In this study we analyzed primary human astrocytomas to investigate immune response indicators across tumor subtypes, and in relation to disease outcome. We demonstrate a striking enrichment of immune response-related genes and genes characteristic of myeloid cells including microglia, macrophages, and monocytes in the mesenchymal subtype of adult and pediatric GBM. Importantly, in adult GBMs, microglia/macrophage, monocyte and granulocyte cell signatures were significantly enriched in tumors from patients with the shortest survival. Interestingly, this was not the case for pediatric GBM: i.e., immune cell-specific signature genes were not enriched in children with the shortest survival. In adults, tumors having a gene expression signature associated with high microglia/macrophage numbers exhibited enhanced expression of characteristic myeloid, granulocyte and CD4 T cell genes and were enriched for tumors of the mesenchymal subtype. These data support marked inter-tumoral differences in the microglia/macrophage response.
Molecular stratification of adult GBM into subtypes has provided a framework for investigations into differential disease pathogenesis and tumor response to therapy 
. However, to date, tumor profiling of GBM has concentrated on analysis of bulk tumor with little attention directed to the non-neoplastic elements, including immune cells, present in the tumor. In the current study we examined the pattern of gene expression signatures characteristic of specific immune cell populations. In adult GBM, immune response genes, including genes representative of microglia/macrophage populations, were enriched in tumors of the mesenchymal subtype. This pattern of gene expression was consistent with the increased numbers of microglia/macrophages identified in mesenchymal as compared to non-mesenchymal GBMs. Furthermore, we identified enrichment of microglia/macrophage-related signature genes in patients with the shortest survival. We suggest microglia/macrophage function may be particularly pronounced in a subset of GBM with aggressive clinical behavior. Further characterization of this subset may be particularly important for identifying patients for clinical trials involving immunomodulatory therapies. Indeed, a recent study of dendritic cell vaccination demonstrated improved responses in patients of the mesenchymal subtype compared to other subtypes 
. PLX3397, an inhibitor of macrophage colony stimulating factor 1, and other such therapies aimed at inhibiting the microglia/macrophage response, may also prove particularly effective in this molecularly defined subset of GBM.
Our data indicate an enrichment of microglia/macrophage-related signature genes in tumors from patients with short survival. As increased gene expression may reflect both differences in cell number and differences in microglia/macrophage phenotype or activation state, we examined microglia/macrophage number in available UCSF tumors. Increased absolute numbers of microglia/macrophages was not associated with short survival in this GBM cohort. This discrepancy with the gene expression data may be a result of intra-tumoral heterogeneity, alternatively it may suggest possible differences in gene expression levels per cell. To identify immune response-related genes most strongly associated with survival we performed CoxBoost modeling on immune response genes identified in a worse prognosis gene signature of adult GBM 
. In addition to established clinical variables, 12 potential immune response-related genes were found to be significantly associated with poor survival. These included genes known to be expressed by microglia (CCL8 
, SPP1 
, IRF7 
, IL1RAP 
), macrophages (IL1RN 
, SPP1 
, PDPN 
, IL1RAP 
, MDK 
, TFRC 
, HRH4 
), and tumor-associated macrophages (IL6 
, SPP1 
). Many of the corresponding gene products are also known to influence monocyte/microglia/macrophage recruitment and activation (IL6 
, SPP1 
, CCL8 
, MDK 
Some of the genes identified by CoxBoost modeling have been previously associated with GBM disease outcome. Notably, PDPN was included in a 9 gene prognostic signature defined by Colman et al. 
, the inflammatory cytokine IL6 has been linked to poor prognosis in GBM 
and high serum levels of the secreted glycoprotein SPP1 (osteopontin) correlate with poor survival in GBM 
. Our studies, similar to all studies that analyze expression data from bulk tumor, are limited by the inability to definitively identify the cellular source of a particular transcript. However, analysis of gene expression data from purified human tumor-associated microglia/macrophages demonstrated increased expression of some of these genes (8/12) relative to bulk tumor. Thus total expression levels of many of these survival-related immune genes is likely derived in part from the microglia/macrophage infiltrate.
To elucidate other factors associated with a high microglia/macrophage infiltrate, adult tumors were stratified by microglia/macrophage number into high and low subsets and based on the differentially expressed genes, hierarchical clustering was performed on a large set of profiled GBMs. Tumors with a high microglia/macrophage gene expression signature demonstrated specific enrichment of macrophage, granulocyte and CD4 T cell gene sets. In mammary adenocarcinoma, a significant interplay between CD4+ T cells and macrophages in promoting invasion and metastasis has been demonstrated 
, and it is possible that such interactions may also occur in a subset of GBM. While tumors with a high microglia/macrophage gene expression signature were most commonly tumors of the mesenchymal subtype they also included other tumor subtypes suggesting potential biologic differences within the tumor subtypes.
The gene expression signature associated with high microglia/macrophage number was most strongly driven by two genes: KCNN2 and C14orf139. KCNN2 or SK2, a calcium activated potassium channel, is induced in activated microglia and helps to regulate the microglia respiratory burst function 
. KCNN2 expression has been previously reported in human glioma 
, and in melanoma KCNN2 is thought to help regulate hypoxia-induced cell proliferation 
. The function of C14orf139 is currently unknown.
While there exist some similarities between pediatric and adult high-grade astrocytomas, a substantial literature attests to anatomic, histologic, and molecular differences in adult versus pediatric disease 
. Our data suggest the immune response may represent one of these differences. Recently, pediatric high-grade gliomas have been divided into three subtypes based on patterns of gene expression. While the pediatric HC3/mesenchymal subtype-specific signature genes are not identical to those used for adult tumors 
, they share common elements with the adult mesenchymal subtype of Phillips et al. 
and we demonstrate similarities with the mesenchymal subtype of Verhaak et al. 
. Using these signature genes 
we were able to stratify an independent cohort of pediatric grade II, III and IV astrocytomas into the three defined subtypes. To our knowledge, this is the first study validating the stratification of pediatric astrocytomas into transcriptionally defined classes. These data emphasize the robustness of the gene signatures and subtypes defined by Paugh et al. 
Similar to adult tumors, the pediatric HC3/mesenchymal subtype was enriched in immune response-related genes in both the UCSF and Paugh et al. 
pediatric cohorts. Furthermore, pediatric HC3/mesenchymal GBM were also enriched for gene signatures of microglia/macrophages and monocytes, emphasizing a potential subtype-specific role for these immune cells in adult and pediatric tumors. In contrast to adult GBM, the microglia/macrophage gene signatures were not significantly enriched in tumors from short survival patients among the pediatric GBM cohort. While these results must be interpreted with caution given the relatively small number of pediatric tumors analyzed, it emphasizes potentially important differences in tumor biology between adult and pediatric patients.
Taken together, our study demonstrates increased expression of immune response related genes, including microglia/macrophage signature genes, in a subset of adult and pediatric GBM. Understanding the factors that drive this differential immune response and its implications for therapeutic decision-making is critical. Future studies will be designed to elucidate these differences.