Summary of RNA Sequencing Detection of EGFRvIII.

<p>^a Sample 21b is a separate RNA extraction from specimen 21.</p><p>^b Sample 35b is a separate RNA extraction from specimen 35.</p><p>^c Sample 836b is a separate RNA extraction from specimen 836.</p><p>Summary of RNA Sequencing Detection of EGFRvIII.</p

PCR amplification of EGFR for genomic alterations leading to EGFRvIII transcript.

<p>A. Schematic of sequencing primers and areas of interest. Arrows indicate the location of the primers used to detect EGFRvIII in genomic DNA and unspliced RNA. Bars with diamond caps indicate areas amplified for splice donor and acceptor mutations. The shaded area is lost in EGFRvIII. B. Representative PCR amplification of the splice donor/acceptor sites of <i>EGFR</i> exons 1, 2, 7 and 8 in an EGFRvIII positive HNSCC DNA sample. These bands were excised and sequenced for mutations. C. Representative long-range PCR amplification of <i>EGFR</i> intron 1 for a single EGFRvIII positive HNSCC DNA sample. L: base pair marker, W: water control, a-j: primer sets.</p

HNSCC long range PCR of EGFR intron 1.

<p>^a The NCBI reference sequence for EGFR GRCh37.p10 was used with NT_033968.6 for DNA and NM_005228.3 for mRNA</p><p>^b mRNA with EGFRvIII transcript is denoted as “+”, wtEGFR only transcript is denoted as “-“</p><p>^c Tumor samples with EGFR gene amplification are denoted by “+”</p><p>^d Tumor samples with Exon 1 to 8 joining at the pre mRNA level are denoted as “+”.</p><p>HNSCC long range PCR of EGFR intron 1.</p

EGFRvIII correlation with EGFR amplification and HPV.

<p>A. Twenty five HNSCC tumors with known EGFR gene amplification status (via FISH) were tested for EGFRvIII positivity (28 tumors are shown, three tumors marked with N did not have gene amplification data). RNA was isolated and EGFRvIII and GAPDH were RT-PCR amplified as described in Materials and Methods. 4 of 12 EGFR amplified samples contained EGFRvIII, 5 of 12 of samples without EGFR amplification expressed EGFRvIII. All EGFRvIII bands were excised and sequenced to verify exon 1 to exon 8 joining (samples with confirmed EGFRvIII are denoted by “+”). B. A fisher’s exact test showed a lack of association between EGFRvIII expression and EGFR amplification (p = 0.56). C. A fisher’s exact test showed a lack of association between EGFRvIII expression and p16 expression (p = 0.27).</p

Presence of Exon 1 to 8 joining in DNA and total RNA.

<p>^a(+/-) indicate EGFRvIII status of tumor sample by RT-PCR</p><p>^bND: EGFRvIII not detected</p><p>^cvIII: EGFRvIII detected.</p><p>Presence of Exon 1 to 8 joining in DNA and total RNA.</p

GBM long range PCR of EGFR intron 1.

<p>^a The NCBI reference sequence for EGFR GRCh37.p10 was used with NT_033968.6 for DNA and NM_005228.3 for mRNA</p><p>^b mRNA with EGFRvIII transcript is denoted as “+”, wtEGFR only transcript is denoted as “-“</p><p>^c Tumor samples with EGFR gene amplification are denoted by “+”</p><p>^d Tumor samples with Exon 1 to 8 joining at the pre mRNA level are denoted as “+”.</p><p>GBM long range PCR of EGFR intron 1.</p

Determination of gene expression signature for high microglia/macrophage infiltrate and association with cell-specific gene signatures and molecular subtypes.

<p>(<b>A</b>) Variation in number of tumor-associated microglia/macrophage in 34 GBM tumors. Each dot represents an individual patient tumor, red denotes tumors defined to have high numbers of microglia/macrophages (greater than 67.5 microglia/macrophages per 400× field), blue dots denote tumors defined to have low microglia/macrophage numbers (less than 44.6 microglia/macrophages per 400× field). A representative image of microglia/macrophage staining (Iba1) for each group is shown (scale bars = 50 µm). (<b>B</b>) Q-Q plot of genes differentially expressed in high microglia/macrophage tumors (n = 9) and low microglia/macrophage tumors (n = 9), as denoted in (A). (<b>C</b>) Unsupervised hierarchical clustering of adult GBMs (TCGA, n = 573), based on the six differentially expressed genes between high and low microglia/macrophage tumors as denoted in (A) and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043339#pone-0043339-t002" target="_blank">Table 2</a>, identified four distinct clusters (red, increased expression and green, decreased expression). Low microglia/macrophage tumors were primarily in Cluster 2 (6/9) and high microglia/macrophage tumors were predominantly in Cluster 3 (8/9). (<b>D</b>) Enrichment of cell-specific gene signatures was analyzed in Cluster 3 tumors (surrogate high microglia/macrophage) as compared to Cluster 2 tumors (surrogate low microglia/macrophage). Bars denote normalized enrichment score for each cell-specific gene signature. Black bars represent gene signatures with significant enrichment (FDR<25%), white bars are those which did not reach significance (FDR>25%). GIM: glioma-infiltrating microglia/macrophages, HSC: hematopoietic stem cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043339#pone.0043339.s013" target="_blank">Table S7</a> for complete GSEA data output. (<b>E</b>) Distribution of molecular subtypes across microglia/macrophage signature clusters. The numbers within the bars express the number of tumors within each subtype/cluster combination.</p

Immune response-related genes most strongly associated with survival in adult GBM^a.

a<p>CoxBoost modeling was performed on 58 immune response-related genes from the TCGA worst prognosis signature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043339#pone.0043339-Carro1" target="_blank">[9]</a>.</p>b<p>Coefficient for the standardized covariates in the Cox proportional hazards model.</p

Genes differentially expressed in tumors with high versus low numbers of microglia/macrophage.

a<p>Mean gene expression (Log₂) for microglia/macrophage high (n = 9) and low tumors (n = 9) in adult GBM.</p

Enrichment of microglia/macrophage gene signatures in the mesenchymal subtype.

<p>Immune cell-specific gene expression signatures were used to suggest dominant inflammatory cell populations across tumor subtypes. GSEA analysis comparing the mesenchymal (Mes) to non-mesenchymal (Non-Mes) subtypes was performed in (<b>A</b>) adult GBM (TCGA, n = 173) and (<b>B</b>) pediatric GBM (grade IV, n = 38) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043339#pone.0043339-Paugh1" target="_blank">[11]</a>. Bars show the normalized enrichment score for each cell-specific gene signature. Black bars represent gene signatures with significant enrichment (FDR<25%), white bars represent gene signatures which did not reach significance (FDR>25%). GIM: glioma-infiltrating microglia/macrophages, HSC: hematopoietic stem cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043339#pone.0043339.s007" target="_blank">Table S1</a> for signature gene lists and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043339#pone.0043339.s010" target="_blank">Table S4</a> for complete GSEA data output. (<b>C</b>) Numbers of microglia/macrophages per 400× field, as determined by immunostaining for Iba1, across tumor subtypes in adult GBM (n = 24). Each dot represents an individual patient tumor and the mean is denoted by the line. Microglia/macrophage cell numbers are significantly higher in the mesenchymal subtype compared to the non-mesenchymal subtype (* Mann-Whitney, p = 0.04). Representative images of Iba1 staining for the mesenchymal and non-mesenchymal subtypes are shown (Iba1 cell counts of 66.8 and 44.6, respectively), scale bars = 50 µm. (<b>D–E</b>) The extent of (D) hypoxia, as determined by CA9 immunostaining (n = 22), and (E) vascularity, as determined by CD34 (n = 26), was compared across tumor subtypes in adult GBM. Each dot represents an individual patient tumor and the mean is denoted by the line. The level of hypoxia is significantly higher in the mesenchymal compared to non-mesenchymal subtype (* Mann-Whitney, p = 0.027), however vascularity was not significantly different (Mann-Whitney, p = 0.173).</p