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 tumorassociated macrophages (TAM) correlates with the medulloblastoma molecular subgroups and contributes to a diagnostic signature.Methods: Gene-expression profiling using human exon array (n ¼ 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 ¼ 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.

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 &lt; 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 &lt; 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&lt;0.001 for Groups 3 and 4; CSF1R, t-test p&lt;0.01 for all groups). To validate these findings, we performed immunohistochemical analysis of 51 primary and 3 recurrent medulloblastoma tumors using antibodies directed against CD163, which were then blindly graded by two neuropathologists. There were significantly greater numbers of macrophages observed in tumors of the SHH subgroup compared to those in Group 3 and Group 4 (t test p&lt;0.001 for both groups). Interestingly, macrophages in the desmoplastic or SHH tumors were concentrated in the poorly differentiated and highly proliferative internodular areas suggesting a tumor-proliferating role for these TAMs. 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.

<p>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</p>

<p>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 <0.15. Supplemental Figure 3. HuEx expression levels of CSF1R are significantly higher in SHH tumors compared to Group 3 or Group 4 subgroups (p<0.0001 respectively). Supplemental Figure 4. Progression free survival (PFS) and overall survival (OS) analyses using Kaplan-Meier plots and log rank tests for samples analyzed using TLDA 31-gene signature (A) PFS by molecular subgroup. (B) OS by molecular subgroup. Supplemental Figure 5. TLDA expression levels of CD163 are similar among histologic subtypes of SHH medulloblastomas Supplemental Figure 6. Progression free survival (PFS) and overall survival (OS) analyses using Kaplan-Meier plots and log rank tests for SHH tumors based on median TLDA expression of CD163.(A) PFS of high- vs low-expressers. (B) OS of high- vs low-expressers. Children who died of treatment-related toxicity without evidence of disease progression are excluded from the analyses (n=3). Supplemental Figure 8. Representative IHC images of SHH tumors with desmoplastic histology stained with anti-Ki-67 antibody demonstrating increased cell proliferation in internodular areas corresponding to the presence of macrophages. Supplemental Figure 7. Additional representative (2 per group) of CD163 IHC images of SHH with desmoplastic histology, SHH with classic histology, Group 3 and Group 4 tumors. Supplemental Figure 8. Representative IHC images of SHH tumors with desmoplastic histology stained with anti-Ki-67 antibody demonstrating increased cell proliferation in internodular areas corresponding to the presence of macrophages. Supplemental Figure 9. Unsupervised clustering of the 40 most variable macrophage related genes. Heatmap demonstrating clustering of SHH and WNT group of tumors compared to Group 3/4 tumors. Rows represent names of genes and HuEx probeset identification. White to red coloring indicates expression level of the gene (log2).</p>