Heterogeneity Of Neuroblastoma Research Articles

Abstract LB015: Differential expression of deubiquitinating enzymes and its related DNA damage response genes in neuroblastoma heterogeneity

Abstract SK-N-AS and SK-N-DZ are two cell lines of pediatric solid tumors neuroblastomas well characterized to evaluate heterogeneity between different neuroblastomas and are presumed as models for differences between different neuroblastoma. Using this model system, we recently evaluated if radiation could improve efficacy of radiation therapy and to follow these studies later with animal models to determine decrease of tumor burden. Differences between the N-type and S-type neuroblastoma cell lines SK-N-DZ and SK-N-AS, respectively were observed to be high and their responses to radiation stress were equally heterogeneous. Ubiquitin signaling plays a key role during the DNA damage response in protein recruitment and protein interactions. Deubiquitinating enzymes (DUBs) deconjugate ubiquitin from ubiquitylated substrate proteins to control their activity and stability and are shown to regulate various biological processes. DUBs may play a role in regulating the radiosensitivity of cancer cells by modulating DNA damage response and repair pathways. Determination of specific DUBs that are involved in regulating radiosensitivity in cancer cells will provide opportunity for drug targeting and visa-vis enhance the efficacy of radiation therapy in neuroblastoma cancer treatment. Here, using bioinformatic tools, the differentially regulated DUBs were identified. The relationship between DEGs-DUBs with the genes involved in various DNA repair pathways (NHEJ, HR and Fanconi anemia pathway; KEGG pathway database) are described. We identified 32 DEGs (14 upregulated and 18 downregulated) with these neuroblastoma cell lines. USP22 and USP41 DUBs and its differential expression in these cell lines denotes the importance of such analysis. Especially considering USP22 a potentially biomarker for predicting the outcome of patients with neuroblastoma. These DEG-DUBs were likewise correlated with 91 DNA damage response (DDR) genes belonging to three different pathways (NHEJ, HR and Fanconi anemia pathway). Out of which 44 of the DDR genes showed significant correlation with DEG-DUBs. Further studies are needed to determine the mechanisms underlying the complex relationship of these DUBs and to determine the critical DUBs for drug targeting and to enhance the efficacy of radiation therapy in cancer treatment. Citation Format: Vaibhav Vaibhav, Fahika Nazeerulla, Tatjana Paunesku, Ekta Tripathi, Gayle Woloschak, Prashanthi Karyala, Rao V. Papineni. Differential expression of deubiquitinating enzymes and its related DNA damage response genes in neuroblastoma heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB015.

Neuroblastoma and its Target Therapies: A Medicinal Chemistry Review.

Neuroblastoma (NB) is a childhood malignant tumour belonging to a group of embryonic tumours originating from progenitor cells of the sympathoadrenal lineage. The heterogeneity of NB is reflected in the survival rates of those with low and intermediate risk diseases who have survival rates ranging from 85 to 90 %. However, for those identified with high-risk Stage 4 NB, the treatment options are much more limited. For this group, current treatment consists of immunotherapy (monoclonal antibodies) in combination with anti-cancer drugs and has a 40 to 50 % survival rate. The purpose of this review is to summarise NB research from a medicinal chemistry perspective and to highlight advances in targeted drug therapy in the field. The review examines the medicinal chemistry of a number of drugs tested in research, some of which are currently under clinical trial. It concludes by proposing that future medicinal chemistry research into NB should consider other possible target therapies and adopt a multi-target drug approach rather than a one-drug-one-target approach for improved efficacy and less drug-drug interaction for the treatment of NB Stage 4 (NBS4) patients.

Abstract 2847: Study of spatial and temporal epigenetic heterogeneity in neuroblastoma suggests role of H3K27me3-mediated repression

Abstract Introduction: Trimethylation of Histone 3 Lysine 27 (H3K27) is widely understood to be a mark associated with gene repression and linked to gene silencing during development. Neuroblastoma, a developmental pediatric extracranial solid malignancy, has a relatively silent mutational landscape. Therefore, epigenetic modifications, including H3K27me3 mediated repression, could explain underlying developmental origins and the wide clinical heterogeneity observed. While previous studies have focused on the role of distinct super enhancer histone marks and their core regulatory circuits, less is understood about repressive control. We aimed to therefore investigate the role of H3K27me3-mediated repression in Neuroblastoma patients. Methods: Genome-wide H3K27me3 ChIP-Seq profiles were obtained from 48 diagnostic tissue biopsies covering all clinical and molecular patient subgroups. Transcriptome sequencing, ChIP-Seq of additional histone marks and WES/WGS was also performed on the same samples and on a panel of Neuroblastoma cell line models, with multiomics integrative analysis to validate findings. HMCAN, deepTools, DiffBind and ROSE packages were used to analyze the ChIP-seq data. Results: A comparison of H3K27me3 regions across all patient ChIP-Seq profiles, identified commonly repressed genes specific to Neuroblastoma. These genes are enriched in development and differentiation pathways, with significantly lower RNA expression levels than other publicly available pediatric tumor datasets. When compared to publicly available single cell RNAseq of the healthy adrenal medulla, we can further highlight a potential role of H3K27me3-mediated repression in regulating cell lineage commitment and differentiation ability. In addition, we identified that patient samples cluster into their clinical subgroups based on H3K27me3 patterns and differentially repressed genes, specifically between MYCN-amplified and non-amplified high-risk groups. A CRISPR screen to further validate the functional role of these repressed genes is being carried out. Conclusions: To our knowledge, this study shows for the first time that understanding of H3K27me3 mediated repression in Neuroblastoma, can uncover new insights into both disease development and clinical subclassifications. This knowledge will be important when considering future potential novel treatment options. Citation Format: Charlotte Butterworth, Alexandra Saint-Charles, Yasmine Iddir, Jaydutt Bhalshankar, Angela Bellini, Charles Bobin, Elisa Wecht, Young-Gyu Park, Caroline Louis, Virginie Raynal, Dominique Plantaz, Hervé Sartelet, Valérie Combaret, Gaelle Pierron, Isabelle Janoueix-Lerosey, Kai-Oliver Henrich, Olivier Delattre, Frank Westermann, Gudrun Schleiermacher. Study of spatial and temporal epigenetic heterogeneity in neuroblastoma suggests role of H3K27me3-mediated repression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2847.

Mutational topography reflects clinical neuroblastoma heterogeneity

Neuroblastoma is a pediatric solid tumor characterized by strong clinical heterogeneity. Although clinical risk-defining genomic alterations exist in neuroblastomas, the mutational processes involved in their generation remain largely unclear. By examining the topography and mutational signatures derived from all variant classes, we identified co-occurring mutational footprints, which we termed mutational scenarios. We demonstrate that clinical neuroblastoma heterogeneity is associated with differences in the mutational processes driving these scenarios, linking risk-defining pathognomonic variants to distinct molecular processes. Whereas high-risk MYCN-amplified neuroblastomas were characterized by signs of replication slippage and stress, homologous recombination-associated signatures defined high-risk non-MYCN-amplified patients. Non-high-risk neuroblastomas were marked by footprints of chromosome mis-segregation and TOP1 mutational activity. Furthermore, analysis of subclonal mutations uncovered differential activity of these processes through neuroblastoma evolution. Thus, clinical heterogeneity of neuroblastoma patients can be linked to differences in the mutational processes that are active in their tumors.

Molecular Heterogeneity in Neuroblastoma and Its Clinical Significance

Molecular Heterogeneity in Neuroblastoma and Its Clinical Significance

Identification of the molecular subtypes and construction of risk models in neuroblastoma

The heterogeneity of neuroblastoma directly affects the prognosis of patients. Individualization of patient treatment to improve prognosis is a clinical challenge at this stage and the aim of this study is to characterize different patient populations. To achieve this, immune-related cell cycle genes, identified in the GSE45547 dataset using WGCNA, were used to classify cases from multiple datasets (GSE45547, GSE49710, GSE73517, GES120559, E-MTAB-8248, and TARGET) into subgroups by consensus clustering. ESTIMATES, CIBERSORT and ssGSEA were used to assess the immune status of the patients. And a 7-gene risk model was constructed based on differentially expressed genes between subtypes using randomForestSRC and LASSO. Enrichment analysis was used to demonstrate the biological characteristics between different groups. Key genes were screened using randomForest to construct neural network and validated. Finally, drug sensitivity was assessed in the GSCA and CellMiner databases. We classified the 1811 patients into two subtypes based on immune-related cell cycle genes. The two subtypes (Cluster1 and Cluster2) exhibited distinct clinical features, immune levels, chromosomal instability and prognosis. The same significant differences were demonstrated between the high-risk and low-risk groups. Through our analysis, we identified neuroblastoma subtypes with unique characteristics and established risk models which will improve our understanding of neuroblastoma heterogeneity.

MYCN Amplification, along with Wild-Type RB1 Expression, Enhances CDK4/6 Inhibitors' Efficacy in Neuroblastoma Cells.

Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB's mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB's cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.

Potential Prognostic Value of FDG PET/CT-derived Intratumoral Metabolic Heterogeneity in Neuroblastoma.

Potential Prognostic Value of FDG PET/CT-derived Intratumoral Metabolic Heterogeneity in Neuroblastoma.

Abstract 3194: Cancer-associated fibroblast heterogeneity in neuroblastoma

Abstract Extracellular matrix (ECM) remodeling is a critical process within the solid tumor microenvironment. Cancer associated fibroblasts (CAFs) are the primary producers of matrix proteins and remodeling enzymes that cross-link and degrade matrix proteins. CAFs are enigmatic in that they can promote or impede tumor growth, suggesting heterogeneity that is not completely understood. How CAF heterogeneity evolves over tumor progression and how this evolution impacts ECM remodeling within solid tumors remain an outstanding question across cancer models. Recent studies have focused on CAF function in models of adult cancer, but little attention has been directed toward understanding CAF heterogeneity and ECM remodeling in childhood cancers, which are etiologically different diseases. Neuroblastoma (NB) is a childhood cancer of neuroendocrine cells that accounts for 15% of pediatric cancer deaths. Like other solid tumors, the NB tumor microenvironment includes malignant cells, CAFs, and immune cells. Our laboratory studies NB using a penetrant, spontaneous model of NB (TH-MYCN, AlkF1178L/+) in immunocompetent mice, allowing in vivo investigation of the tumor immune microenvironment from tumorigenesis to progressive disease. Using this model, we previously demonstrated the pro-tumorigenic role of macrophages and CD4+ T cells by showing that their depletion significantly reduced the formation of tumors and extended survival. In this study, 10X Chromium gene expression analysis of stromal cells from nascent and mature tumors identified two distinct subsets of CAFs characterized by alpha smooth muscle actin (Acta2) and Wilms tumor 1 (Wt1) expression. Interestingly, only Wt1 CAFs expressed chemokines and cytokines involved in recruitment of myeloid and T cells. Wt1 CAFs were enriched in the early tumor microenvironment and expressed genes encoding ADAMTS enzymes involved in degradation of the proteoglycan versican. Our preliminary findings indicate that Wt1 CAFs may be a crucial early recruiter of pro-tumorigenic immune cells in our model of neuroblastoma. Citation Format: Nicolas B. Peterson, Paul G. Thomas. Cancer-associated fibroblast heterogeneity in neuroblastoma [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 3194.

Circulating tumor cells in neuroblastoma: Current status and future perspectives.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 10% to 20% of deaths of pediatric malignancies. Due to the poor prognosis and significant biological heterogeneity of neuroblastoma, it is essential to develop personalized therapeutics and monitor treatment response. Circulating tumor cells (CTCs), as one of the important analytes for liquid biopsy, could facilitate response assessment and outcome prediction for patients in a non-invasive way. Several methods and platforms have been used for the enrichment and detection of CTCs. The enumeration of CTCs counts and evaluation of tumor-specific mRNA transcript levels could provide prognostic information at diagnosis, during or after chemotherapy, and during the process of disease progression. So far, studies into neuroblastoma CTCs are only in the preliminary stages. The quality-controlled large prospective cohort studies are needed to evaluate the clinical significance and statistical rigor of CTC detection methods. Moreover, there remains a lot to be explored and investigated in genotyping characterization of neuroblastoma (NB) CTCs and construction of in-vitro or in-vivo functional models. CTCs and circulating tumor DNA (ctDNA) analysis will be complementary in understanding tumor heterogeneity and evolution over the course of therapy for patients with NB in the future.

Genetic and Histopathological Heterogeneity of Neuroblastoma and Precision Therapeutic Approaches for Extremely Unfavorable Histology Subgroups.

Peripheral neuroblastic tumors (neuroblastoma, ganglioneuroblastoma and ganglioneuroma) are heterogeneous and their diverse and wide range of clinical behaviors (spontaneous regression, tumor maturation and aggressive progression) are closely associated with genetic/molecular properties of the individual tumors. The International Neuroblastoma Pathology Classification, a biologically relevant and prognostically significant morphology classification distinguishing the favorable histology (FH) and unfavorable histology (UH) groups in this disease, predicts survival probabilities of the patients with the highest hazard ratio. The recent advance of neuroblastoma research with precision medicine approaches demonstrates that tumors in the UH group are also heterogeneous and four distinct subgroups—MYC, TERT, ALT and null—are identified. Among them, the first three subgroups are collectively named extremely unfavorable histology (EUH) tumors because of their highly aggressive clinical behavior. As indicated by their names, these EUH tumors are individually defined by their potential targets detected molecularly and immunohistochemically, such as MYC-family protein overexpression, TERT overexpression and ATRX (or DAXX) loss. In the latter half on this paper, the current status of therapeutic targeting of these EUH tumors is discussed for the future development of effective treatments of the patients.

Abstract PO-041: Multiplexed ion beam imaging to describe tumor-immune microenvironment and tumor heterogeneity in neuroblastoma

Abstract Neuroblastoma is a tumor of the peripheral sympathetic nervous system, accounting for 10% of pediatric cancer associated deaths and the most common cancer diagnosed during infancy(1,2). This neural crest derived malignancy is characterized by clinical heterogeneity ranging from spontaneous regression in some patients to treatment resistance, metastasis and death in others(3,4). Here, we report the application of Multiplexed Ion Beam Imaging (MIBI), a single-cell, high-dimensional imaging method to determine the expression and co-expression of antigens on neuroblastoma cells and describe the local immune environment. We have optimized a 40-antibody panel to capture adrenergic or mesenchymal identity(5,6), infiltrating immune cells, tissue architecture and known neuroblastoma proteins. We optimized our antibody staining and determined tissue specificity using a unique tissue microarray which included 32 NBL cores but also additional childhood solid tumors including osteosarcoma, glioblastoma, Ewing’s sarcoma, rhabdomyosarcoma, atypical teratoid rhabdoid tumor, and Wilms tumor. Healthy pediatric tissues from various organs including kidney, liver, and tonsil were also included. We have uncovered divergent neuroblastoma cell populations differentiated by co-expression of CD57, Ki67 and GPC2 as well as mesenchymal (FN1 and SNAI2/SLUG) and adrenergic (TH, GATA3 and PHOX2B) antibodies, demonstrating promising early results of our application of MIBI. We will describe infiltrating immune populations and spatial relationships to these unique tumor cell populations. Our work demonstrates the feasibility of using MIBI to make translatable discoveries in neuroblastoma and other childhood cancers. With this we will lay the foundation for future discovery and validation of novel targets for immunotherapy in neuroblastoma. Citation Format: Marte B. Kammersgaard, Marc Bosse, Daniel Martinez, Kristopher R. Bosse, John M. Maris, Crystal L. Mackall, Robert M. Angelo, Kara L. Davis. Multiplexed ion beam imaging to describe tumor-immune microenvironment and tumor heterogeneity in neuroblastoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-041.

Neuroblastoma and Stem Cell Therapy: An Updated Review.

Neuroblastoma (NBM) is the second leading pediatric cancer that develops from the precursors of the sympathetic nervous system. To date, surgery, chemotherapy, and radiation serve as the first-line treatment against NBM in high-risk patients. However, few of these approaches have severe side effects. Recently, numerous studies have also reported that high chemotherapy doses, along with stem cell rescue, improvise event-free survival in patients. In this review, the authors attempted to discuss the pathogenesis associated with NBM and how stem cell therapy can be employed for the treatment of NBM. Stem cells are a group of multipotent, undifferentiated cells that are capable of producing all cells in a particular tissue, organ, or organism. They have an endogenous self-renewal property. This property is tightly modulated for the normal homeostasis within the body. However, the failure of this process leads to carcinogenesis, including NBM. As these properties are modulated via various intrinsic as well as extrinsic pathways, the arrest of these pathways via various drugs may help in controlling various carcinomas, including NBM. Recently, stem cells were utilized for the diagnosis and treatment of NBM. Nevertheless, most of the studies conducted to date are mainly designed on bulk-cell analysis, which in turn provides little information about the population of cells. Thus, the authors believe that, by employing single-cell RNA sequencing technologies and computational approaches, we can unmask the tumor heterogeneity in NBM in a more comprehensive way. In the near future, this information will be highly useful for the identification of biomarkers and treatment associated with NBM in humans.

Hypoxia Predicts Poor Prognosis in Neuroblastoma Patients and Associates with Biological Mechanisms Involved in Telomerase Activation and Tumor Microenvironment Reprogramming

The biological and clinical heterogeneity of neuroblastoma (NB) demands novel biomarkers and therapeutic targets in order to drive the most appropriate treatment for each patient. Hypoxia is a condition of low-oxygen tension occurring in poorly vascularized tumor tissues. In this study, we aimed to assess the role of hypoxia in the pathogenesis of NB and at developing a new clinically relevant hypoxia-based predictor of outcome. We analyzed the gene expression profiles of 1882 untreated NB primary tumors collected at diagnosis and belonging to four existing data sets. Analyses took advantage of machine learning methods. We identified NB-hop, a seven-gene hypoxia biomarker, as a predictor of NB patient prognosis, which is able to discriminate between two populations of patients with unfavorable or favorable outcome on a molecular basis. NB-hop retained its prognostic value in a multivariate model adjusted for established risk factors and was able to additionally stratify clinically relevant groups of patients. Tumors with an unfavorable NB-hop expression showed a significant association with telomerase activation and a hypoxic, immunosuppressive, poorly differentiated, and apoptosis-resistant tumor microenvironment. NB-hop defines a new population of NB patients with hypoxic tumors and unfavorable prognosis and it represents a critical factor for the stratification and treatment of NB patients.

Abstract 5216: Atypical PKC inhibitors ICA-1S and ζ-Stat show inhibition of neuroblastoma cell proliferation

Abstract Neuroblastoma, the pediatric cancer of the early nerve cells, has the tendency to become resistant to traditional therapeutics due to its highly heterogeneous nature. Atypical Protein Kinase C-iota (PKC- ι) has been found to promote cell proliferation in neuroblastoma cells through the PKC- ι /Cdk7/Cdk2 signaling pathway. In this study we utilized various cell lines; I-type (intermediate) CHP-212& BE(2)-C, N-types (neuroblastic); BE(2)-M17& SH-SY5Y and S-types (substrate adherent); SK-N-BE(2) & LAI-5s, representing neuroblastoma heterogeneity to investigate the use of aPKC inhibitors as potential neuroblastoma therapeutics. The aPKC inhibitors, 5-amino-1-2,3-dihydroxy-4-(methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) (PKC- ι specific) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) (PKC- ζ specific), both display the ability to reduce neuroblastoma proliferation in a cell type specific manner. ICA-1S was shown to reduce proliferation of BE(2)-C, BE(2)-M17, LAI-5s and SK-N-BE(2) to varying degrees. ζ-Stat was able to consistently reduce proliferation of BE(2)-C, BE(2)-M17, SK-N-BE(2), LAI-5s. The degree of proliferation inhibition varied by drug and cell line. Increasing concentration of ICA-1S displayed an inverse relationship between increasing concentration and a decrease in cell proliferation in the four cell lines previously mentioned. ζ-Stat showed a similar relationship to ICA-1S, however had a lower level inhibition than ICA-1S. WST-1 analysis showed that both drugs displayed varying degrees of toxicity in a cell line dependent manner. The results show that I-type cell lines showed the most linearly proportional toxicity to ICA-1S and ζ-Stat in that increasing concentration increased drug toxicity. N-type cell lines displayed a disproportional relationship between concentration and toxicity. The S-type cell lines followed suit with the N-type in their varying degrees of toxicity. Western blot analysis shows that both inhibitors were effective in reducing the expression of total aPKC levels. We observed the downregulation of various mesenchymal markers using the inhibitors. In particular, ICA-1S appears to have a high percentage of decreased expression of N-cadherin. We also accessed the degree of phosphorylation of Vimentin, S33, S39 and S56 positions which are crucial for its activation. In summary both inhibitors prove to be promising potential neuroblastoma therapeutics. The drugs appear to behave in a more cytostatic manner inhibiting the proliferation of neuroblastoma cells. Further western blots ae still ongoing to determine other pathways through which the drugs act to decrease proliferation. Ongoing experiments include the analysis of tumors collected from mouse models to ascertain the effects of the drugs in vivo as well as the analysis of human tumor samples to ascertain the expression of aPKCs and other markers in patient populations. Experiments also include the use of gene expression knockdowns to compare with the effects from the inhibitors as well as the use of microscopy techniques; fluorescent and electron miscopy to observe the localization of mesenchymal and proliferative markers. Citation Format: Sloan Breedy, Wishrawana S. Ratnayake, Christopher Apostolatos, Avijit Dey, Mildred Acevedo-Duncan. Atypical PKC inhibitors ICA-1S and ζ-Stat show inhibition of neuroblastoma cell proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5216.

Noninvasive MRI Native T1 Mapping Detects Response to MYCN-targeted Therapies in the Th-MYCN Model of Neuroblastoma.

Noninvasive early indicators of treatment response are crucial to the successful delivery of precision medicine in children with cancer. Neuroblastoma is a common solid tumor of young children that arises from anomalies in neural crest development. Therapeutic approaches aiming to destabilize MYCN protein, such as small-molecule inhibitors of Aurora A and mTOR, are currently being evaluated in early phase clinical trials in children with high-risk MYCN-driven disease, with limited ability to evaluate conventional pharmacodynamic biomarkers of response. T1 mapping is an MRI scan that measures the proton spin-lattice relaxation time T1. Using a multiparametric MRI-pathologic cross-correlative approach and computational pathology methodologies including a machine learning-based algorithm for the automatic detection and classification of neuroblasts, we show here that T1 mapping is sensitive to the rich histopathologic heterogeneity of neuroblastoma in the Th-MYCN transgenic model. Regions with high native T1 corresponded to regions dense in proliferative undifferentiated neuroblasts, whereas regions characterized by low T1 were rich in apoptotic or differentiating neuroblasts. Reductions in tumor-native T1 represented a sensitive biomarker of response to treatment-induced apoptosis with two MYCN-targeted small-molecule inhibitors, Aurora A kinase inhibitor alisertib (MLN8237) and mTOR inhibitor vistusertib (AZD2014). Overall, we demonstrate the potential of T1 mapping, a scan readily available on most clinical MRI scanners, to assess response to therapy and guide clinical trials for children with neuroblastoma. The study reinforces the potential role of MRI-based functional imaging in delivering precision medicine to children with neuroblastoma. SIGNIFICANCE: This study shows that MRI-based functional imaging can detect apoptotic responses to MYCN-targeted small-molecule inhibitors in a genetically engineered murine model of MYCN-driven neuroblastoma.

Abstract SY24-02: Neuroblastoma: A developmental twist

Abstract Intra-tumor heterogeneity is considered as a source of drug-resistant cells that can escape therapy and form relapses. Several tumor types display a structured intra-tumor heterogeneity, consisting of immature tumor cells and lineage-committed tumor cells. The immature populations of some tumor types have been implicated in drug resistance. The pediatric tumor neuroblastoma includes besides lineage committed adrenergic (ADRN) cells a minority of undifferentiated mesenchymal (MES) tumor cells which diverge in gene expression and epigenetic modifications (van Groningen et al., Nature Genetics 2017). MES cells are relatively resistant to chemotherapy and are enriched in post-treatment samples, suggesting selection and a role in relapse development. However, the co-existence of two tumor cell populations, their differential drug sensitivity and their relation to normal development remain enigmatic. Here we show how MES-type neuroblastoma cells fundamentally differ from ADRN-type cells and resemble normal precursor cells of the adrenergic lineage, including expression of metabolic routes providing resistance to therapy. MES cells and ADRN cells share the same genetic defects, but strongly differ in super enhancers. Each cell type is marked by about 200 specific super enhancers that are absent in the other cell type. Analysis of the genes under their control identified about 20 MES-specific transcription factors and 20 ADRN-specific transcription factors. Chip-seq analysis showed that they conform to a model of MES-specific or ADRN-specific core regulatory circuitries (CRC). Such circuitries were proposed to form a feed-forward system that imposes lineage specification (Hnisz et al., Cell 2012). In agreement with a cell-type specifying role for the CRC, we found that induced expression of MES-specific CRC transcription factors could reprogram ADRN-type cells into MES-type cells. This included a shift towards MES-type super enhancers and expression of associated MES-type genes. Neuroblastoma are tumors of the peripheral adrenergic lineage, which is neural crest derived. MES-type cells lack expression of typical differentiation markers of this lineage, like enzymes for the adrenalin synthesis route TH and DBH. We therefore analyzed whether MES cells resemble precursor cell types of this lineage. Schwann Cell Precursors (SCP) were recently implicated as migratory precursor cells of the adrenergic lineage (Furlan et al., Science 2017). We found that MES cells are highly similar to SCP cells. We therefore analyzed whether the drug resistance of MES cells relate to their normal developmental counterparts. Indeed we observed that the similarity of SCP and MES cells extends to metabolic routes that affect resistance to drugs used in neuroblastoma therapy. Our analyses show that heterogeneity in neuroblastoma is structured and resembles cell types of the normal differentiation program of the adrenergic lineage. MES and ADRN cell types have conserved gene expression programs that determine their response to therapy. MES cells are thereby unresponsive to clinically used therapeutic drugs and may survive therapy. Targeting of lineage-differentiated as well as immature tumor cell types may therefore be a promising strategy to attenuate development of therapy-resistant tumor relapses. Citation Format: Rogier Versteeg, Linda Valentijn, Jan Koster, Tim van Groningen, Mohamed Hamdi, Ellen Westerhout, Johan van Nes. Neuroblastoma: A developmental twist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY24-02.

Dynamics of Minimal Residual Disease in Neuroblastoma Patients.

Neuroblastoma is a common extracranial solid tumor of neural crest (NC) origin that accounts for up to 15% of all pediatric cancer deaths. The disease arises from a transient population of NC cells that undergo an epithelial-mesenchymal transition (EMT) and generate diverse cell-types and tissues. Patients with neuroblastoma are characterized by their extreme heterogeneity ranging from spontaneous regression to malignant progression. More than half of newly diagnosed patients present highly metastatic tumors and are stratified into a high-risk group with dismal outcome. As many as 20% of high-risk patients have residual disease that is refractory or progressive during induction chemotherapy. Although a majority of high-risk patients achieve remission, larger part of those patients has minimal residual disease (MRD) that causes relapse even after additional consolidation therapy. MRD is composed of drug-resistant tumor cells and dynamically presented as cancer stem cells (CSCs) in residual tumors, circulating tumor cells (CTCs) in peripheral blood (PB), and disseminated tumor cells (DTCs) in bone marrow (BM) and other metastatic sites. EMT appears to be a key mechanism for cancer cells to acquire MRD phenotypes and malignant aggressiveness. Due to the restricted availability of residual tumors, PB and BM have been used to isolate and analyze CTCs and DTCs to evaluate MRD in cancer patients. In addition, recent technical advances make it possible to use circulating tumor DNA (ctDNA) shed from tumor cells into PB for MRD evaluation. Because MRD can be detected by tumor-specific antigens, genetic or epigenetic changes, and mRNAs, numerous assays using different methods and samples have been reported to detect MRD in cancer patients. In contrast to the tumor-specific gene-rearrangement-positive acute lymphoblastic leukemia (ALL) and the oncogenic fusion-gene-positive chronic myelogenous leukemia (CML) and several solid tumors, the clinical significance of MRD remains to be established in neuroblastoma. Given the extreme heterogeneity of neuroblastoma, dynamics of MRD in neuroblastoma patients will hold a key to the clinical validation. In this review, we summarize the biology and detection methods of cancer MRD in general and evaluate the available assays and clinical significance of neuroblastoma MRD to clarify its dynamics in neuroblastoma patients.

Dissecting Intra-Tumor Heterogeneity by the Analysis of Copy Number Variations in Single Cells: The Neuroblastoma Case Study.

Tumors often show intra-tumor heterogeneity because of genotypic differences between all the cells that compose it and that derive from it. Recent studies have shown significant aspects of neuroblastoma heterogeneity that may affect the diagnostic-therapeutic strategy. Therefore, we developed a laboratory protocol, based on the combination of the advanced dielectrophoresis-based array technology and next-generation sequencing to identify and sort single cells individually and carry out their copy number variants analysis. The aim was to evaluate the cellular heterogeneity, avoiding overestimation or underestimation errors, due to a bulk analysis of the sample. We tested the above-mentioned protocol on two neuroblastoma cell lines, SK-N-BE(2)-C and IMR-32. The presence of several gain or loss chromosomal regions, in both cell lines, shows a high heterogeneity of the copy number variants status of the single tumor cells, even if they belong to an immortalized cell line. This finding confirms that each cell can potentially accumulate different alterations that can modulate its behavior. The laboratory protocol proposed herein provides a tool able to identify prevalent behaviors, and at the same time highlights the presence of particular clusters that deviate from them. Finally, it could be applicable to many other types of cancer.

The challenge of defining “ultra‐high‐risk” neuroblastoma

Given the biological and clinical heterogeneity of neuroblastoma, risk stratification is vital to determining appropriate treatment. Historically, most patients with high-risk neuroblastoma (HR-NBL) have been treated uniformly without further stratification. Attempts have been made to identify factors that can be used to risk stratify these patients and to characterize an "ultra-high-risk" (UHR) subpopulation with particularly poor outcome. However, among published data, there is a lack of consensus in the definition of the UHR population and heterogeneity in the endpoints and statistical methods used. This review summarizes our current understanding of stratification of HR-NBL and discusses the complex issues in defining UHR neuroblastoma.