Prognostic Marker In Neuroblastoma Research Articles

Whole-tumoral metabolic heterogeneity in 18F-FDG PET/CT is a novel prognostic marker for neuroblastoma

BackgroundNeuroblastoma (NB) is a highly heterogeneous tumor, and more than half of newly diagnosed NB are associated with extensive metastases. Accurately characterizing the heterogeneity of whole-body tumor lesions remains clinical challenge. This study aims to quantify whole-tumoral metabolic heterogeneity (WMH) derived from whole-body tumor lesions, and investigate the prognostic value of WMH in NB.MethodsWe retrospectively enrolled 95 newly diagnosed pediatric NB patients in our department. Traditional semi-quantitative PET/CT parameters including the maximum standardized uptake value (SUVmax), the mean standardized uptake value (SUVmean), the peak standardized uptake value (SUVpeak), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) were measured. These PET/CT parameters were expressed as PSUVmax, PSUVmean, PSUVpeak, PMTV, PTLG for primary tumor, WSUVmax, WSUVmean, WSUVpeak, WMTV, WTLG for whole-body tumor lesions. The metabolic heterogeneity was quantified using the areas under the curve of the cumulative SUV-volume histogram index (AUC-CSH index). Intra-tumoral metabolic heterogeneity (IMH) and WMH were extracted from primary tumor and whole-body tumor lesions, respectively. The outcome endpoints were overall survival (OS) and progression-free survival (PFS). Survival analysis was performed utilizing the univariate and multivariate Cox proportional hazards regression. The optimal cut-off values for metabolic parameters were obtained by receiver operating characteristic curve (ROC).ResultsDuring follow up, 27 (28.4%) patients died, 21 (22.1%) patients relapsed and 47 (49.5%) patients remained progression-free survival, with a median follow-up of 35.0 months. In survival analysis, WMTV and WTLG were independent indicators of PFS, and WMH was an independent risk factor of PFS and OS. However, IMH only showed association with PFS and OS. In addition to metabolic parameters, the International Neuroblastoma Staging System (INSS) was identified as an independent risk factor for PFS, and neuron-specific enolase (NSE) served as an independent predictor of OS.ConclusionWMH was an independent risk factor for PFS and OS, suggesting its potential as a novel prognostic marker for newly diagnosed NB patients.

HIF and MYC signaling in adrenal neoplasms of the neural crest: implications for pediatrics.

Pediatric neural crest-derived adrenal neoplasms include neuroblastoma and pheochromocytoma. Both entities are associated with a high degree of clinical heterogeneity, varying from spontaneous regression to malignant disease with poor outcome. Increased expression and stabilization of HIF2α appears to contribute to a more aggressive and undifferentiated phenotype in both adrenal neoplasms, whereas MYCN amplification is a valuable prognostic marker in neuroblastoma. The present review focuses on HIF- and MYC signaling in both neoplasms and discusses the interaction of associated pathways during neural crest and adrenal development as well as potential consequences on tumorigenesis. Emerging single-cell methods together with epigenetic and transcriptomic analyses provide further insights into the importance of a tight regulation of HIF and MYC signaling pathways during adrenal development and tumorigenesis. In this context, increased attention to HIF-MYC/MAX interactions may also provide new therapeutic options for these pediatric adrenal neoplasms.

Ferroptosis-related gene signatures in neuroblastoma associated with prognosis.

Background: Ferroptosis, a form of regulatory cell death, has been linked to the development of various tumors. Peripheral neuroblastoma (NB) is one of the most common extracranial solid tumors in children, and it has been proposed that regulating tumor cell ferroptosis may be a future treatment for NB. However, it is unclear how ferroptosis contributes to NB development. Methods: Expression data were collected from two independent cohorts (GEO and Arrayexpress databases). Univariate Cox analysis, multivariate Cox analysis, and the least absolute shrinkage and selection operator (Lasso) algorithm were applied to create a prognostic signature, whose performance was quantified using the area under the receiver operating characteristic curve (AUC) and Kaplan–Meier curves. A prognostic meta-analysis was used to test the suitability and stability of the FRG signature. Drug sensitivity analyses were performed using the data collected from Cell Miner™. Results: PROM2, AURKA, STEAP3, CD44, ULK2, MAP1LC3A, ATP6V1G2, and STAT3 are among the eight genes in the FRG prognostic signature, all of which were highly expressed in stage 1 NB, except AURKA. Furthermore, the high-risk group, which was stratified by signature, had a lower overall survival rate than the low-risk group. GSEA revealed that high-risk groups have more biological processes related to ferroptosis. Conclusion: Ferroptosis-related genes are expressed differently between stages 1 and 4 NB. The FRG signature successfully stratified NB patients into two risk groups and can accurately predict the overall survival in NB. In addition, we found that the gene AURKA might have the potential to be a prognostic marker in NB.

Abstract 1276: Nucleolin has prognostic value in neuroblastoma patients

Abstract Background: Neuroblastoma (NB) represents the most frequent form of extracranial solid tumor of infants, responsible for 15% of childhood cancer deaths. Nucleolin (NCL) prognostic value in NB was here investigated. Methods: NCL protein expression was evaluated in NB patients at diagnosis and after induction chemotherapy. NCL mRNA prognostic value was first assessed in a cohort of 20 stage M NB patients and confirmed in the MultiPlatform786 including 786 NB patients of all stages. Overall and event-free survival curves were plotted by Kaplan-Meier method and compared by log-rank test. Findings: NCL protein resulted significantly overexpressed in NB tumors compared to the non tumoral counterpart, and higher in stage M compared to stage L patients. In the stage M cohort and in MultiPlatform786 dataset, patients with high NCL mRNA expression revealed a significant lower survival probability than those with low NCL expression. In MultiPlatform786 dataset, NCL mRNA expression was significantly higher in patients with age >18 months, in stage M and in MYCN amplified tumors than in patients with age ˂18 months and in stage L or MS and with MYCN non amplified tumors, respectively. Multivariate analysis suggested NCL has a significant prognostic value even in the model adjusted for established prognostic markers. NCL significantly stratified patients with age <18 and age >18, stage M, >18 months and stage M tumor, stages L or MS, and with MYCN not amplified. A significant correlation between NCL and MYCN, MYC, and TERT was found in two independent datasets. Gene set enrichment analysis revealed a significant positive enrichment of MYC target genes and genes involved in telomerase maintenance. NCL protein resulted down-modulated after chemotherapy, in association with morphological features of neuroblastic differentiation. Interpretation: NCL is a novel and independent prognostic marker for NB. Funding: IMH-EuroNanoMed II-2015 (ER-2015-2360441-Eranet) and AIRC IG n. 24397 to PF. Citation Format: Davide Cangelosi, Chiara Brignole, Veronica Bensa, Roberto Tamma, Fabiana Malaguti, Barbara Carlini, Enzo Calarco, Patrizia Perri, Domenico Ribatti, Nuno A. Fonseca, Joao N. Moreira, Alessandra Eva, Loredana Amoroso, Massimo Conte, Alberto Garaventa, Angela R. Sementa, Maria V. Corrias, Mirco Ponzoni, Fabio Pastorino. Nucleolin has prognostic value in neuroblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1276.

E2F3 gene expression is a potential negative prognostic marker for localised and MYCN not-amplified neuroblastoma: Results of in silico analysis of 786 samples.

Neuroblastoma (NB) is an enigmatic childhood malignancy characterised by a wide range of clinical behaviour. Many potential oncogenes for NB have recently been identified. Among them, E2 transcription factor 3 (E2F3) expression was associated with a poor survival in 134 stage 4S patients, but evidence for other stage groups remains poorly investigated. We have analysed the expression of E2F3 gene from a database of 786 NB samples. Overall and event-free survivals (EFS) were assessed by the Kaplan-Meier method, splitting the data on the median and tertile expression values. The Cox model was applied to control for the confounding by stage, age and MYCN amplification. Validation was performed by an in silico analysis of an independent cohort of 283 NB patients. Furthermore, an immunofluorescence analysis on 48 formalin-fixed, paraffin-embedded NB specimens was also performed. E2F3 overexpression was associated with a poor survival (EFS=84%, 95% CI: 79%-95%, for low expression levels; EFS=62%, 95% CI: 56%-68% for middle levels; EFS=30%, 95% CI: 24%-36%, for high levels, p<.001). This association was confirmed in multivariable analysis and was more evident in patients with MYCN not-amplified and localised stages. Immunofluorescence results and the validation on an independent cohort of NB primary samples confirmed these findings. E2F3 is a new potential prognostic marker in NB with favourable characteristics at diagnosis. Further studies are needed to elucidate the potential role of E2F3 in NB oncogenesis and progression, in order to identify new targets for therapeutic interventions.

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 Prognostic Genes in Neuroblastoma in Children by Weighted Gene Coexpression Network Analysis.

Background Neuroblastoma is a malignant neuroendocrine tumor from the sympathetic nervous system, the most common extracranial tumor in children. Identifying potential prognostic markers of neuroblastoma can provide clues for early diagnosis, recurrence, and treatment. Methods RNA sequence data and clinical features of 147 neuroblastomas were obtained from the TARGET (Therapeutically Applicable Research to Generate Effective Treatments project) database. Application weighted gene coexpression network analysis (WGCNA) was used to construct a free-scale gene coexpression network, to study the interrelationship between its potential modules and clinical features, and to identify hub genes in the module. We performed Lasso regression and Cox regression analyses to identify the three most important genes and develop a new prognostic model. Data from the GSE85047 cohort verified the predictive accuracy of the prognostic model. Results 14 coexpression modules were constructed using WGCNA. Brown coexpression modules were found to be significantly associated with disease survival status. Multivariate Cox analysis was performed on genes from univariate Cox regression and Lasso regression analyses using the Cox proportional hazards regression model. Finally, we constructed a three-gene prognostic model: risk score = (0.003812659∗CKB) + (−0.152376975∗expDST) + (0.032032815∗expDUT). The prognosis of samples in the high-risk group was significantly poorer than that of samples in the low-risk group (P=1.225e − 06). The risk model was also regarded as an independent predictor of prognosis (HR = 1.632; 95% CI = 1.391–1.934; P < 0.001). Conclusion Our study constructed a neuroblastoma coexpressing gene module and identified a prognostic potential risk model for prognosis in neuroblastoma.

Neuroblastoma Associated Genes Are Enriched in Trunk Neural Crest

Neural crest is a remarkable transient cell population present during embryogenesis, with the ability to differentiate into a multitude of cell types. Depending on anterior to posterior position, neural crest derivatives (cranial, vagal, trunk and sacral, respectively) migrate and commit to restricted lineages. Neuroblastoma is a tumor of infancy and locates along sympathetic ganglia. Based on tumor location and tumor cell gene expression, neuroblastoma is believed to arise from cells of the trunk neural crest-derived sympathetic nervous system. Here we establish the gene expression pattern of trunk enriched- as well as neuroblastoma associated genes over time during early embryogenesis. We show that genes associated with trunk neural crest development as well as neuroblastoma progression are highly expressed at time points when trunk neural crest cells delaminate from the neural tube, undergo epithelial-to-mesenchymal transition and migrate throughout the embryonic body. Using recently established crestosphere cultures – in vitro maintenance of multipotent and self-renewing neural crest stem cells – we confirm that diagnostic and prognostic markers of neuroblastoma are highly enriched in trunk- as compared to cranial neural crest. Our data strengthen a trunk neural crest origin for neuroblastoma.

Sequence-dependent trafficking and activity of GDE2, a GPI-specific phospholipase promoting neuronal differentiation.

ABSTRACTGDE2 (also known as GDPD5) is a multispanning membrane phosphodiesterase with phospholipase D-like activity that cleaves select glycosylphosphatidylinositol (GPI)-anchored proteins and thereby promotes neuronal differentiation both in vitro and in vivo. GDE2 is a prognostic marker in neuroblastoma, while loss of GDE2 leads to progressive neurodegeneration in mice; however, its regulation remains unclear. Here, we report that, in immature neuronal cells, GDE2 undergoes constitutive endocytosis and travels back along both fast and slow recycling routes. GDE2 trafficking is directed by C-terminal tail sequences that determine the ability of GDE2 to cleave GPI-anchored glypican-6 (GPC6) and induce a neuronal differentiation program. Specifically, we define a GDE2 truncation mutant that shows aberrant recycling and is dysfunctional, whereas a consecutive deletion results in cell-surface retention and gain of GDE2 function, thus uncovering distinctive regulatory sequences. Moreover, we identify a C-terminal leucine residue in a unique motif that is essential for GDE2 internalization. These findings establish a mechanistic link between GDE2 neuronal function and sequence-dependent trafficking, a crucial process gone awry in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.

The presence of Y674/Y675 phosphorylated NTRK1 via TP53 repression of PTPN6 expression as a potential prognostic marker in neuroblastoma

The tumor suppressor TP53 promotes nerve growth factor receptor (NTRK1) -Y674/Y675 phosphorylation (NTRK1-pY674/pY675) via repression of the NTRK1 phosphatase PTPN6 in a ligand-independent manner, resulting in suppression of breast cancer cell proliferation. Moreover, NTRK1-pY674/pY675 together with low levels of PTPN6 and TP53 expression is associated with favorable disease-free survival of breast cancer patients. We determined whether in neuroblastoma this protein expression pattern impacts relapse-free survival (RFS). NTRK1-pY674/pY675, PTPN6, and TP53 expression was assessed in 98 neuroblastoma samples by immunohistochemistry. Association between expression levels and RFS was investigated by multivariate and Kaplan-Meier analysis. Mutant or wild-type TP53 was identified by sequencing tumor DNA. Tumors expressing NTRK1-pY674/pY675 and low or undetectable levels of PTPN6 and TP53 were significantly associated with 5-year RFS (P = .014) when the dataset was stratified by MYCN amplification, segmental chromosomal abnormalities and histology. Similar results were observed with tumors expressing wild-type TP53, NTRK1-pY674/pY675 and low or undetectable levels of PTPN6. Kaplan-Meier analysis demonstrated a significant correlation (P = .004), with a 50% probability of RFS (median survival 4.73 years) when present compared with 19.51% (median survival 11.63 months) when absent. Similar results were seen with non-amplified MYCN or unfavorable/undifferentiating samples and tumors from patients aged 18 months or less. Importantly, NTRK1-pY674/pY675 is an independent predictor of improved RFS. These results strongly suggest that NTRK1-pY674/pY675 together with wild-type TP53 and undetectable or low levels of PTPN6 expression is a potential biomarker of improved RFS of neuroblastoma patients. The predictive value of NTRK1-pY674/pY675 together with wild-type TP53 and low PTPN6 expression could contribute to neuroblastoma patient prognosis.

The deubiquitinating enzyme UCHL1 is a favorable prognostic marker in neuroblastoma as it promotes neuronal differentiation

BackgroundNeuroblastoma (NB) is the most common pediatric solid tumor that originates from neural crest-derived sympathoadrenal precursor cells that are committed to development of sympathetic nervous system. The well differentiated histological phenotype of NB tumor cells has been reportedly associated with favorable patient outcome. Retinoic acid (RA) can effectively induce NB cell differentiation, thereby being used in the clinic as a treatment agent for inducing the differentiation of high-risk NB. However, the underlying molecular mechanisms of regulating differentiation remain elusive.MethodsThe correlation between clinical characteristics, survival and the deubiquitinating enzyme ubiquitin C-terminal hydrolase 1 (UCHL1) expression were assessed using a neuroblastic tumor tissue microarray, and then validated in three independent patient datasets. The different expression of UCHL1 in ganglioneuroblastoma, ganglioneuroma and NB was detected by immunohistochemistry, mass spectra and immunoblotting analysis, and the correlation between UCHL1 expression and the differentiated histology was analyzed, which was also validated in three independent patient datasets. Furthermore, the roles of UCHL1 in NB cell differentiation and proliferation and the underlying mechanisms were studied by using short hairpin RNA and its inhibitor LDN57444 in vitro.ResultsBased on our neuroblastic tumor tissue microarrays and three independent validation datasets (Oberthuer, Versteeg and Seeger), we identified that UCHL1 served as a prognostic marker for better clinical outcome in NB. We further demonstrated that high UCHL1 expression was associated with NB differentiation, indicated by higher UCHL1 expression in ganglioneuroblastomas/ganglioneuromas and well-differentiated NB than poorly differentiated NB, and the positive correlation between UCHL1 and differentiation markers. As expected, inhibiting UCHL1 by knockdown or LDN57444 could significantly inhibit RA-induced neural differentiation of NB tumor cells, characterized by decreased neurite outgrowth and neural differentiation markers. This effect of UCHL1 was associated with positively regulating RA-induced AKT and ERK1/2 signaling activation. What’s more, knockdown of UCHL1 conferred resistance to RA-induced growth arrest.ConclusionOur findings identify a pivotal role of UCHL1 in NB cell differentiation and as a prognostic marker for survival in patients with NB, potentially providing a novel therapeutic target for NB.

Innate immune sensor laboratory of genetics and physiology 2 suppresses tumor cell growth and functions as a prognostic marker in neuroblastoma.

The innate immune receptors, such as toll‐like receptor 3 (TLR3), melanoma differentiation‐associated 5 (MDA5) and retinoic acid‐inducible gene‐I (RIG‐I), have been shown to be differentially expressed in neuroblastoma (NB) and promote dsRNA poly (I:C)‐induced NB suppression in vitro and in vivo. However, the role of another important innate immune cytosolic sensor, laboratory of genetics and physiology 2 (LGP2), in the cancer behavior of NB remains unclear. Here, we demonstrated that the expression levels of LGP2 were either low or undetectable in all NB cell lines tested with or without MYCN amplification. LGP2 expression levels were significantly increased only in NB cells without MYCN amplification, including SK‐N‐AS and SK‐N‐FI after poly (I:C) treatment in vitro and in mouse xenograft models. Ectopic expression of LGP2 in NB cells significantly enhanced poly (I:C)‐induced NB cell death associated with downregulation of MDA5, RIG‐I, MAVS and Bcl‐2, as well as upregulation of Noxa and tBid. By immunofluorescence analyses, LGP2 localized mainly in the cytoplasm of NB cells after poly (I:C) treatment. In human NB tissue samples, cytoplasmic LGP2 expression was positively correlated with histological differentiation and inversely correlated with MYCN amplification. Positive cytoplasmic LGP2 expression in tumor tissues could predict a favorable outcome in NB patients independent of other prognostic factors. In short, LGP2 was effective in promoting poly (I:C)‐induced NB suppression and cytoplasmic LGP2 can serve as an independent favorable prognostic factor in NB patients.

ZNF281 inhibits neuronal differentiation and is a prognostic marker for neuroblastoma

Derangement of cellular differentiation because of mutation or inappropriate expression of specific genes is a common feature in tumors. Here, we show that the expression of ZNF281, a zinc finger factor involved in several cellular processes, decreases during terminal differentiation of murine cortical neurons and in retinoic acid-induced differentiation of neuroblastoma (NB) cells. The ectopic expression of ZNF281 inhibits the neuronal differentiation of murine cortical neurons and NB cells, whereas its silencing causes the opposite effect. Furthermore, TAp73 inhibits the expression of ZNF281 through miR34a. Conversely, MYCN promotes the expression of ZNF281 at least in part by inhibiting miR34a. These findings imply a functional network that includes p73, MYCN, and ZNF281 in NB cells, where ZNF281 acts by negatively affecting neuronal differentiation. Array analysis of NB cells silenced for ZNF281 expression identified GDNF and NRP2 as two transcriptional targets inhibited by ZNF281. Binding of ZNF281 to the promoters of these genes suggests a direct mechanism of repression. Bioinformatic analysis of NB datasets indicates that ZNF281 expression is higher in aggressive, undifferentiated stage 4 than in localized stage 1 tumors supporting a central role of ZNF281 in affecting the differentiation of NB. Furthermore, patients with NB with high expression of ZNF281 have a poor clinical outcome compared with low-expressors. These observations suggest that ZNF281 is a controller of neuronal differentiation that should be evaluated as a prognostic marker in NB.

Transcription factor activating protein 4 is synthetically lethal and a master regulator of MYCN-amplified neuroblastoma

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target. We demonstrate that TFAP4 is a direct target of MYCN in neuroblastoma cells, and that its expression and activity strongly negatively correlate with neuroblastoma patient survival. Silencing TFAP4 selectively inhibits MYCN-amplified neuroblastoma cell growth both in vitro and in vivo, in xenograft mouse models. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as evidenced by increased neurite outgrowth and upregulation of neuronal markers. Taken together, our results demonstrate that TFAP4 is a key regulator of MYCN-amplified neuroblastoma and may represent a valuable novel therapeutic target.

Targeting of PHOX2B expression allows the identification of drugs effective in counteracting neuroblastoma cell growth.

The pathogenic role of the PHOX2B gene in neuroblastoma is indicated by heterozygous mutations in neuroblastoma patients and by gene overexpression in both neuroblastoma cell lines and tumor samples.PHOX2B encodes a transcription factor which is crucial for the correct development and differentiation of sympathetic neurons.PHOX2B overexpression is considered a prognostic marker for neuroblastoma and it is also used by clinicians to monitor minimal residual disease. Furthermore, it has been observed that neuronal differentiation in neuroblastoma is dependent on down-regulation of PHOX2B expression, which confirms that PHOX2B expression may be considered a target in neuroblastoma.Here, PHOX2B promoter or 3′ untranslated region were used as molecular targets in an in vitro high-throughput approach that led to the identification of molecules able to decrease PHOX2B expression at transcriptional and likely even at post-transcriptional levels. Further functional investigations carried out on PHOX2B mRNA levels and biological consequences, such as neuroblastoma cell apoptosis and growth, showed that chloroquine and mycophenolate mofetil are most promising agents for neuroblastoma therapy based on down-regulation of PHOX2B expression.Finally, a strong correlation between the effect of drugs in terms of down-regulation of PHOX2B expression and of biological consequences in neuroblastoma cells confirms the role of PHOX2B as a potential molecular target in neuroblastoma.

Characterization of Chromosomal Aberrations in Neuroblastoma Formalin-Fixed Paraffin- Embedded Specimens with Standard ArrayCGH Procedure - Preliminary Experience

Background: The broad spectrum of neuroblastoma (NB) clinical behavior depends on a genomic landscape of tumor cells. The amplification of MYCN oncogene is the most powerful negative prognostic marker in NB. Moreover, segmental chromosomal alterations are also associated with a poor outcome. Therefore, the comprehensive characterization of tumor genetic features is obligatory for NB patients. These features determine the risk stratification and therapeutic decisions in treatment. Purpose: Our report focuses on a possibility to use standard microarray procedure to demonstrate critical structural chromosomal alteration in archival samples of NB tumors. Methods and Findings: Formalin-fixed paraffin-embedded tissue samples from 8 NB primary tumors have been analyzed by cytogenetic microarrays. It achieved a very good quality of genomic DNA from fixed samples. Chromosomal abnormalities were detected in 7 out of 8 cases. It was not an incidence of MYCN amplification. Conclusion: The results demonstrate that it is possible to obtain reliable and highquality microarray data from archival samples.

Retracted: PHOX2B Is Associated with Neuroblastoma Cell Differentiation

Neuroblastoma is a common pediatric malignancy that accounts for ∼15% of tumor-related deaths in children. The tumor is generally believed to originate from neural crest cells during early sympathetic neurogenesis. As the degree of neuroblastoma differentiation has been correlated with clinical outcome, clarifying the molecular mechanisms that drive neuroblastoma progression and differentiation is important for increasing the survival of these patients. In a previous study, the authors identified paired-like homeobox 2b (PHOX2B) as a key mediator of neuroblastoma pathogenesis in a TH-MYCN mouse model. In the present study, they aimed to define whether PHOX2B is also associated with proliferation and differentiation of human neuroblastoma cells. PHOX2B expression in neuroblastoma cells was evaluated by immunoblot analyses, and the effects of PHOX2B on the proliferation of neuroblastoma cells in vitro were determined using clonogenic and sphere formation assays. Xenograft experiments in NOD/SCID mice were used to examine the in vivo response to PHOX2B knockdown. Their data demonstrated that PHOX2B acts as a prognostic marker in neuroblastoma and that retinoic acid-induced neuronal differentiation downregulates PHOX2B expression, thereby suppressing the self-renewal capacity of neuroblastoma cells and inhibiting tumorigenicity. These findings confirmed that PHOX2B is a key regulator of neuroblastoma differentiation and stemness maintenance and indicated that PHOX2B might serve as a potential therapeutic target in neuroblastoma patients.

Abstract 2083: TFAP4 inhibits differentiation of MYCN-amplified neuroblastoma

Abstract Background. Despite the identification of MYCN-amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed. An alternative approach is to identify genes synthetically lethal to MYCN-amplified neuroblastoma. Through a whole genome shRNA screen, we have previously identified transcription factor AP4 (TFAP4) as a synthetic lethal gene of MYCN-amplified neuroblastoma. Silencing of TFAP4, both in vitro and in vivo inhibited the growth of MYCN-amplified neuroblastoma. TFAP4 has previously reported to repress neuronal specific genes in non-neuronal cells (Kim et al, 2006). Here, we investigate the function of TFAP4 in inhibiting differentiation of MYCN-amplified neuroblastoma. Methods: Master regulators specifically associated to MYCN amplified patients were identified by MARINA (Master Regulator Inference algorithm). TFAP4 expression was measured by real-time PCR, Western blot and immunohistochemistry. GAP43 expression was detected by Western blot. Gene expression changes after silencing of TFAP4 or retinoic acid treatment was determined by RNAseq. Results: TFAP4 was identified as a master regulator specifically in MYCN amplified neuroblastoma patients in both NCI-TARGET dataset and a published GEO dataset (Kocak et al, 2013). We determined that TFAP4 is a direct target of MYCN and TFAP4 expression was highly correlated with Stage 4, MYCN-amplified neuroblastoma in both patient datasets. Neuronal markers TUBB3 and GAP43 expression were inversely correlated with TFAP4 expression in TARGET (P = 9.77e-03 and P = 7.78e-08). Silencing TFAP4 in MYCN amplified cells induced a differentiated phenotype, as shown by markedly increased the number of neurites, and an increase in the neuronal differentiation marker GAP43. Forty hours after silencing TFAP4, we observed 415 genes that were significantly up-regulated and 457 genes down-regulated (P&amp;lt;0.01). Genes with increased expression were enriched for gene ontology terms associated with neuronal activity. Gene signature of TFAP4 also showed a strong correlation with the treatment signature of the differentiation agent retinoic acid in SKNDZ. Conclusions. We demonstrated that TFAP4 is an important downstream effector of MYCN, that inhibits the neuronal differentiatin of MYCN-amplified neuroblastoma. This may provide novel targets for the treatment of neuroblastoma. Citation Format: Shuobo Zhang, Gonzalo Lopez, Jiyang Yu, Jose Silva, Angela Kadenhe-Chiweshe, Andrea Califano, Darrell Yamashiro. TFAP4 inhibits differentiation of MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2015-2083

Abstract 5000: The heterogeneous expression and functional relationship of ALK and NLRR1 in neuroblastoma

Abstract Introduction: Neuroblastoma (NB) is one of the most common extracranial solid tumors in childhood, which arises from neural crest cells in sympathoadrenal lineage. Although recent studies have revealed that anaplastic lymphoma kinase (ALK) signal is aberrantly activated in high-risk group of NB due to nonsynonymous mutations, gene amplification and mutation-independent overexpression, the regulatory mechanism of the signal remains elusive. Recently, we have identified neuronal leucine rich repeat 1 (NLRR1), a type I transmembrane protein, as a poor prognostic marker in NB and, notably, it accelerates epidermal growth factor receptor (EGFR) signal to enhance cell proliferation. Here we have revealed that NLRR1 functionally affects ALK signal and vice versa in NB. Material &amp; Methods: Cell growth of NB cell lines was evaluated by WST-8 assay or living cell imaging system. Immunoprecipitation was performed to check protein-protein interactions and concentrate secreted proteins. Further binding studies were performed by utilizing alkaline phosphatase-tagged and Fc-tagged proteins. Quantification of secreted proteins was assessed by sandwich ELISA assay. For ALK and EGFR signal inhibition, NB cells were treated with crizotinib, alectinib and erlotinib. The expression levels of ALK and NLRR1 were evaluated in embryonic Nlrr1 knockout mice as well as in the NB clinical samples by immunohistochemistry. Results: In the binding assays including immunoprecipitation, we found that NLRR1 extracellular domain physically interacted with the full-length ALK. Unexpectedly, NLRR1 expression suppressed ALK phosphorylation and cell proliferation in ALK-positive NB cells. Similar inhibitory effect was also observed by the treatment of the conditioned medium from the culture of NLRR1-expressing cells, suggesting a soluble form of NLRR1 protein. Certainly, the additional investigation confirmed that NLRR1 was cleaved to generate a soluble fragment of the extracellular domain. Conversely ALK inhibitors (ALKi) treatment resulted in up-regulation of NLRR1 and EGFR. Furthermore, EGF treatment restored ALKi-induced cell growth suppression. Additional erlotinib treatment certainly competed with the EGF rescue effect against ALKi. Finally, immunohistochemical analysis revealed that NLRR1 and ALK showed mutually exclusive expression pattern in NB tissues and murine embryonic dorsal root ganglia. Conclusion: We have identified the bilateral regulatory system between ALK and NLRR1, in which NLRR1 suppresses ALK phosphorylation whereas the inhibition of ALK results in the elevated expression of NLRR1 and EGFR. These findings in part support their mutually exclusive expression pattern in developing neural tissues and human NB. Further study should clarify the pathological meaning of the heterogeneity between ALK and NLRR1/EGFR pathways to develop a novel dual inhibition therapy in NB. Citation Format: Shunpei Satoh, Atsushi Takatori, Atsushi Ogura, Miki Ohira, Shamim Md. Hossain, Katsuyoshi Koh, Hiroshi Kishimoto, Ryoji Hanada, Akira Nakagawara. The heterogeneous expression and functional relationship of ALK and NLRR1 in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5000. doi:10.1158/1538-7445.AM2015-5000

PROGNOSTIC SIGNIFICANCE OF MDM2 GENE EXPRESSION IN CHILDHOOD NEUROBLASTOMA

To investigate the association of MDM2 expression at the mRNA levels in neuroblastoma with clinical features and unfavorable disease factors to determine the possibility of it usage as a prognostic marker of neuroblastoma. Total RNA and DNA were extracted from tumor tissue samples of total 91 neuroblastoma patients (mean age: 39.45 ± 4.81 months). MDM2 mRNA levels were detected with Q-PCR. TP53 gene deletion was detected with FISH method. MYCN amplification was detected with -Q-PCR analysis in fresh tumor samples and FISH in FFPE samples. We investigated the association of MDM2 mRNA expression with clinical outcome in neuroblastoma patients (n = 91). Kaplan - Meier curves showed a significant association of high MDM2 expression with poor event-free survival (p < 0.001). Clinical outcome of patients without MYCN amplification with low MDM2 expression was associated with better event-free survival than with high MDM2 expression (p < 0.001). Overexpression of MDM2 can be used as significant prognostic marker for patient stratification on risk groups and treatment optimization. Our results showed that the high expression of MDM2 at mRNA levels is an important factor of neuroblastoma prognosis. It may be a valuable additional molecular marker in guiding specific therapy in MYCN non-amplified NB patients without TP53 gene deletion.