Supplemental Methods and Supplemental Figures 1-9 from Tumor-Associated Macrophages in SHH Subgroup of Medulloblastomas

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).