Clinically useful Next Generation Sequencing in Glioblastoma

Jennifer Morrissette, PhD Clinical Director, Center for Personalized Diagnosis

Brain tumors (and all cancers) have a genetic component

• Molecular pathways involved in tumor growth and progression are often activated by genetic alterations

• Anticancer agents targeting many oncogenic pathways continue to enter clinical trials and become FDA approved

• Genomics technologies enable robust tumor genomic profiling in the clinical arena

Evolution of panel sequencing

Current: Solid Version 2: 153 ABL1,AKT1,AKT2,AKT3,ALK,APC,AR,ARAF,ARID1A,ARID2, ATM, ATRX,AURKA, Solid panel version 1: 47 genes BAP1, BRAF,BRCA1,BRCA2, BRIP, BTK, CBP, CCND1, CCND2, CCND3 , CCNE1, CDH1, CDK4, CDK6, CDKN2A, CHEK2,CIC, CRKL, CSF1R, CTNNB1, DAXX, ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, DDR2, DNMT3A,EGFR, EIF1Ax, EPHA3,ERBB2, ERBB3, ERBB4, ERCC2, ERG, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, ESR1, ESR2, EZH2, FBXW7, FGF3, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, FUBP1, FGFR3, FLT3, GNA11, GNAQ, GNAS, GATA3, GNA11, GNAQ, GNAS, HRAS, H3F3A, IDH1, IDH2, IGF1R, JAK1, JAK2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, JAK3, KCNG1, KDM5A, KDM5C, KDM6A, KDR, KIT, KMT2C, KRAS, LRRK2, KIT, KRAS, MET, MLH1, MPL, NOTCH1, MAP2K1, MAP2K2,MAP2K4 ,MAPK1 ,MAPK3, MAX, MCL1, MDM2 NPM1, NRAS, PDGFRA, PIK3CA, PTEN, ,MDM4,MED12, MEN1, MET, MITF, MLH1, MRE11A, MSH2, MSH6, MTOR, PTPN11, RB1, RET, SMAD4, SMARCB1, MYC, MYCN, NBN, NF1, NF2, NKRT1, NKRT2, NKRT3, NKX2-1, NOTCH1, SMO, SRC, STK11, TP53, VHL NOTCH2, NOTCH3, NRAS, EP300, PAK1, PALB2, PBRM1, PDGFRA, PIK3CA, PIK3CB, PIK3R1, PTCH1, PTEN, PTPN11, RAB35, RAC1, RAD50, RAD51, Retired 9/19/16 RAD51B, RAD51C, RAD51D, RAF1, RB1, RET,RHOA, RNF43, SETD2, SF3B1, SLIT2, SMAD4, SMARCA4, SMO, SPOP, SRC, STAG2, STK11, SUFU, SUZ12, SYK, TERT, TET2, TGFBR2, TP53, TRAF7, TSC1, TSC2, TSHR, U2AF1, VHL, WT1, XRCC2 Genomic information available for a wider variety of tumor types

Tumor Type % of Total

Anaplastic Astro EGFRvIII Status in Tested 10% Oligo Tumors 6%

Glioma 6% Positive Meningioma 15% 3% Astrocytoma 3% GBM Gliosarcoma 68% 2% Negative Pineal 85% 2% Detection of additional gene mutations aids in diagnosis, prognosis and treatment

47 gene panel EGFRvIII is often co-occurs with other EGFR alterations 14 Negative Positive

12

10

8

6

4

2

0 Number of mutations in individual tumors vary by tumor type

Likelihood of neoantigen formation

Khalil et al. Nature Reviews Clinical Oncology, 2016

More than mutations: mutational tumor load

Data from 71 patients at Penn The presence of a deleterious PTEN • Median estimated mutational burden: mutation was associated with higher 12 mutations/Mb (range, 0-66; IQR, 8-16) mutational load

Outliers Translocation panel: July 2017

• RNA isolated from FFPE tissue • 15 genes: partner agnostic • Anchored sequencing to detect unknown partners with known driver mutations: identification of novel fusions with known targets

ALK, BRAF, EGFR*, ERG, FGFR1, *Identifies splicing changes FGFR2, FGFR3, MYC, MET*, NTRK1, (EGFRvIII) in addition to NTRK2, NTRK3, RET, ROS1, TMPRSS2 translocations

Design of panel allows for detection of unique partners

Fusion partner Target gene on panel cDNA with fusion partner

Amplification with generic sequencing primer for reverse

Sequencing occurs in parallel to detect expressed fusion gene with biologically relevant target Detection of a known EGFRvIII alteration and a novel AGAP2-EGFR rearrangement

In-frame translocation with EGFR AGAP2: mediates anti- apoptotic effects of nerve growth factor via PI3K activation. Overexpressed in cancer cells and promotes cell invasion. Detection of a novel rearrangements

ALK fusion in a brain tumor: ALK-FXR1

ALK targeted therapy: common in lung adenocarcinoma Acknowledgements Paul Hess, MD, PhD

CPD development team

Robyn Sussman, PhD R&D Coordinator

Eva Klinman, PhD, Derek Oldridge, MD Candidate MD,PhD Jason Rosenbaum, MD

MacLean Nasrallah, Carmela Paolillo, PhD Zev Binder, MD, PhD Stephen Bagley, MD Amanda Oran, PhD MD,PhDNeuropathology