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Purpose: Medulloblastoma in children can be categorized into at least four molecular subgroups, offering the potential for targeted therapeutic approaches to reduce treatment-related morbidities. Little is known about the role of tumor microenvironment in medulloblastoma or its contribution to these molecular subgroups. Tumor microenvironment has been shown to be an important source for therapeutic targets in both adult and pediatric neoplasms. In this study, we investigated the hypothesis that expression of genes related to tumor-associated macrophages (TAM) correlates with the medulloblastoma molecular subgroups and contributes to a diagnostic signature.Methods: Gene-expression profiling using human exon array (n = 168) was analyzed to identify medulloblastoma molecular subgroups and expression of inflammation-related genes. Expression of 45 tumor-related and inflammation-related genes was analyzed in 83 medulloblastoma samples to build a gene signature predictive of molecular subgroups. TAMs in medulloblastomas (n = 54) comprising the four molecular subgroups were assessed by immunohistochemistry (IHC).Results: A 31-gene medulloblastoma subgroup classification score inclusive of TAM-related genes (CD163 and CSF1R) was developed with a misclassification rate of 2%. Tumors in the Sonic Hedgehog (SHH) subgroup had increased expression of inflammation-related genes and significantly higher infiltration of TAMs than tumors in the Group 3 or Group 4 subgroups (P < 0.0001 and P < 0.0001, respectively). IHC data revealed a strong association between location of TAMs and proliferating tumor cells.Conclusions: These data show that SHH tumors have a unique tumor microenvironment among medulloblastoma subgroups. The interactions of TAMs and SHH medulloblastoma cells may contribute to tumor growth revealing TAMs as a potential therapeutic target. Clin Cancer Res; 21(6); 1457–65. ©2014 AACR.

Supplemental Methods and Supplemental Figures 1-9 Supplemental Figure 1. Overview of samples studies and 31-gene signature development Supplemental Figure. 2. Non-negative matrix factorization (NMF) analysis of the combined cohort of medulloblastoma samples (n=168) (A) Cophenetic correlation coefficient based on 2000 clustering runs for 2-10 clusters utilizing genes obtained at varying coefficients of variation; the data demonstrate that the highest cophenetic correlation coefficient was obtained at 4 clusters with 369 genes. (B) NMF consensus heatmap of 4 subgroups using the dataset containing 369 genes with high coefficient of variation. (C) Silhouette plot for identification of outliers. Outliers (n=31) were defined as samples with a silhouette width

Supplemental Tables 1-6 Supplemental Table 1. Patient and tumor characteristics Supplemental Table 2. TLDA design (45 genes) Supplemental Table 3. Information for samples evaluated using HuE Supplemental Table 4. TLDA 31-gene molecular classification of core samples Supplemental Table 5. TLDA 31-gene classification for samples without HuEx data Supplemental Table 6. TLDA 31-gene Confusion Matrix

Purpose: Medulloblastoma in children can be categorized into at least four molecular subgroups, offering the potential for targeted therapeutic approaches to reduce treatment-related morbidities. Little is known about the role of tumor microenvironment in medulloblastoma or its contribution to these molecular subgroups. Tumor microenvironment has been shown to be an important source for therapeutic targets in both adult and pediatric neoplasms. In this study, we investigated the hypothesis that expression of genes related to tumor-associated macrophages (TAM) correlates with the medulloblastoma molecular subgroups and contributes to a diagnostic signature. Methods: Gene-expression profiling using human exon array (n = 168) was analyzed to identify medulloblastoma molecular subgroups and expression of inflammation-related genes. Expression of 45 tumor-related and inflammation-related genes was analyzed in 83 medulloblastoma samples to build a gene signature predictive of molecular subgroups. TAMs in medulloblastomas (n = 54) comprising the four molecular subgroups were assessed by immunohistochemistry (IHC). Results: A 31-gene medulloblastoma subgroup classification score inclusive of TAM-related genes (CD163 and CSF1R) was developed with a misclassification rate of 2%. Tumors in the Sonic Hedgehog (SHH) subgroup had increased expression of inflammation-related genes and significantly higher infiltration of TAMs than tumors in the Group 3 or Group 4 subgroups (P < 0.0001 and P < 0.0001, respectively). IHC data revealed a strong association between location of TAMs and proliferating tumor cells. Conclusions: These data show that SHH tumors have a unique tumor microenvironment among medulloblastoma subgroups. The interactions of TAMs and SHH medulloblastoma cells may contribute to tumor growth revealing TAMs as a potential therapeutic target. Clin Cancer Res; 21(6); 1457–65. ©2014 AACR.

Background: Neuroblastoma, a clinically heterogeneous childhood tumor, presents as high-risk disease in 40% of diagnoses and is fatal in roughly half of these patients. In an effort to elucidate the genetic basis of high-risk disease, our lab previously undertook a genome-wide association study (2101 cases, 4202 controls), identifying a cluster of single-nucleotide polymorphisms on chromosome 6p associate with an increased risk of developing of high-risk disease (p < 1e-15). Here we utilized fine mapping to demonstrate that the highly most significant single nucleotide polymorphism (SNP) associations reside within a long intergenic non-coding RNA, CASC15, which we hypothesize plays a critical role in the development of high-risk neuroblastoma through the dysregulation of neuronal growth and differentiation pathways. Methods: CASC15 expression data was obtained from patient samples (n = 251, exon-level expression arrays), cell lines (qRT-PCR) and 108 primary neuroblastomas (RNA-Seq). Subcellular localization of CASC15-S was determined bioinformatically, as well as by RNA-FISH and 5′/3′ RACE. Depletion was carried out using both siRNA and shRNA constructs, and cell viability and growth kinetics were measured using the CellTiter-Glo and Xcelligence platforms. Gene expression analyses of neuroblastoma cell lines stably silenced for CASC15 was accomplished via Transcriptome 2.0 arrays followed by gene set enrichment and Ingenuity pathway analysis. Results: Regional imputation of the 6p22.3 locus refined our initial GWAS signal, and resulted in the validation CASC15-S as a functional gene isoform in neuroblastoma. Subsequent stratification of these imputed polymorphisms identified a functional SNP upstream of CASC15-S, rs9295534, which exhibits enhancer activity. Patients with high-risk disease express substantially less CASC15-S than low-risk patients (p < 0.0001), and lower CASC15-S levels correlate with poor overall survival (adj. p = 3.2e-06). CASC15-S depletion increases cellular proliferation, with ectopic overexpression of the CASC15-S transcript sufficient to revert this phenotype. Cells stably depleted of CASC15-S demonstrate overt morphological changes suggestive of a poorly differentiated phenotype, and gene expression pathway analyses corroborate these observations as cells significantly downregulated proneural gene pathways, while increasing cell motility and growth pathways. Lastly, consistent with lncRNA effects on proximal genes involved in development, CASC15-S expression is highly correlated with neighboring SOX4 mRNA levels (r = 0.76), and chromatin looping suggests putative interactions between these two genomic regions. Conclusions: These data suggest that common genetic variation at 6p22 influences CASC15-S expression, thus impacting neural growth and differentiation pathways as well as impacting neuroblastoma initiation and progression. Citation Format: Mike R. Russell, Annalise Penikis, Derek Oldridge, Juan R. Alvarez-Dominguez, Lee McDaniel, Maura Diamond, Olivia Padovan, Pichai Raman, Yimei Li, Jun Wei, Shile Zhang, Janahan Gnanachandran, Robert Seeger, Shahab Asgharzadeh, Javed Khan, Sharon Diskin, John Maris, Kristina Cole. CASC15 is a tumor suppressor lncRNA at the 6p22 neuroblastoma susceptibility locus. [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 144. doi:10.1158/1538-7445.AM2015-144

The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancer. Tumor associated macrophages (TAMs), one of the main contributors to the tumor microenvironment, have been identified in many adult malignancies and in the MYCN non-amplified high risk neuroblastomas. TAMs have been shown to promote cancer via multiple mechanisms including tumor cell growth and survival, invasion, metastasis, angiogenesis, inflammation, and immune regulation. Recent studies in adult gliomas have demonstrated increased expression of macrophage/microglia associated genes in subtypes of gliomas, however little is known about the role of inflammation in medulloblastomas, the most common malignant childhood brain tumor. In this study we examined the expression of inflammation-related genes and presence of TAMs in molecular subgroups of pediatric medulloblastoma. The molecular subgroups of medulloblastomas were initially identified using Human Exon Array (HuEx) microarray data from 168 samples (65 patients profiled at Children's Hospital Los Angeles and 103 from previously published cohort) using a modified algorithm based on non-negative matrix factorization. We then examined the inflammation and immunology related genes that were differentially expressed among the molecular subgroups and discovered greater expression of inflammation-related genes in tumors of the SHH molecular subgroup compared with those of the Group 3 and Group 4 subgroups. Gene expression analysis was then performed on 83 medulloblastoma samples using a custom-built TaqMan Low Density Array (TLDA) card containing 41 tumor-related and 4 inflammation-related genes that were significantly deregulated among medulloblastoma subgroups in the HuEx microarray analyses. Thirty-one of these genes were used to develop a signature predictive of molecular subgroup. The internal cross-validted error rate of the TLDA 31-gene signature was 8%, with predominant misclassifications occurring between Groups 3 and 4. The expression levels of CD163 and CSF1R, two markers associated with TAMs, were higher in tumors of the SHH subgroup compared to those in the WNT, Group 3, and Group 4 subgroups (CD163, t-test p=.02 for WNT and p Our study reports the first evidence of the presence of TAMs in medulloblastomas and provides a novel 31-gene TLDA signature that accurately determines molecular subgroups of medulloblastomas. The increase in expression of macrophage related genes and intratumoral macrophages was strongly associated with the SHH subgroup of patients. The identification of TAMs in medulloblastomas provides opportunity for testing new therapies directed against TAMs such as CSF1R inhibitors, and the recognition of the importance of tumor microenvironment in childhood brain tumors. Citation Format: Ashley Margol, Janahan Gnanachandran, Nathan Robison, Long Hung, Rebekah Kennedy, Marzieh Vali, Girish Dhall, Jonathan Finlay, Anat Epstein, Mark Krieger, Kathleen Dorris, Maryam Fouladi, Floyd Gilles, Alexander Judkins, Richard Sposto, Shahab Asgharzadeh. Presence of tumor-associated macrophages in SHH medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B18.

Background: The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers. Our group has demonstrated the significance of inflammation-related genes in poor outcome of children diagnosed with metastatic MYCN non-amplified neuroblastoma (NBL-NA). We investigated the effect of TAMs on neuroblastoma growth using a novel murine model. Methods: We have established a novel 100% penetrant NBL-NA murine model driven by SV40's Large T-antigen (NBL-Tag) and characterized expression of immune-related genes in tumors at various developmental stages. Single cell suspensions of murine tumors were analyzed using flow cytometry for determination of immune cells frequency and IL6 levels. NBL-Tag mice were crossed with IL6 knockout mice (NBL-Tag/IL6-/-) to investigate the role of IL6 in tumor pathogenesis. Results: NBL-Tag tumor growth coincided with IL6 levels becoming detectable and increasing in blood. Immune-related gene expression analysis of NBL-Tag adrenal glands in mice at pre-tumor development age (4, 8 and 12 week old) showed age-dependent increase in expression of IL6 and CCL2 (MCP1) compared to wild-type adrenal glands demonstrating early monocyte chemotaxis and establishment of a pro-inflammatory niche. FACS of tumors showed high expression of IL6, and infiltration by IL6-producing TAMs. In vitro co-culture of peritoneal macrophages from wild type mice increased proliferation of a NBL-Tag mice driven cell line (NBT2) by average of 50% over its basal rate, as measured by Brdu incorporation. This effect coincided with two-fold increase in IL6 protein level in the co-cultured media. However, the proliferative changes were only partially reversed by blocking IL6, suggesting other soluble factors also contribute to the observed effects. IL6 genetic ablation studies were conducted to generate NBL-Tag/IL6-/- mice, which convincingly demonstrated IL6 was not required for tumor growth and development. Macrophages from IL6-/- mice increased in vitro proliferation of tumor to similar levels as those from wild-type mice, while IHC staining of NBL-Tag/IL6-/- tumors revealed similar level of TAM infiltration and positivity for phospo-STAT3, indicating an IL6-independent pathway for STAT3 activation. Lastly, gene expression analyses revealed upregulation of MYC in tumor cells during co-culture and was confirmed by western analysis. Conclusions: This study demonstrates the recruitment and growth-promoting effects of TAMs in early pathogenesis of neuroblastoma in a novel MYCN non-amplified murine model. The macrophage and neuroblastoma cell cross-talk lead to tumor cell proliferation with phosphorylation of STAT3 and MYC upregulation in tumor cells. These effects were found to be independent of IL6 activity. Citation Format: Michael D. Hadjidaniel, Soheila Shirinbak, Sakunthala Muthugounder, Long Hung, Michael Sheard, Janahan Gnanachandran, Jin Kim, Richard Sposto, Hiroyuki Shimada, Shahab Asgharzadeh. Tumor-associated macrophages (TAMs) promote murine neuroblastoma tumor growth through upregulation of MYC and independent of IL6 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3668. doi:10.1158/1538-7445.AM2014-3668

Background: The tumor microenvironment is important in the prognosis of Neuroblastoma (NBL), a common childhood cancer of the sympathetic nervous system. However little is know about the role of T regulatory-cells (Treg cells) in NBL. We hypothesize that Treg cells may play an important role in NBL development and immunotherapy targeted to Treg cells may modify the natural course of murine model of neuroblastoma driven by SV40's large T-antigen (NBL-Tag). Methods: Treg cells (positive for CD25/FOXP3/CD4/CD3ϵ) along with other common leukocytes were identified by flow cytometry in single-cell suspensions of adrenal tumors, lymph nodes, spleens, bone marrow and blood of NBL-Tag mice at 14-22 weeks of age. Age dependent pattern of T-cell infiltration and expression of 45 inflammation-relate genes (including CD4) were assessed by CD4 immunohistochemical staining (IHC), and RT-PCR (TaqMan-Low-Density-Array:TLDA), respectively. A luciferase-expressing NBL-Tag cell line (NBTH-Luc) was used to establish a subcutaneous neuroblastoma models in C57Bl/6 mice and tumors were profiled using similar flow cytometry strategies as above. Animals with subcutaneous tumors or transgenic animals were subjected to immunotherapy using anti-CTLA-4, anti-PD-1, or combination of the two (with appropriate isotype controls) in low and high tumor burden scenarios. Results: IHC showed presence and increasing number of CD4+ cells in tumors of NBL-Tag mice over time. In comparison, no CD4+ cells were identified in wild-type adrenal glands. TLDA analysis also showed on average a 4-fold increased expression of CD4 cells in tumors compared to wild-type adrenal glands. Flow cytometry data demonstrated that the CD4+CD25+FOXP3+ Treg cells infiltrate NBL-Tag tumors at all ages (mean=2.9% of all CD45+ cells, n=17 mice). Subcutaneous syngeneic tumors were also infiltrated with CD4+CD25+FOXP3+ Treg cells (mean=8.6% of all CD45+ cells, n=7 mice). Combination of anti-CTLA-4 or anti-PD-1 was not effective in well-established tumors in either NBL-Tag transgenic model or subcutaneous model (p=NS). However, combination immunotherapy prevented formation of subcutaneous tumors (minimal residual disease model) compared to control group (p Conclusion and Future Directions: Our results demonstrate presence of Treg cells in NBL-Tag tumors. Treatment with anti-CTLA-4 and anti-PD-1 prevented formation of neuroblastoma tumors in a syngeneic subcutaneous model. Our data provides pre-clinical evidence that Ipilimumab and Nivolumab, recently approved melanoma drugs, may be useful in treating neuroblastoma children with minimal residual disease. Citation Format: Randall Chan, Soheila Shirinbak, Sakunthala Muthugounder, Long Hung, Janahan Gnanachandran, Michael Hajidaniel, Shahab Asgharzadeh. Regulatory T-cells and effects of anti-CTLA4 and anti-PD1 therapy in a transgenic murine model of neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5021. doi:10.1158/1538-7445.AM2014-5021

The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers. Recent studies have demonstrated the prognostic significance of tumor-associated macrophages (TAM) in several cancers. Our group has also demonstrated the significance of TAMs and interleukin-6 receptor in predicting poor outcome in children diagnosed with metastatic MYCN non-amplified neuroblastoma (NBL-NA). Previous in vitro work has shown that co-culture of a human neuroblastoma MYCN non-amplified cell line with monocytes enhanced proliferation, an effect mediated in part by interleukin-6 (IL6). To address the mechanism by which inflammation contributes to tumorigenesis in neuroblastoma, we have established a novel, 100% penetrant, murine model of metastatic NBL-NA. The tumors of these transgenic mice (NBL-Tag) are detected by MRI only after 12 weeks of age, which coincide with IL6 levels becoming detectable and increasing in blood. DNA profiling of tumors reveal no amplification of the MYCN locus, while RNA profiling and FACS analyses of tumors show high expression of IL6, and infiltration by IL6-producing TAMs. Thus, a pro-inflammatory microenvironment mimicking that observed in human metastatic NBL-NA characterizes the growth of NBL-Tag murine tumors. Immune-related gene expression analysis of NBL-Tag adrenal glands in mice at pre-tumor development age (4, 8 and 12 weeks old) show age-dependent increase in expression of CCL2 (aka monocyte chemotactic protein 1) and fibronectin (FN1) compared to wildtype adrenal glands demonstrating early monocyte chemotaxis and establishment of a pro-inflammatory niche. In vitro co-culture of spleen or peritoneal macrophages from wildtype mice increases proliferation of a NBL-Tag cell line (NBT2) by 30 - 50% over its basal rate, as measured by Brdu incorporation. This effect coincides with two-fold increase in IL6 protein level in the co-cultured media. However, these proliferative changes do not require cell-cell contact and are only partially reversed by blocking IL6, suggesting other soluble factors also contribute to the observed effects. Co-cultured NBT2 cells also increase their expression of MYC by two-fold, providing insight into the tumor proliferative mechanism facilitated by TAMs. In summary, this study demonstrates the recruitment and growth-promoting effects of IL6 producing TAMs in early pathogenesis of neuroblastoma in a novel MYCN non-amplified murine model. The NBL-Tag mice provide a robust transgenic model to study neuroblastoma pathogenesis and provide a platform to assess therapies targeting tumor cells and tumor-associated inflammatory cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 383. doi:1538-7445.AM2012-383