1. Molecular diagnostics in pediatric glial tumors Joon-Hyung Kim, MSIV Weill Cornell Medical College

2. Tumor typeMolecular phenotypeAssociation Oligodendroglioma1p/19q codeletionAdults > Peds GangliogliomaBRAF V600EPeds > Adults PXABRAF V600EPeds = Adults Mesenchymal histology Pilocytic astrocytomaBRAF-KIAA1549Tumor location/resectability (Cerebellar > Non-cerebellar) DIPG1q gain, 17p loss H3F3A, HIST1H3B mutations (K27M) Peds > Adults DIPG > Non-Brainstem pGBM pGBMH3F3A mutation ATRX, DAXX mutation ATRX mutation Peds > Adults GBM > WHO I,II,III gliomas Peds > Adults Type of H3F3A mutation (G34R/V > K27M) Key molecular alterations in pediatric glial tumors

3. 1p/19q codeletion 1p/19q codeletion with polysomy “relative deletion” No 1p/19q codeletion Snuderl et al. Clin Cancer Res 2009. Progression Free SurvivalOverall Survival Anaplastic oligodendroglioma in adults (n=64)

4. 1p and 19q deletions are infrequent in pediatric oligodendroglioma SeriesNAge1p del 19q del 1p/19q del MGMT met IDH1 mut TP53 mut Pollack et al., 20038NA12 Raghavan et al. 2003152-9000NA 1110-18203NA Kreiger et al., 2005135-18100NA Suri et al., 201173-18000500 Creach et al., 2012151-18NA 2 Total691-184 (7.4%)2 (3.7%)5 (8.2%)5 (71%)00

5. Tumor typeMolecular phenotypeAssociation Oligodendroglioma1p/19q codeletionAdults > Peds GangliogliomaBRAF V600EPeds > Adults PXABRAF V600EPeds = Adults Mesenchymal histology Pilocytic astrocytomaBRAF-KIAA1549Tumor location/resectability (Cerebellar > Non-cerebellar) DIPG1q gain, 17p loss H3F3A, HIST1H3B mutations (K27M) Peds > Adults DIPG > Non-Brainstem pGBM pGBMH3F3A mutation ATRX, DAXX mutation ATRX mutation Peds > Adults GBM > WHO I,II,III gliomas Peds > Adults Type of H3F3A mutation (G34R/V > K27M)

6. MacConaill et al. 2009 BRAF V600E in Ganglioglioma in Children

7. Schindler et al. 2011

8. Pediatric Ganglioglioma% BRAF V600E MacConaill et al., 200957% (8/14) Dougherty et al., 201050% (9/18) Schindler et al., 201113% (3/24) Total36% (20/56) Pediatric PXA Schindler et al., 201178% (28/36) Dias-Santagata et al, 201157% (4/7) Total74% (32/43) BRAF V600E in Ganglioglioma and Pleomorphic Xanthoastrocytoma in Children < 18 years Initially described in melanoma, colon and papillary thyroid carcinoma Vemurafenib (“V600E mutated BRAF inhibitor”) – FDA approved for late-stage or unresectable melanoma (Aug 2011)

9. Tumor typeMolecular phenotypeAssociation GangliogliomaBRAF V600EPeds > Adults PXABRAF V600EPeds = Adults Mesenchymal histology Pilocytic astrocytomaBRAF-KIAA1549Tumor location/resectability (Cerebellar > Non-cerebellar) Oligodendroglioma1p/19q codeletionAdults > Peds DIPG1q gain, 17p loss H3F3A, HIST1H3B mutations (K27M) Peds > Adults DIPG > Non-Brainstem pGBM pGBMH3F3A mutation ATRX, DAXX mutation ATRX mutation Peds > Adults GBM > WHO I,II,III gliomas Peds > Adults Type of H3F3A mutation (G34R/V > K27M) Unlike in adults, EGFR amplification, PTEN deletion, IDH1 mutations are rarely observed in pGBM.

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11. Schwartzentruber et al., 2012 H3F3A mutations are exclusive to high grade tumors and occur in the pediatric setting. H3F3A, ATRX, and DAXX mutations distinguish pediatric from adult GBM.

12. Tumor typeMolecular phenotypeAssociation GangliogliomaBRAF V600EPeds > Adults PXABRAF V600EPeds = Adults Mesenchymal histology Pilocytic astrocytomaBRAF-KIAA1549Tumor location/resectability (Cerebellar > Non-cerebellar) Oligodendroglioma1p/19q codeletionAdults > Peds DIPG1q gain, 17p loss H3F3A, HIST1H3B mutations (K27M) Peds > Adults DIPG > Non-Brainstem pGBM pGBMH3F3A mutation ATRX, DAXX mutation ATRX mutation Peds > Adults GBM > WHO I,II,III gliomas Peds > Adults Type of H3F3A mutation (G34R/V > K27M) H3F3A is located on chromosome 1q, a region of large-scale chromosomal gain in DIPG.

13. AdultsPeds EpigenomeMGMT promoter methylation in adult GBM (associated with pseudoprogression s/p TMZ/RT and improved survival) H3.3-ATRX-DAXX chromatin remodeling pathway in pGBM Chromosome1p/19q codeletion in adult OGD1q gain, 17p loss in DIPG GeneAdult AA with EGFR amplification behaves like GBM EGFR amp, PTEN del are rare in pGBM/DIPG PDGFRA amp in DIPG NucleotideIDH1 mutation (R132H) in secondary GBM and LGG in adults BRAF mutation (V600E) in ganglioglioma and PXA ProteinBRAF-KIAA1549 in cerebellar PA PARP-1 overexpression in DIPG

14. Tumor-derived Exosomes Exosomes are small membrane vesicles (30-100 nm) derived from luminal membranes of multivesicular bodies and released constitutively by fusion with cell membrane Released from tumor cells, exosomes mediate local and systemic cell communication through horizontal transfer of information, such as mRNA, miRNA, proteins, DNA Electron microscopy of exosomes derived from U87 cells Research questions: 1. Can exosomes in peripheral blood of glioma patients serve as biomarkers of tumor progression? 2. Can exosomes in patient plasma reliably predict parent tumor mutational status in brain? -IDH1 R132H mutation -BRAF V600E mutation

15. gDNA exoDNA - -++ DNase 3kb 10kb 1kb DNA is present in tumor cell derived exosomes. ExoDNA exists predominantly as methylated, single stranded DNA Courtesy of Haiying Zhang and David Lyden exoDNA-1 + S1 nuclease 3kb 10kb 1kb - exoDNA-2 + - gDNA + - exoDNA gDNA Anti-5’metCytosine Anti-DNA

16. Acknowledgements Neurosurgery Jeffrey Greenfield, MD, PhD Michael Kaplitt, MD, PhD Philip Stieg, MD, PhD Pediatrics David Lyden, MD, PhD Neurosurgery Philip Gutin, MD Pathology Jason Huse, MD, PhD Neuroradiology Andrei Holodny, MD