Prognostic Factor In Neuroblastoma Research Articles

Migration, invasion, and metastasis are mediated by P-Rex1 in neuroblastoma.

Neuroblastoma accounts for approximately 15% of pediatric cancer-related deaths despite intensive multimodal therapy. This is due, in part, to high rates of metastatic disease at diagnosis and disease relapse. A better understanding of tumor biology of aggressive, pro-metastatic phenotypes is necessary to develop novel, more effective therapeutics against neuroblastoma. Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) has been found to stimulate migration, invasion, and metastasis in several adult malignancies. However, its role in neuroblastoma is currently unknown. In the present study, we found that P-Rex1 is upregulated in pro-metastatic murine models of neuroblastoma, as well as human neuroblastoma metastases. Correspondingly, silencing of P-Rex1 was associated with decreased migration and invasion in vitro. This was associated with decreased AKT-mTOR and ERK2 activity, dysregulation of Rac, and diminished secretion of matrix metalloproteinases. Furthermore, increased P-Rex1 expression was associated with inferior relapse-free and overall survival via tissue microarray and Kaplan-Meier survival analysis of a publicly available clinical database. Together, these findings suggest that P-Rex1 may be a novel therapeutic target and potential prognostic factor in neuroblastoma.

Immune microenvironment heterogeneity reveals distinct subtypes in neuroblastoma: insights into prognosis and therapeutic targets.

Neuroblastoma (NB) is a childhood cancer originating from immature nerve cells in the sympathetic nervous system. Current clinical and molecular subtyping methods for NB have limitations in providing accurate prognostic information and guiding treatment decisions. To overcome these challenges, we explored the microenvironment of NB based on the knowledge-based functional gene expression signatures (Fges), which revealed heterogeneous subtypes. Consensus clustering of Fges activity scores identified three subtypes (Cluster 1, Cluster 2, and Cluster 3) that demonstrated significant differences in prognosis compared to mainstream subtypes. We assessed the immune infiltration, immunogenicity, CD8T cytotoxicity, and tumor purity of these subtypes, uncovering their distinct biological functions. Cluster 1 and Cluster 2 exhibited higher immunoreactivity, while Cluster 3 displayed higher tumor purity and poor prognosis. Gene ontology annotation and pathway analysis identified immune activation in Cluster 1, epithelial-mesenchymal transition (EMT) in Cluster 2, and cell cycle processes in Cluster 3. Notably, the impact of EMT activity on prognosis may vary across NB subtypes. A classification model using XGBoost accurately predicted subtypes in independent NB cohorts, with significant prognostic differences. GPR125, CDK4, and GREB1 emerged as potential therapeutic targets in Cluster 3. CD4K inhibitors showed subtype-specific responses, suggesting tailored treatment strategies. Single-cell analysis highlighted unfavorable clinical features in Cluster 3, including high-risk classification and reduced cytotoxicity. Suppressed interactions between monocytes, macrophages, and regulatory T cells were observed, affecting immune regulation and patient prognosis. To summarize, we have identified a new independent prognostic factor in NB that underscores the significant correlation between tumor phenotype and immune contexture. These findings deepen our understanding of NB subtypes and immune cell interactions, paving the way for more effective treatment approaches.

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 (<2Y age, P = 2.8E-02; >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.

Improved risk stratification by PET-based intratumor heterogeneity in children with high-risk neuroblastoma.

The substratification of high-risk neuroblastoma is challenging, and new predictive imaging biomarkers are warranted for better patient selection. The aim of the study was to evaluate the prognostic role of PET-based intratumor heterogeneity and its potential ability to improve risk stratification in neuroblastoma. Pretreatment 18F-FDG PET/CT scans from 112 consecutive children with newly diagnosed neuroblastoma were retrospectively analyzed. The primary tumor was segmented in the PET images. SUVs, volumetric parameters including metabolic tumor volume (MTV) and total lesion glycolysis (TLG), and texture features were extracted. After the exclusion of imaging features with poor and moderate reproducibility, the relationships between the imaging indices and clinicopathological factors, as well as event-free survival (EFS), were assessed. The median follow-up duration was 33months. Multivariate analysis showed that PET-based intratumor heterogeneity outperformed clinicopathological features, including age, stage, and MYCN, and remained the most robust independent predictor for EFS [training set, hazard ratio (HR): 6.4, 95% CI: 3.1-13.2, p<0.001; test set, HR: 5.0, 95% CI: 1.8-13.6, p=0.002]. Within the clinical high-risk group, patients with a high metabolic heterogeneity showed significantly poorer outcomes (HR: 3.3, 95% CI: 1.6-6.8, p=0.002 in the training set; HR: 4.4, 95% CI: 1.5-12.9, p=0.008 in the test set) compared to those with relatively homogeneous tumors. Furthermore, intratumor heterogeneity outran the volumetric indices (MTVs and TLGs) and yielded the best performance of distinguishing high-risk patients with different outcomes with a 3-year EFS of 6% vs. 47% (p=0.001) in the training set and 9% vs. 51% (p=0.004) in the test set. PET-based intratumor heterogeneity was a strong independent prognostic factor in neuroblastoma. In the clinical high-risk group, intratumor heterogeneity further stratified patients with distinct outcomes.

Predictors of differential outcomes according to response to induction chemotherapy in high-risk neuroblastoma.

10032 Background: Response to induction chemotherapy has been shown to predict outcome in patients with high risk neuroblastoma, with those achieving a complete response (CR) having superior outcomes. Little is known about what factors impact survival within groups of patients with favorable and unfavorable end-induction response. We evaluated whether conventional prognostic factors remain prognostic in subsets of patients defined by response to induction. Methods: Patients from four COG high risk trials (A3973, ANBL02P1, ANBL0532, and ANBL12P1) were included. End-induction response was determined according to the 1993 International Neuroblastoma Response Criteria (INRC). Patients were categorized as having end-induction responses of CR, partial response (PR) or better, less than PR without progressive disease (PD), and PD. Univariate Cox models calculated OS hazard ratios for clinical and biological variables in subsets defined by response category. Results: 1,244 patients were included. Among all patients, age &gt;5 years, INSS stage 4 disease, adrenal primary site and unfavorable histology by INPC were associated with inferior OS (see Table). Among patients who achieved a CR, stage 4 disease was the only factor that remained significantly associated with worse OS. Among those who achieved PR or better, age &gt;5 years, stage 4 disease and unfavorable histology remained significantly associated with inferior OS. For those with less than PR but without PD, adrenal primary site, MYCN amplification and 1p LOH were significantly associated with inferior OS. For those with PD, MYCN amplification and 1p LOH were associated with worse OS, but older age was associated with better OS. Conclusions: Specific prognostic factors in neuroblastoma are associated with differential survival in groups defined by response to induction. Age, stage, and histology appear to be associated with OS for patients with more favorable response to induction, whereas MYCN and 1p LOH play a greater role in patients with unfavorable response to induction. These data can help to further define prognosis for patients with variable responses to induction. [Table: see text]

Long non-coding RNA NHEG1/hsa-miR-665/HMGB1 axis is involved in the regulation of neuroblastoma progression

ABSTRACT Long non-coding (lncRNA) neuroblastoma highly expressed 1 (NHEG1) has been reorganized as a prognostic factor in neuroblastoma (NB), but the molecular mechanisms in the suppression of neuroblastoma remain to be elucidated. In our study, we explored the functional roles of lncRNA NHEG1 in neuroblastoma and the underlying molecular mechanism. We collected NB tumor samples and adjacent normal tissues to compare lncRNA NHEG1 expression. Through bioinformatic target prediction, we selected potential downstream effectors of lncRNA NHEG1 for functional validation in NB cell lines. We observed that lncRNA NHEG1 was significantly upregulated in NB tissues as compared to the normal tissues. In NB tissues, lncRNA NHEG1 expression showed an inverse correlation with hsa-miR-665 (miR-655), but a positive correlation with high mobility group box 1 (HMGB1). In NB cell lines, lncRNA NHEG1 knockdown caused the upregulation of miR-665 and the downregulation of HMGB1. Through a series of functional assays, we further demonstrated that lncRNA Nheg1 knockdown suppressed cell proliferation, migration and invasion of NB cells, which could be rescued by miR-665 inhibitor and HMGB1 overexpression. Together, our data demonstrated that lncRNA NHEG1 serves as a competitive partner to negatively regulate the activity of miR-665, which relieves the inhibition on HMGB1 expression and promotes the aggressive phenotype of neuroblastoma cells. Our study indicates that lncRNA NHEG1/miR-665/HMGB1 axis may play an important role in regulating the aggressiveness and the progression of neuroblastoma.

Prognostic significance of NT5E/CD73 in neuroblastoma and its function in CSC stemness maintenance.

Cluster of differentiation 73 (CD73), a cell surface enzyme that catalyzes adenosine monophosphate (AMP) breakdown to adenosine, is differentially expressed in cancers and has prognostic significance. We investigated its expression profile in neuroblastoma (NB), its association with NB clinical outcomes, and its influence in the regulation of cancer stem cells' (CSCs) stemness maintenance. RNA-Seq data mining (22 independent study cohorts, total n =3836) indicated that high CD73 can predict good NB prognosis. CD73 expression (immunohistochemistry) gauged in an NB patient cohort (n =87) showed a positive correlation with longer overall survival (OS, P = 0.0239) and relapse-free survival (RFS, P = 0.0242). Similarly, high CD73 correlated with longer OS and RFS in advanced disease stages, MYCN non-amplified (MYCN-na), and Stage-4-MYCN-na subsets. Despite no definite association in children < 2years old (2Y), high CD73 correlated with longer OS (P = 0.0294) and RFS (P = 0.0315) in children > 2Y. Consistently, high CD73 was associated with better OS in MYCN-na, high-risk, and stage-4 subsets of children > 2Y. Multivariate analysis identified CD73 as an independent (P = 0.001) prognostic factor for NB. Silencing CD73 in patient-derived (stage 4, progressive disease) CHLA-171 and CHLA-172 cells revealed cell-line-independent activation of 58 CSC stemness maintenance molecules (QPCR profiling). Overexpressing CD73 in CHLA-20 and CHLA-90 cells with low CD73 and silencing in CHLA-171 and CHLA-172 cells with high CD73 showed that CD73 regulates epithelial to mesenchymal transition (E-Cadherin, N-Cadherin, Vimentin), stemness maintenance (Sox2, Nanog, Oct3/4), self-renewal capacity (Notch), and differentiation inhibition (leukemia inhibitory factor, LIF) proteins (confocal-immunofluorescence). These results demonstrate that high CD73 can predict good prognosis in NB, and further suggest that CD73 regulates stemness maintenance in cells that defy clinical therapy.

Neuronal Differentiation-Related Epigenetic Regulator ZRF1 Has Independent Prognostic Value in Neuroblastoma but Is Functionally Dispensable In Vitro.

Simple SummaryNeuroblastoma is the most common pediatric solid tumor occurring outside the brain, and it is thought to arise from cells that acquire errors during the normal process of embryonal development. Today, we know that embryonal development is regulated by epigenetics, a mechanism that determines which genes need to be expressed in each cell type and developmental step. Epigenetic errors, therefore, are considered contributory to the appearance and progression of tumors such as neuroblastoma. Here, we aimed at finding whether ZRF1, a known epigenetic regulator, could play a significant role in the aggressiveness of neuroblastoma. Our results suggest that ZRF1 does not seem to have any relevant function in neuroblastoma cells; however, the levels of this epigenetic regulator are related to the prognostic of neuroblastoma patients and could be used to predict their progression and improve the diagnosis.Neuroblastoma is a pediatric tumor of the peripheral nervous system that accounts for up to ~15% of all cancer-related deaths in children. Recently, it has become evident that epigenetic deregulation is a relevant event in pediatric tumors such as high-risk neuroblastomas, and a determinant for processes, such as cell differentiation blockade and sustained proliferation, which promote tumor progression and resistance to current therapies. Thus, a better understanding of epigenetic factors implicated in the aggressive behavior of neuroblastoma cells is crucial for the development of better treatments. In this study, we characterized the role of ZRF1, an epigenetic activator recruited to genes involved in the maintenance of the identity of neural progenitors. We combined analysis of patient sample expression datasets with loss- and gain-of-function studies on neuroblastoma cell lines. Functional analyses revealed that ZRF1 is functionally dispensable for those cellular functions related to cell differentiation, proliferation, migration, and invasion, and does not affect the cellular response to chemotherapeutic agents. However, we found that high levels of ZRF1 mRNA expression are associated to shorter overall survival of neuroblastoma patients, even when those patients with the most common molecular alterations used as prognostic factors are removed from the analyses, thereby suggesting that ZRF1 expression could be used as an independent prognostic factor in neuroblastoma.

Radiogenomics prediction for MYCN amplification in neuroblastoma: A hypothesis generating study.

MYCN amplification represents a powerful prognostic factor in neuroblastoma (NB) and may occasionally account for intratumoral heterogeneity. Radiomics is an emerging field of advanced image analysis that aims to extract a large number of quantitative features from standard radiological images, providing valuable clinical information. In this retrospective study, we aimed to create a radiogenomics model by correlating computed tomography (CT) radiomics analysis with MYCN status. NB lesions were segmented on pretherapy CT scans and radiomics features subsequently extracted using a dedicated library. Dimensionality reduction/features selection approaches were then used for features procession and logistic regression models have been developed for the considered outcome. Seventy-eight patients were included in this study, as training dataset, of which 24 presented MYCN amplification. In total, 232 radiomics features were extracted. Eight features were selected through Boruta algorithm and two features were lastly chosen through Pearson correlation analysis: mean of voxel intensity histogram (p=.0082) and zone size non-uniformity (p=.038). Five-times repeated three-fold cross-validation logistic regression models yielded an area under the curve (AUC) value of 0.879 on the training set. The model was then applied to an independent validation cohort of 21 patients, of which five presented MYCN amplification. The validation of the model yielded a 0.813 AUC value, with 0.85 accuracy on previously unseen data. CT-based radiomics is able to predict MYCN amplification status in NB, paving the way to the in-depth analysis of imaging based biomarkers that could enhance outcomes prediction.

Overexpression of Macrophage Migration Inhibitory Factor and Its Homologue D-Dopachrome Tautomerase as Negative Prognostic Factor in Neuroblastoma.

Neuroblastoma (NB) represents one of the most frequent pediatric solid tumors. Macrophage migration inhibitory factor (MIF) is a cytokine exerting multiple biological functions. More recently, a second member of the MIF family of cytokine has been identified, the D-dopachrome tautomerase (DDT), that exerts several overlapping functions with MIF. Growing evidence suggests a key role for MIF and DDT in the development of cancer. The aim of this study is to characterize the prognostic value of MIF and DDT in NB. We show that higher expression levels of MIF and DDT in Stage 4 NB samples are associated with a poorer prognosis, independently of the presence of MYCN amplification. Moreover, higher levels of MIF are mostly enriched by Th1 cells, while lower levels of MIF are associated with an increased proportion of B cells, Cytotoxic T cells, Dendritic cells and Natural Killer T cells. We also show that treatment with the histone deacetylase (HDAC) inhibitor, vorinostat, of the NB cell line, SH-SY5Y, determines a significant reduction in the expression of both MIF and DDT. Finally, MIF and DDT inhibition by short interfering RNA is able to revert vincristine sensitivity in vitro. Overall, our data suggest that MIF exert pro-tumorigenic properties in NB, likely by dampening antigen presentation and cytotoxic immune responses, and we propose the HDAC inhibitors as a potential therapeutic strategy for NB patients.

The role of N-Myc gene amplification in neuroblastoma childhood tumour - single-centre experience.

Aim of the studyNeuroblastoma (NBL) is one of the most common extracranial tumours occurring in children with N-Myc gene amplification, acknowledged as a marker of poor prognosis. We assessed the frequency of N-Myc amplification and its impact on NBL markers and on the treatment outcome.Material and methodsAmong 160 children with NBL treated from 1991 to 2015 in one centre 140 patients had known N-Myc gene status, and they were enrolled in the study. The analysed group was divided into two subgroups: with and without N-Myc amplification (25 and 115 children, respectively). Association of N-Myc amplification with stage of the disease, levels of biochemical parameters, overall survival (OS) and failure-free survival (FFS) were analysed.ResultsThe frequency of N-Myc amplification was 17.9%. Most children with N-Myc amplification (64%) were classified to stage 4 NBL. The levels of biochemical markers of NBL: ferritin, dopamine, NSE, and LDH were significantly higher in the group with N-Myc amplification, whereas the levels of VMA and HVA were lower. OS and FFS were significantly lower in children with N-Myc amplification in comparison to children from the control group (OS 53% vs. 76%, p = 0.03; FFS 50% vs. 72%, p = 0.03). The impact of N-Myc amplification on the treatment outcome was significant in patients with stage 4 NBL and children under one year of age.ConclusionsN-Myc amplification is a crucial prognostic factor in neuroblastoma, which is associated with almost all features related with poor prognosis and a higher probability of unfavourable outcome.

Novel Structural Abnormalities Involving Chromosomes 1, 17 and 2 Identified by Fluorescence In Situ Hybridization (FISH) and/or Cytogenetic Karyotyping in Kelly and SH-SY5Y Human Neuroblastoma Cell Lines, Respectively

Abstract: MYCN amplification and 1p36 deletion are important poor prognostic factors in neuroblastoma. 1p36 deletion and unbalanced translocations involving chromosomes 1, 17 and 2p were often reported in neuroblastoma cell lines. We aimed to investigate novel chromosomal abnormalities that are likely to affect neuroblastoma progression in Kelly and SH-SY5Y cell lines. Therefore, we analyzed the metaphase chromosomes using fluorescence in situ hybridization (FISH) method with probes specific to the chromosome bands 2p24 and 1p36 in SH-SY5Y and Kelly, respectively. Moreover, the rearrangements of chromosomes 1 and 17 from Kelly were re-examined by cytogenetic karyotyping. FISH analysis shows duplication of chromosome 2p24 on the long arm of a partner chromosome resulted from an unbalanced translocation der(?9)t(2;?9)(p24;q?34) in SH-SY5Y, suggesting that duplication of 2p24 locus containing MYCN gene may contribute to triggering MYCN amplification in neuroblastoma. On the other hand, FISH and karyotype analyses reveal three copies of chromosome 1 that consist of an intact chromosome 1, an extra derivative chromosome 1 with terminal and interstitial deletions (:p32→q25::q41→qter), and another including only interstitial deletion (pter→q25::q41→qter), leading to monosomy of the long arm segment 1q25-q41 in Kelly. These results suggest that chromosome 1q25-q41 may contain one or more tumour suppressor genes important for neuroblastoma progression. Together, FISH analysis shows that an additional 1p36 locus in Kelly is translocated to the short arm of extra chromosome 17, where the p53 tumour suppressor gene is located. Consequently, these novel structural abnormalities involving chromosomes 1, 17 and 2 could be contributed to the tumourigenicity of neuroblastoma cells.

A Myc Activity Signature Predicts Poor Clinical Outcomes in Myc-Associated Cancers.

Myc transcriptional activity is frequently deregulated in human cancers, but a Myc-driven gene signature with prognostic ability across multiple tumor types remains lacking. Here, we selected 18 Myc-regulated genes from published studies of Myc family targets in epithelial ovarian cancer (EOC) and neuroblastoma. A Myc family activity score derived from the 18 genes was correlated to MYC/MYCN/MYCL1 expression in a panel of 35 cancer cell lines. The prognostic ability of this signature was evaluated in neuroblastoma, medulloblastoma, diffuse large B-cell lymphoma (DLBCL), and EOC microarray gene expression datasets using Kaplan-Meier and multivariate Cox regression analyses and was further validated in 42 primary neuroblastomas using qPCR. Cell lines with high MYC, MYCN, and/or MYCL1 gene expression exhibited elevated expression of the signature genes. Survival analysis showed that the signature was associated with poor outcome independently of well-defined prognostic factors in neuroblastoma, breast cancer, DLBCL, and medulloblastoma. In EOC, the 18-gene Myc activity signature was capable of identifying a group of patients with poor prognosis in a "high-MYCN" molecular subtype but not in the overall cohort. The predictive ability of this signature was reproduced using qPCR analysis of an independent cohort of neuroblastomas, including a subset of tumors without MYCN amplification. These data reveal an 18-gene Myc activity signature that is highly predictive of poor prognosis in diverse Myc-associated malignancies and suggest its potential clinical application in the identification of Myc-driven tumors that might respond to Myc-targeted therapies. Cancer Res; 77(4); 971-81. ©2016 AACR.

MYCN overexpression is associated with unbalanced copy number gain, altered nuclear location, and overexpression of chromosome arm 17q genes in neuroblastoma tumors and cell lines.

MYCN amplification and MYCN overexpression are poor prognostic factors in neuroblastoma. Tumors with unbalanced chromosome arm 17q gain are often associated with MYCN amplification; however, the relationship between chromosome 17 copy number status and MYCN expression is not known. We investigated the relationship between MYCN expression and chromosome 17 copy number, nuclear location, and gene expression. By performing dual-colored fluorescence in situ hybridization on 16 primary neuroblastomas, we found that those with unbalanced gain of 17q have high MYCN expression, those with no gain have medium expression, and those with numerical gain have low expression (P < 0.0001). We also found that the nuclear location of 17q correlates with chromosome 17 copy number status: copies in tumors with unbalanced gain and no gain of chromosome 17 occupy a more central location than those in tumors with balanced gain (P < 0.0001). We show that a more central nuclear location of 17q coincides with increased expression of genes found within this chromosome arm. To further understand the association between MYCN expression and chromosome 17, we overexpressed MYCN in two low-expressing MYCN cell lines, SHEP and GIMEN. We found that both cell lines had an unbalanced gain of chromosome 17q, a more central nuclear location of the region and increased expression of the 17q genes. Therefore, this study indicates, for the first time, a functional relationship between MYCN overexpression and the gain of 17q in neuroblastoma.

IGF2BP1 harbors prognostic significance by gene gain and diverse expression in neuroblastoma.

Chromosomal 17q21-ter gain in neuroblastoma is both a common and prognostically significant event. The insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) gene is located near the proximal edge of this region. Here, its prognostic value is evaluated in neuroblastoma. The mRNA expression of IGF2BP family members was first evaluated by microarray data sets. In addition, in a separate cohort of 69 tumors, IGF2BP1 gene copy number, mRNA, and protein abundance were determined and compared with clinical parameters. In two independent microarray data sets, 77% to 100% of tumors had substantial IGF2BP1 mRNA levels measured. High IGF2BP1 transcript abundance was significantly associated with stage 4 tumors (P < .001) and decreased patient survival (P < .001). IGF2BP1 was also associated with MYCN gene amplification and MYCN mRNA abundance. In the 69 neuroblastoma samples, IGF2BP1 DNA copy number (increased in 84% of tumors), mRNA, and protein abundance were significantly higher in stage 4 compared with stage 1 tumors. Importantly, IGF2BP1 protein levels were associated with lower overall patient survival (P = .012) and positively correlated with MYCN mRNA, even when excluding MYCN-amplified tumors. Moreover, IGF2BP1 clearly affected MYCN expression and neuroblastoma cell survival in vitro. In neuroblastoma, IGF2BP1 was expressed in the majority of neuroblastoma specimens analyzed and was associated with lower overall patient survival and MYCN abundance. These data demonstrate that IGF2BP1 is a potential oncogene and an independent negative prognostic factor in neuroblastoma.

Endoglin (CD 105) as a potential prognostic factor in neuroblastoma.

Endoglin (CD105) is a cytokine that modulates angiogenesis by regulating different cellular functions, including endothelial proliferation, differentiation, migration and formation of microvessels. CD105 is expressed strongly in the tumor vasculature, and intratumoral microvessel density (IMVD), as determined by the use of antibodies to CD105, it has been found to be an important prognostic indicator for outcome in various malignances. This study aims to determine if the clinical outcome of children with neuroblastoma is correlated with IMVD, as determined by CD105 staining and other prognostic factors. Tumor tissue specimens from 38 patients with peripheral neuroblastic tumors who underwent surgical resection or biopsy of their primary tumor without any preoperative therapy were retrospectively reviewed. IMVD was identified immunohistochemically using monoclonal antibodies against CD105. Prognostic factors, such as the MYCN oncogene, disease stage, histopathology and age, were correlated with outcome. Among 38 examined specimens, the median IMVD value was 23.2 (15.1-28.4). The IMVD identified by CD105 was significantly higher in patients with unfavorable histology, metastatic disease, MYCN amplification and COG high risk group. ROC analysis was used to find significant IMVD level regarding EFS. The cut-off >18 was selected according to the greatest sensitivity (100%) and specificity (68.42%). The multivariate Cox proportional hazards analysis demonstrated that MYCN amplification and IMVD were significant prognostic factors in predicting EFS (hazard ratio for MYCN amplification: 3.61; 95% CI: 1.20-10.90; P = 0.023 and for IMVD: 1.05; 95% CI: 1.00-1.09; P = 0.037). IMVD determined by CD105 appeared to be an independent prognostic factor for neuroblastoma.

Frequency of the ALK gene and its prognostic value in neuroblastoma by FISH

Introduction The anaplastic lymphoma kinase (ALK) gene has been identified as a major neuroblastoma (NB) predisposition gene. Furthermore, there are controversies on the correlation between ALK gene aberration and clinical outcome in neuroblastoma (NBL). Materials and methods We evaluated N-MYC/ALK gene copy number by fluorescence in situ hybridization and analyzed 32 bone marrow samples infiltrated by NB and analyzed their association with the clinical outcome of the patients. Results Although an increase in ALK gene aberration is a recurrent genetic abnormality of NB (50%, 16/32), ALK amplification was only present in one NB (3.2%, 1/32). In addition, ALK positivity also significantly correlates with N-MYC gene copy number ( P P Conclusion ALK aberrations in NB were correlated with advanced tumor types and an increase in both N-MYC/ALK gene aberrations. ALK aberrations predict an inferior prognosis, which can be used as a prognostic factor in NB in clinical practice.

Aryl Hydrocarbon Receptor Downregulates MYCN Expression and Promotes Cell Differentiation of Neuroblastoma

Neuroblastoma (NB) is the most common malignant disease of infancy. MYCN amplification is a prognostic factor for NB and is a sign of highly malignant disease and poor patient prognosis. In this study, we aimed to investigate novel MYCN-related genes and assess how they affect NB cell behavior. The different gene expression found in 10 MYCN amplification NB tumors and 10 tumors with normal MYCN copy number were analyzed using tissue oligonucleotide microarrays. Ingenuity Pathway Analysis was subsequently performed to identify the potential genes involved in MYCN regulation pathways. Aryl hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, was found to be inversely correlated with MYCN expression in NB tissues. This correlation was confirmed in a further 14 human NB samples. Moreover, AHR expression in NB tumors was found to correlate highly with histological grade of differentiation. In vitro studies revealed that AHR overexpression in NB cells induced spontaneous cell differentiation. In addition, it was found that ectopic expression of AHR suppressed MYCN promoter activity resulting in downregulation of MYCN expression. The suppression effect of AHR on the transcription of MYCN was compensated for by E2F1 overexpression, indicating that E2F1 is involved in the AHR-regulating MYCN pathway. Furthermore, AHR shRNA promotes the expression of E2F1 and MYCN in NB cells. These findings suggest that AHR is one of the upstream regulators of MYCN. Through the modulation of E2F1, AHR regulates MYCN gene expression, which may in turn affect NB differentiation.

Changes in MYCN expression in human neuroblastoma cell lines following cisplatin treatment may not be related to MYCN copy numbers

Neuroblastoma is a tumor accounting for approximately 10% of all childhood malignancies and 50% of all childhood cancer-related deaths. MYCN gene copy number variation represents the most important prognostic factor in neuroblastoma. Prognostic significance of MYCN gene expression is more complicated and may depend on other factors such as MYCN gene copy number status. In the present study, we assessed MYCN gene expression using real-time RT-PCR following cisplatin treatment in three human neuroblastoma cell lines (UKF-NB-3, UKF-NB-4 and SK-N-AS) and their cisplatin-resistant counterparts. We also examined MYCN gene status and copy number (gain and amplification) variations using interphase and metaphase fluorescent insitu hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA). Only cisplatin-sensitive UKF-NB-4 cells exhibited decreased MYCN expression following treatment with cisplatin. Other sensitive neuroblastoma cells did not exhibit a change in MYCN expression. In contrast, cisplatin-resistant UKF-NB-4 and SK-N-AS cells exhibited increased MYCN expression irrespective of the number of MYCN copies or concentration of cisplatin in the medium. In MYCN-amplified neuroblastoma cells we did not observe any significant change in the number of MYCN copies after cisplatin treatment, whereas MYCN-non-amplified SK-N-AS cells revealed during cisplatin treatment an increased number of MYCN gene copies caused by 2p gain in the majority of cells by FISH. We postulated that cisplatin treatment does not result directly in altered transcription of MYCN. A functional change in MYCN mRNA levels and increased MYCN expression in cisplatin-resistant neuroblastoma cells do not have a clear relationship to MYCN copy numbers. These findings may further contribute to the understanding of cisplatin chemotherapy in connection with MYCN expression, and the possible copy number variations in MYCN neuroblastoma cells may be of importance since targeting of MYCN is being tested as neuroblastoma therapy.

On the significance of germline cytogenetic rearrangements at MYCN locus in neuroblastoma

BackgroundMYCN oncogene amplification is the most important prognostic factor in neuroblastoma. 25% neuroblastoma tumors have somatic amplifications at this locus but little is known about its constitutional aberrations and their potential role in carcinogenesis. Here, we have performed an array-CGH and qPCR characterization of two patients with constitutional partial 2p trisomy including MYCN genomic region.ResultsOne of the patients had congenital neuroblastoma and showed presence of minute areas of gains and losses within the common fragile site FRA2C at 2p24 encompassing MYCN. The link between 2p24 germline rearrangements and neuroblastoma development was reassessed by reviewing similar cases in the literature.ConclusionsIt appears that constitutional rearrangements involving chromosome 2p24 may play role in NB development.