Additional file 1 of Global activation of oncogenic pathways underlies therapy resistance in diffuse midline glioma
Creators: name: M.-M. Georgescu
name: M. Z. Islam
name: Y. Li
name: M. L. Circu
name: J. Traylor
name: C. M. Notarianni
name: C. N. Kline
name: D. K. Burns
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1 : Supplemental Table S1. Primary antibodies for WB. Supplemental Table S2. Autopsy gross parameters. Supplemental Table S3. IHC results. Supplemental Table S4. Mutations in DIPG. Supplemental Table S5. CNVs. Supplemental Figure S1. Radiologic characteristics of DIPG cases. A. F5 MRI: axial (left and center) and coronal (right) T2W-FLAIR images showing hyperintense diffusely infiltrating pontine mass extending posteriorly into the right cerebellum and rostrally into the midbrain and subthalamus. Images were acquired 2.5 months pre-mortem. B-C. F10 MRI at initial diagnosis (B) and 10 months later (C) showing rim-enhancing pontocerebellar mass with initial extension into the left cerebellum (B) and subsequent extension into the right cerebellum and frontal periventricular region (C). Yellow arrows show post-contrast enhancing tumor foci. D. F12 axial and sagittal T2W-FLAIR images acquired 1-month pre-mortem showing the frontal and cerebellar secondary foci with yellow arrows. Supplemental Figure S2. Example of histologic examination for H&amp;E quantified analysis. F12 H&amp;E sections are shown at the levels schematically illustrated on a normal brain coronal section. Higher magnification of areas marked with small blue squares are shown in adjacent pictures with blue borders. Note massive infiltration of periventricular areas, white matter tracts, septum pellucidum, hippocampus, brainstem and cerebellum (red arrow shows the invasive tumor front in the left cerebellum) by neoplastic cells with pleomorphic nuclei resembling glioblastoma. LV, lateral ventricle; R, right; L, left. Supplemental Figure S3. Variable ultrastructural morphology of neoplastic cells in DIPG. A. Variable nuclear morphology of F5 neoplastic cells, showing elongated/fibrillary forms (yellow arrows) admixed with larger forms (blue arrows). The red arrow indicates a myelinated axon “hugged” by a fibrillary neoplastic cell. B. F10 larger neoplastic astrocyte with abundant cytoplasm (blue arrow) dissecting the myelinated axons (red arrow) of the corpus callosum. C. F12 neoplastic astrocytes with nuclear pleomorphism (blue arrows) within the frontal white matter (myelinated axons shown with red arrows and oligodendroglia shown with green arrow). D. F12 large pontine fibrillary neoplastic astrocyte with prominent nucleolus. Supplemental Figure S4. Ganglioglioma-like pontine focus in DIPG. F10 H&amp;E of a focal proliferation of large binucleated dysplastic ganglion cells, interspersed with small astrocytic neoplastic cells, conferring a typical ganglioglioma appearance to a limited pontine area of the tumor. IHC and NGS following microdissection of this area revealed histone H3 K27M mutation in all the neoplastic cells, consistent with DMG/K27M. Supplemental Figure S5. IHC profiles in DIPG. IHC with indicated antibodies of the pontine tumor areas from F5, F10 biopsy and autopsy, and F12. The F10 autopsy focus of dysplastic binucleated ganglion cells (red arrows) is shown. These cells express GFAP but not vimentin, whereas the small neoplastic cells express both types of intermediate filaments. Supplemental Figure S6. Quantitative protein expression analysis in DIPG. A-B. Bar graphs representing the indicated protein levels from various foci of the 3 DIPG autopsies. The quantification of the WB bands was performed by densitometric analysis, as described in Material and Methods. Individual densitometric values were normalized to the corresponding actin values. In (B), the phosphoprotein values were normalized to the corresponding total unphosphorylated protein values, as indicated. Results are expressed as fold-increase or fold-decrease in comparison to normal control. The WBs for each antibody were repeated at least twice, with similar results. The labeling is as in Fig. 4: T, tumor; N, normal; primary pontine (P) foci indicated in red; secondary cerebellar (C) and frontal (Fr) foci, in green. Sample laterality: R, right; L, left. For the tri-methylated histone H3 K27 residue (H3K27Me3) antibody in (A) that showed undetectable expression in both normal and tumor samples, the WB was repeated in the presence of a positive control from an autopsy case of glioblastoma, IDH-mutant, WHO grade IV, known to harbor increased levels of H3 K27 tri-methylation.