Meier OS curves were generated based on site of relapse. Results: The analytic cohort included 1,431 (DIN = 1,327; ISO = 104) patients. Among DIN patients, 492 relapsed, many in &gt; 1 site. In the randomized cohort (n = 248), 122 relapsed (DIN = 68/144; ISO = 54/104). The frequencies (DIN; ISO) by site of relapse in the randomized cohort were: bone (53%; 54%), CNS (16%; 11%), lymph node (13%; 17%), abdominal (10%; 17%), paraspinal (6%; 2%), liver (3%; 4%), other soft tissue (22%; 7%). A higher proportion of ISO patients had marrow relapse (29.4% DIN; 48.2% ISO); however, the proportion of DIN patients with lung relapses appeared higher (9% vs 2%). Among all relapsed patients, the proportion with bone relapse did not appear to differ between treatment groups, regardless of MYCN status. Among patients with MYCN amplified disease, the proportion with marrow relapse did not appear to differ based on treatment [21/149 (14.1%) DIN; 3/20 (15.0%) ISO]; however, among patients with MYCN non-amplified disease, the proportion with marrow relapse appeared higher in the ISO group [16/23; 69.6%] vs the DIN group [52/193 (26.9%)]. Conclusions: In this exploratory analysis of patients on COG ANBL0032, the pattern for site of relapse appears to differ between patients treated with DIN vs ISO. While immunotherapy remains the treatment of choice in this population, the findings from this retrospective exploratory analysis warrant further investigation to decrease the risk for post-immunotherapy relapse. Clinical trial information: NCT00026312.

CT-based morphologic and radiomics features for the classification of MYCN gene amplification status in pediatric neuroblastoma.

MYCN onco-gene amplification in neuroblastoma confers patients to the high-risk disease category for which prognosis is poor and more aggressive multimodal treatment is indicated. This retrospective study leverages machine learning techniques to develop a computed tomography (CT)-based model incorporating semantic and non-semantic features for non-invasive prediction of MYCN amplification status in pediatric neuroblastoma. From 2009 to 2020, 54 pediatric patients treated for neuroblastoma at a specialized children's hospital with pre-treatment contrast-enhanced CT and MYCN status were identified (training cohort, n = 44; testing cohort, n = 10). Six morphologic features and 107 quantitative gray-level texture radiomics features extracted from manually drawn volume-of-interest were analyzed. Following feature selection and class balancing, the final predictive model was developed with eXtreme Gradient Boosting (XGBoost) algorithm. Accumulated local effects (ALE) plots were used to explore main effects of the predictive features. Tumor texture maps were also generated for visualization of radiomics features. One morphologic and 2 radiomics features were selected for model building. The XGBoost model from the training cohort yielded an area under the receiver operating characteristics curve (AUC-ROC) of 0.930 (95% CI, 0.85-1.00), optimized F1-score of 0.878, and Matthews correlation coefficient (MCC) of 0.773. Evaluation on the testing cohort returned AUC-ROC of 0.880 (95% CI, 0.64-1.00), optimized F1-score of 0.933, and MCC of 0.764. ALE plots and texture maps showed higher "GreyLevelNonUniformity" values, lower "Strength" values, and higher number of image-defined risk factors contribute to higher predicted probability of MYCN amplification. The machine learning model reliably classified MYCN amplification in pediatric neuroblastoma and shows potential as a surrogate imaging biomarker.

Whole-tumour apparent diffusion coefficient (ADC) histogram analysis to identify MYCN-amplification in neuroblastomas: preliminary results.

To determine the role of apparent diffusion coefficient (ADC) histogram analysis in the identification of MYCN-amplification status in neuroblastomas. We retrospectively evaluated imaging records from 62 patients with neuroblastomas (median age: 15 months (interquartile range (IQR): 7-24 months); 38 females) who underwent magnetic resonance imaging at our institution before the initiation of any therapy or biopsy. Fourteen patients had MYCN-amplified (MYCNA) neuroblastoma. Histogram parameters of ADC maps from the entire tumour was obtained from the baseline images and the normalised images. The Mann-Whitney U test was used to compare the absolute and normalised histogram parameters amongst neuroblastomas with and without MYCN-amplification. Receiver operating characteristic (ROC) curves and area under the curves (AUC) were generated for the statistically significant histogram parameters. Cut-offs obtained from the ROC curves were evaluated on an external validation set (n-15, MYCNA-6, F-7, age 24 months (10-60)). A logistic regression model was trained to predict MYCNA by combining statistically significant histogram parameters and was evaluated on the validation set. MYCN-amplified neuroblastomas had statistically significant higher maximum ADC and lower minimum ADC than non-amplified neuroblastomas. They also demonstrated higher entropy, variance, energy, and lower uniformity than non-amplified neoplasms (p > 0.05). Energy, entropy, and maximum ADC had AUC of 0.85, 0.79, and 0.82, respectively. Whole tumour ADC histogram analysis of neuroblastomas can differentiate between tumours with and without MYCN-amplification. These parameters can help identify areas for targeted biopsies or can be used to predict subtypes of these high-risk tumours before biopsy results are available. • MYCN-amplification significantly affects treatment decisions in neuroblastomas. • MYCN-amplified neuroblastomas had significantly different ADC histogram metrics as compared to tumours without amplification. • ADC histogram metrics can be used to predict MYCN-amplification status based on imaging.

SAP30, an oncogenic driver of progression, poor survival, and drug resistance in neuroblastoma

Neuroblastoma is the most devastating extracranial solid malignancy in children. Despite an intense treatment regimen, the prognosis for high-risk neuroblastoma patients remains poor, with less than 40% survival. So far, MYCN amplification status is considered the most prognostic factor but corresponds to only ∼25% of neuroblastoma patients. Therefore, it is essential to identify a better prognosis and therapy response marker in neuroblastoma patients. We applied robust bioinformatic data mining tools, such as weighted gene co-expression network analysis, cisTarget, and single-cell regulatory network inference and clustering on two neuroblastoma patient datasets. We found Sin3A-associated protein 30 (SAP30), a driver transcription factor positively associated with high-risk, progression, stage 4, and poor survival in neuroblastoma patient cohorts. Tumors of high-risk neuroblastoma patients and relapse-specific patient-derived xenografts showed higher SAP30 levels. The advanced pharmacogenomic analysis and CRISPR-Cas9 screens indicated that SAP30 essentiality is associated with cisplatin resistance and further showed higher levelsin cisplatin-resistant patient-derived xenograft tumor cell lines. Silencing of SAP30 induced cell death invitro and led to a reduced tumor burden and size invivo. Altogether, these results indicate that SAP30 is a better prognostic and cisplatin-resistance marker and thus a potential drug target in high-risk neuroblastoma.

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

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

Identification of a Five-Gene Signature Derived From MYCN Amplification and Establishment of a Nomogram for Predicting the Prognosis of Neuroblastoma.

Background: Neuroblastoma (NB), the most common solid tumor in children, exhibits vastly different genomic abnormalities and clinical behaviors. While significant progress has been made on the research of relations between clinical manifestations and genetic abnormalities, it remains a major challenge to predict the prognosis of patients to facilitate personalized treatments. Materials and Methods: Six data sets of gene expression and related clinical data were downloaded from the Gene Expression Omnibus (GEO) database, ArrayExpress database, and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. According to the presence or absence of MYCN amplification, patients were divided into two groups. Differentially expressed genes (DEGs) were identified between the two groups. Enrichment analyses of these DEGs were performed to dig further into the molecular mechanism of NB. Stepwise Cox regression analyses were used to establish a five-gene prognostic signature whose predictive performance was further evaluated by external validation. Multivariate Cox regression analyses were used to explore independent prognostic factors for NB. The relevance of immunity was evaluated by using algorithms, and a nomogram was constructed. Results: A five-gene signature comprising CPLX3, GDPD5, SPAG6, NXPH1, and AHI1 was established. The five-gene signature had good performance in predicting survival and was demonstrated to be superior to International Neuroblastoma Staging System (INSS) staging and the MYCN amplification status. Finally, a nomogram based on the five-gene signature was established, and its clinical efficacy was demonstrated. Conclusion: Collectively, our study developed a novel five-gene signature and successfully built a prognostic nomogram that accurately predicted survival in NB. The findings presented here could help to stratify patients into subgroups and determine the optimal individualized therapy.

Growth factor signaling predicts therapy resistance mechanisms and defines neuroblastoma subtypes

Neuroblastoma (NB) has a low frequency of recurrent mutations compared to other cancers, which hinders the development of targeted therapies and novel risk stratification strategies. Multikinase inhibitors have shown potential in treating high-risk NB, but their efficacy is likely impaired by the cancer cells’ ability to adapt to these drugs through the employment of alternative signaling pathways. Based on the expression of 48 growth factor-related genes in 1189 NB tumors, we have developed a model for NB patient survival prediction. This model discriminates between stage 4 NB tumors with favorable outcomes (>80% overall survival) and very poor outcomes (<10%) independently from MYCN-amplification status. Using signaling pathway analysis and gene set enrichment methods in 60 NB patients with known therapy response, we identified signaling pathways, including EPO, NGF, and HGF, upregulated in patients with no or partial response. In a therapeutic setting, we showed that among six selected growth factors, EPO, and NGF showed the most pronounced protective effects in vitro against several promising anti-NB multikinase inhibitors: imatinib, dasatinib, crizotinib, cabozantinib, and axitinib. Mechanistically kinase inhibitors potentiated NB cells to stronger ERK activation by EPO and NGF. The protective action of these growth factors strongly correlated with ERK activation and was ERK-dependent. ERK inhibitors combined with anticancer drugs, especially with dasatinib, showed a synergistic effect on NB cell death. Consideration of growth factor signaling activity benefits NB outcome prediction and tailoring therapy regimens to treat NB.