Genomic Approaches to High Grade Gliomas I SEVE BALLESTEROS FOUNDATION TRANSLATIONAL NEURONCOLOGY SUMMIT Will Parsons, M.D., Ph.D. Madrid, Spain 29 October 2010

Overview Background and overview of GBM and genome studies Lessons learned from completed analysis of GBM The role of IDH genes in malignant gliomas

Overview Background and overview of GBM and genome studies Lessons learned from completed analysis of GBM The role of IDH genes in malignant gliomas

Objective To translate our developing understanding of the genetics and genomics of gliomas into improved patient care Tumor Biology Clinical Application Diagnosis & Classification Prognosis Therapeutics

Glioblastoma multiforme (GBM, malignant glioma, grade IV astrocytoma) Most common and lethal primary brain tumor Occurs in both adults and children Categorized into two groups (does not currently impact treatment) Primary (>90%) Secondary (<10%): have evidence of pre-existing lower-grade glioma Gleevec (imatinib) ( i-MAT-in-ib) Herceptin (trastuzumab - tras too' zoo mab), Irressa (gefitinib) ge-FI-tye-nib), Tarceva (erlotinib) Imatinib had shown unparalleled results in patients with advanced chronic myelogenous leukemia (remission rates approaching 98%), and the first GIST patients treated with imatinib demonstrated dramatic response rates unseen with other therapeutic modalities. Thousands of patients worldwide with advanced GIST have been treated with imatinib, with the demonstration of significant response rates, prolongation of survival, and improvement in quality of life. Trastuzumab has had an enormous impact on the clinical management of breast cancer: the survival of Her-2-positive metastatic breast cancer patients has improved significantly and tumor Her-2 status has been built into the decision-making tree for primary breast cancer patients. gefitinib

Example: colorectal cancer Cancer is a genetic disease Example: colorectal cancer

Somatic (tumor-specific) mutations drive cancers Normal *

Examples of genotype-directed cancer therapies Gleevec (imatinib) Chronic myelogenous leukemia (BCR-ABL) Gastrointestinal Stromal Tumors (c-KIT) Herceptin (trastuzumab) Breast Cancer (HER-2) Iressa (gefitinib) and Tarceva (erlotinib) NSCLC (EGFR) Gleevec (imatinib) ( i-MAT-in-ib) Herceptin (trastuzumab - tras too' zoo mab), Irressa (gefitinib) ge-FI-tye-nib), Tarceva (erlotinib) Imatinib had shown unparalleled results in patients with advanced chronic myelogenous leukemia (remission rates approaching 98%), and the first GIST patients treated with imatinib demonstrated dramatic response rates unseen with other therapeutic modalities. Thousands of patients worldwide with advanced GIST have been treated with imatinib, with the demonstration of significant response rates, prolongation of survival, and improvement in quality of life. Trastuzumab has had an enormous impact on the clinical management of breast cancer: the survival of Her-2-positive metastatic breast cancer patients has improved significantly and tumor Her-2 status has been built into the decision-making tree for primary breast cancer patients. gefitinib

What we know about cancer genetics

From gene to genome pre-2003 1 gene 2003 100 genes 2005 250 genes 2006 2007 18,000 genes 2008 20,000 genes

Bioinformatic analysis of Identification of candidate Integrated genomic analysis Copy number Mutation Expression Hybridization to high density oligonucleotide arrays Serial analysis of gene expression using next generation sequencing Bioinformatic analysis of altered genes Identification of candidate cancer genes Identification of mutated pathways

Overview Background and overview of GBM and genome studies Lessons learned from completed analysis of GBM The role of IDH genes in malignant gliomas

So what have we learned from our genomic analysis of GBM?

GBMs contain a complex variety of genetic alterations

Alterations per GBM sample

Frequently-altered GBM genes

Less frequently-altered GBM genes

Landscape of solid tumors

A small number of core pathways are altered in a high fraction of GBMs

Core genetic pathways in GBMs

Individualized cancer pathways

Important cancer genes remain to be discovered

Frequently-altered GBM genes

Overview Background and overview of GBM and genome studies Lessons learned from completed analysis of GBM The role of IDH genes in malignant gliomas

IDH1 mutations are found in ~10% of GBMs

IDH1 mutations

Isocitrate + NAD(P)+ ----------> a-ketoglutarate + CO2 + NAD(P)H Isocitrate dehydrogenases (IDHs) Isocitrate + NAD(P)+ ----------> a-ketoglutarate + CO2 + NAD(P)H NAD(+) Form homodimer Regeneration of NADPH for biosynthetic processes -Defense against oxidative damage? NADP(+) Form heterotetramer a2bg Catalyze rate-limiting step of TCA cycle IDH3A Chr 15 IDH3G Chr X IDH3B Chr 20 IDH1 Chr 2 IDH2 Chr 15 Mitochondria Cytoplasm/peroxisomes

IDH1-mutated GBMs have distinctive clinical and genetic characteristics

IDH1 mutation and patient age

IDH1 mutation and patient survival

IDH1-mutated GBMs

IDH1 mutations define a specific molecular subgroup of GBMs (secondary GBMs)

IDH1 mutations in gliomas target a single amino acid (R132)

IDH1 structure NADP Isocitrate Arg 132

Equivalent IDH2 mutations are found in some gliomas without IDH1 mutations

IDH1/IDH2 mutations are also found in lower grade astrocytomas and oligodendrogliomas

Our improving knowledge of cancer genetics needs to be translated into clinical advances for our patients

Molecular classification of gliomas WHO classification, astrocytic tumors Pilocytic astrocytoma (grade I) Diffuse astrocytoma (grade II) Anaplastic astrocytoma (grade III) Glioblastoma multiforme (grade IV) Molecular classification Astrocytoma with recurrent genetic lesions BRAF gene fusion IDH1/2 mutation EGFR mutation/amplification Other genetic alterations Astrocytoma, therapy related

Targeted inhibition of mutant IDH1/IDH2 may be a therapeutically-useful strategy

IDH1/IDH2 mutations were found to abolish the enzymatic activities of the proteins

Enzymatic activity of wild-type and mutant IDH1 and IDH2 Cell lysates were extracted from HOG cells transfected with vectors encoding the indicated proteins. Expression of wild-type and mutant IDH1 and IDH2 was determined by Western blotting using an anti-flag antibody. The activity levels of these proteins were analyzed by monitoring the production of NADPH. Figure 2. Enzymatic Activity of Wild-Type and Mutant IDH1 and IDH2 Proteins. Cell lysates were extracted from a human oligodendroglioma cell line without IDH1 or IDH2 mutations that had been transfected with vectors encoding the indicated proteins. Panel A shows the expression of proteins encoded by wild-type and mutant IDH1 and IDH2, as determined by Western blotting, with the use of an anti-FLAG antibody. Panel B shows the activity levels of these proteins, as analyzed by monitoring the production of NADPH. GAPDH denotes glyceraldehyde 3-phosphate dehydrogenase. Yan H et al. N Engl J Med 2009;360:765-773

IDH1/IDH2 mutations were found to impart a new function on the enzymes

Conclusions Integration of genome-wide analyses is important to obtain global picture of genetic alterations Cancers display high degree of complexity and heterogeneity Small number of core pathways are altered in high fraction of human cancers Mutated genes and pathways provide potential targets for therapeutic and diagnostic intervention

Conclusions Tumor heterogeneity emphasizes the critical importance of continued investment in tumor banking, creation of model systems Characterization of altered GBM genes “Mountains” and “hills” “Drivers” and “passengers” Functional studies

Conclusions IDH1 and IDH2 mutations are frequent early events in several types of malignant gliomas IDH-mutated gliomas have characteristic clinical and genetic findings Identifies IDH1/IDH2 as a previously unsuspected and potentially-useful target for diagnostics and therapeutics Further functional studies required to clarify role of IDH1/IDH2 mutation in tumorigenesis

Conclusions The identification of characteristic molecular alterations such as IDH1 and IDH2 mutations in gliomas can facilitate molecular classification To allow prognostication and aid decision-making regarding use of currently-available therapies To allow rational application of targeted therapies to specific biological groups Additional analyses of specific subgroups of gliomas (example: pediatric GBM) will be necessary to improve our understanding of these tumors

Acknowledgements