DIFFUSE GLIOMAS Precursor cell Precursor cell

IDH1/2 mutation EGFR amplification/mutation G-CIMP NF1 loss/mutation PIK3R1/PIK3CA muttation WHO 2016 classification of primary brain PTEN loss/mutation TP53 mutation 1p/19q co-deletion TP53 loss ATRX mutation TERT promoter mutation tumors CDK2NA loss CDKN2B loss Felice Giangaspero RB1loss/mututaion CDK4 amplification Grade II-III TERT promoter mutation Grade II-III astrocytoma oligodendroglioma

Grade II-III RB1 loss/mutation CDKN2A loss Astrocytoma wt CDk4/6 amplification PDGFRA amplification

IDH-wt glioblastoma IDH-mut glioblastoma (grade IV) (grade IV)

Major changes in the 2016 CNS WHO Diffuse Gliomas WHO 4 Ed. 2007 WHO 4 Ed.+ 2016 • Formulating concept of how CNS tumor diagnoses are structured in the Diffuse astrocytoma, IDH-1 mutant (II) Diffuse astrocytoma, (II) Gemistocytic astrocytoma, IDH-mutant (II) molecular era Fibrillary astrocytoma Diffuse astrocytoma, IDH-wildtype (II) Gemistocytic astrocytoma Diffuse astrocytoma,NOS (II) • Major restructuring of medulloblastomas, with incorporation of genetically Protoplasmatic astrocytoma defined entities Anaplastic astrocytoma, IDH-mutant (III) Anaplastic astrocytoma, (III) Anaplastic astrocytoma, IDH-wildtype (III) • Major restructuring of diffuse gliomas, with incorporation of genetically Anaplastic astrocytoma, NOS (III) defined entities Glioblastoma, (IV) Giant cell glioblastoma Glioblastoma, IDH-wildtype (IV) • Major restructuring of other embryonal tumors, with incorporation of genetically defined Gliosarcoma Giant cell glioblastoma (IV) entities and removal of the term “primitive neuroectodermal tumor” Gliomatosis cerebri Gliosarcoma (IV) Epithelioid glioblastoma (IV) • Novel approach distinguishing pediatric look-alikes, including designation of Glioblastoma, IDH-mutant (IV) Glioblastoma, NOS (IV) novel, genetically defined entity Oligodendroglioma, (II) • Addition of newly recognized entities, variants and patterns Anaplastic oligodendroglioma, (III) Diffuse midline glioma, H3 K27M-mutant (IV) • Diffuse midline glioma, H3 K27M–mutant (entity) Oligoastrocytoma, (II) Oligodendroglioma, IDH-mutant and 1p/19q-co-deleted (II) • Embryonal tumour with multilayered rosettes, C19MC-altered (entity) Anaplastic oligoastrocytoma, (III) Oligodendroglioma, NOS (II) • Ependymoma, RELA fusion–positive (entity) Anaplastic oligodendroglioma, IDH-mutant and 1p/19q-codeleted (III) • Diffuse leptomeningeal glioneuronal tumor (entity) Anaplastic oligodendroglioma, NOS (III) • Anaplastic PXA (entity) Oligoastrocytoma (II) Anaplastic oligoastrocytoma (III) Acta Neuropathol. 2016;131:803-20

Useful markers for an integrated diagnosis in diffuse gliomas

DIFFUSE GLIOMAS • IDH: IHC/Seq • ATRX:IHC • 1p/19q codeletion: FISH/ aCGH/CNV • TP53:IHC/Seq • TERT :Seq • H3.3K27 (pedGBM):IHC/Seq Integrated Diagnosis by IHC 1p-19q co-deletion

IDH1 ATRX

Diffuse astrocytoma (grade II) IDH1 mutated ATRX mutated

Diffuse Astrocytoma (grade II WHO) Female 27 yrs

IDH1 R132H

IDH2 MUTATION R172G (A>G) ATRX p53

Frequency of specific IDH mutations in gliomas Age distribution of IDH mutations

Lai et al. JCO, 2011 Anaplastic Astrocytoma (grade III WHO) Male 13 yrs Characteristics of H3 K27-mutant gliomas in adults

IDH1 R132H Meyronet et al. Neuro-Oncol 2017, 19:1127-1134

Histone and chromatin modifier mutations High Grade Gliomas H3- G34 Mutated in pHGG and DIPG

ATRX p53

H3F3A G34R GLY ARG

15 years old boy

Jones C, Baker SJ, Nat Rev Cancer. 2014, 14 (10)

Diffuse Midline Glioma, H3 K27M–mutant Imaging of High Grade Gliomas H3- G34 Mutated

DIPG $$*ĺ$7*lysine ĺmethionine)

Vettermann et al. Clin Nucl Med 2018, 43:895-898 anti-H3K27M (#ABE419 Millipore DIFFUSE GLIOMAS Mutational landscape in grade II and III gliomas Precursor cell Precursor cell

IDH1/2 mutation EGFR amplification/mutation G-CIMP NF1 loss/mutation PIK3R1/PIK3CA muttation PTEN loss/mutation TP53 mutation 1p/19q co-deletion TP53 loss ATRX mutation TERT promoter mutation CDK2NA loss CDKN2B loss RB1loss/mututaion CDK4 amplification Grade II-III TERT promoter mutation Grade II-III astrocytoma oligodendroglioma

Grade II-III RB1 loss/mutation CDKN2A loss Astrocytoma wt CDk4/6 amplification PDGFRA amplification

IDH-wt glioblastoma IDH-mut glioblastoma Suzuki et al. Nature Genetics 2015 (grade IV) (grade IV)

1RYHOLPSURYHGJUDGLQJV\VWHP V IRU INFILTRATIVE ASTROCYTOMA IDH-PXWDQWDVWURF\WLFJOLRPDV Histological Parameters for Grading

Nuclear Mitoses Vascular Necrosis atypia prolif.

Grade III Grade IV Grade IV Grade II Shirahata et al. Acta Neuropathol 2018, 136:153-166

Mutational landscape in grade II and III gliomas Diffuse Astrocytoma (Grade II WHO) IDH WT

50 yrs M

Molecular features of GBM (Grade IV)

Suzuki et al. Nature Genetics 2015 EGFR Ampl 10q - Gain 7 IDHwt diffuse astrocytoma stratified for combinations of alterations of chromosomes 7 and 10. cIMPACT-NOW Grade II-III (A-AA) Grade IV (GBM) (Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy—Not Official WHO)

To provide guidelines between WHO updates and to facilitate future WHO classification updates

Stichel et al. Acta Neuropathol 2018

F,03$&7-NOW Adult IDH WT lower grade gliomas stratification recommended GLDJQRVWLF criteria IRU³'LIIXVHastrocytic JOLRPD IDH-wildtypeZLWKmolecular IHDWXUHV RIJOLREODVWRPD:+2 JUDGH,9´

1. EGFR amplification OR 2. Combined whole chromosome 7 gain and whole FKURPRVRPHORVV í OR 3. TERT promoter mutation

Aibadula et al. Neuro- Oncology 19: 1327-37, 2017 Brat et al. Acta Neuropathol (2018) 136:805-810)

'LIIXVH$VWURF\WRPD,'+-:LOGW\SH$'LVVROYLQJ'LDJQRVLV

LOW GRADE GLIOMAS/GLIONEURONAL TUMORS

Hasselblatt et al. J Neuropathol Exp Neurol. 2018;77(6):422-425 Other Astrocytic Tumors Pilocytic astrocytoma GANGLIOGLIOMA Pilomyxoid astrocytoma Subependymal giant cell astrocytoma Pleomorphic xantoastrocytoma Anaplastic pleomorphic xantoastrocytoma* Other gliomas Chordoid glioma of the third ventricle Angiocentic glioma Astroblastoma Neuronal and mixed neuronal-glia tumors ABRESPONSE TO VEMURAFENIB AFTER 3 MS OF TREATMENT Dysembryoplastic neuroepithelial tumour Gangliocytoma Ganglioglioma Anaplastic ganglioglioma Dysplastic cerebellar gangliocytoma Papillary glioneuronal tumour Diffuse leptomeningeal glioneuronal tumor* 2016 4+ Edition Central neurocytoma BRAF mutation V600E (GTG/GAG) Cerebellar liponeurocytoma Paraganglioma From del Bufalo et al. J Transl Med 2014, 12: 356

LGG/GN TUMORS AS A MAPK PATHWAY DISEASE Other Astrocytic Tumors Pilocytic astrocytoma Pilomyxoid astrocytoma Subependymal giant cell astrocytoma Pleomorphic xantoastrocytoma Anaplastic pleomorphic xantoastrocytoma* Other gliomas Chordoid gliomaof the third ventricle Angiocentic glioma Astroblastoma Neuronal and mixed neuronal-glia tumors Dysembryoplastic neuroepithelial tumour Gangliocytoma Ganglioglioma Anaplastic ganglioglioma Dysplastic cerebellar gangliocytoma Papillary glioneuronal tumour Diffuse leptomeningeal glioneuronal tumor* 2016 4+ Edition Central neurocytoma Cerebellar liponeurocytoma Paraganglioma

Distribution of LGG/GN histologies and molecular genetic alterations DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR by anatomic tumor location. (DLGNT)

Syn

Sturm et al. JCO -2377. Perilongo et al. Child’s Nerv Syst, 2002, 18: 505-12 BRAF alterations in DLGNT C11orf95-RELA IXVLRQV in supratentorial ependymoma

Dodgshun et al. J Neurooncol 2016 Rodriguez et. Acta Neuropathol 2015 Parker M., et al. :Nature, 2014. 506(7489)

C11orf95-RELA fusion: nuclear translocation of RELA with activation of NF-kB pathway

EPENDYMOMAS

EPENDYMAL TUMORS RELA fusion: IHC surrogates

WHO 4 Ed. 2007 WHO 4 Ed.+ 2016

Subependymoma Subependymoma Myxopapillary Ependymoma Myxopapillary Ependymoma Ependymoma Ependymoma Cellular Cellular L1CAM Papillary Papillary Clear cell Clear cell Tanycytic Tanycytic Anaplastic Ependymoma Ependymoma RELA fusion positive Anaplastic Ependymoma

NF-kB/p65 Intracranial EPN Subgroups

Mack and 7D\ORUCurr Opin Oncol  J Mol Med (2015) 93:1075–1084

0HGXOOREODVWRPD in adultsthey're not MXVWELJkids

EMBRYONAL TUMORS

Lassaletta & Ramaswamy Neuro Oncol 2016, 18:895.7

Embryonal tumors Medulloblastoma MAJOR CARACTERISTICS OF ADULT MB

WHO 4 Ed. 2007 WHO 4 Ed.+ 2016 • 3 MOLECULAR VARIANTS SHH (62%) Medulloblastoma Medulloblastoma genetically defined Group 4 (28%) Desmoplastic/nodular MB WNT-activated WNT (10%) MBEN MB SHH-activated and TP53-mutant Anaplastic MB MB SHH-activated and TP53-wildtype Large cell MB MB non-WNT/non-SHH • SHH MB group 3 PATCH1 and SMO mutations MB group 4 No TP53 mutation Medulloblastoma histologically defined MB Classic • WNT MB Desmoplastic/nodular MBEN no better prognosis MB Large cell/anaplastic MB NOS Remke et al. J Clin Oncol 2011 Zhao et al. Neuro-Oncology 2016 HGGHG (31%) Embryonal tumors MNGM (1%) 61% EPNEP (5%) ATRTAT (4%) PINEALPI (2%) WHO 4 Ed. 2007 WHO 4 Ed.+ 2016 MBM (3%) CPCCP (1%) EEWSW (2%) PXAPX (1%) CNS PNET Embryonal tumour with multilayered EETMRT (11%) CNS Neuroblastoma rosettes C19MC-altered Non-neoplastic brain CNS Embryonal tumour with multilayered CNS-PNETNET 1 Ganglioneuroblastoma rosettes NOS n.323 Institutional % CNS embryonal tumors NOS Medulloepithelioma Medulloepithelioma diagnosis 15% Ependymoblastoma CNS neuroblastoma Atypical teratoid/rhabdoid CNS ganglioneuroblastoma tumor CNS embryonal tumour NOS Atypical teratoid/rhabdoid tumour CNSCNS NNB-FOXR2B-FOXR2 ((14%)14%) CNS embryonal tumour with rhabdoid CNSC HGNETHGNET-MN1T-MN1 ((3%)3%) 24% CNSC EFT-CICEFTT-CIC (4%) features CNSC HGNET-BCORHGNETT-BCOR ((3%)3%)

6WXUPHWDO&HOO–

DNA Methylation Profiling WHO 2016: no CNS-PNETs anymore! A novel molecular tool • Epigenetic modifications, including DNA methylation, explain how diverse tissues can be derived from the same genome sequence • CNS Embryonal tumor, NOS • In most instances, DNA methylation acts as a ‘fingerprint’ or ‘memory’ of cellular • CNS (Ganglio) Neuroblastoma lineage, making it well suited for classification based on cell of origin

If the following biomarkers are not present: ES cell

Amplification C19MC/LIN28+ ETMR SMARCB1 loss ATRT Progenitor H3.3 /IDH mutation High Grade Glioma RELA-C11orf95 fusion Ependymoma

Astrocytoma Medulloblastoma Brena, Huang & Plass, Nat Genet 2006 Oligodendroglioma

New Brain Tumor Entities Emerge from DNA methylation-based classification of central nervous Molecular Classification of CNS-PNETS system tumours

Sturm et al., 2016, Cell 164, 1060–1072 &DSSHU'et al. Nature 555, –   DNA methylation-based classification of central nervous system tumours

&DSSHU'et al. Nature 555, –  

Machine-learning methods could improve cancer diagnosis.

Wong and Yip .Nature 555, – 

Thank You