MAST BOLOGNA, 25-26 OCTOBER, 2016 DEP DEPArray rray User Meeting - PowerPoint PPT Presentation

MAST • BOLOGNA, 25-26 OCTOBER, 2016

DEP DEPArray rray™ User Meeting Disse ssect cting G g Gen eneti tic and and Epigenetic Heter erog ogen enei eity y in Malignant B Brain Tumors Matija Snuderl, MD Director of Molecular Pathology and Diagnostics Department of Pathology New York University, NY, USA MAST • BOLOGNA, 25-26 OCTOBER, 2016

Precision Medicine: Tailoring the treatment for the patient based on underlying molecular biology Tumour Biopsy Histological Genomic pathology pathology Mutation Mutation Mutation Glioma A B C IDH1 EGFRvIII STAT3 Therapy Modified from - McDermott U, Settleman J: Personalized cancer therapy with selective kinase inhibitors: an emerging paradigm in medical oncology . J Clin Oncol 27:2009; 5650-9 MAST • BOLOGNA, 25-26 OCTOBER, 2016

Molecular changes underlying recurrence and treatment failure: molecular progression and intratumoral heterogeneity WHO II – astrocytoma p53 mut PDGFR Initial diagnosis 5-10 year WHO III – anaplastic astrocytoma 11p/19q loss CDK4/6 amp RB mut 2-3 year Surgery, XTR+TMZ WHO IV– glioblastoma 10q loss PTEN mut 1 year Recurrence MAST • BOLOGNA, 25-26 OCTOBER, 2016

Glioblastoma, WHO Grade IV • The most common primary malignant brain tumor of adults, ~12% of all intracranial tumors • Incidence = 3-4/100,000/yr (Eur, USA) • Peak age of Incidence: 45-75ys • One of the deadliest tumors, with a median survival of 1 yr • GBMs arise de novo or from lower grade lesions • Common genetic alterations: - Receptor tyrosine kinase gene amplification or overexpression (~ 50% of primary GBMs: EGFR, PDGFRA/KIT/VEGFR2, MET ) - TP53 gene mutations (~ 60% of secondary GBMs) - Homozygous CDKN2A deletion (~40% of GBMs) MAST • BOLOGNA, 25-26 OCTOBER, 2016

Glioblastoma, WHO Grade IV, histological criteria Microvascular proliferation Hypercellularity, atypia, mitosis Pseudopallisading necrosis Tumor microenvironment MAST • BOLOGNA, 25-26 OCTOBER, 2016

Molecular Glioblastoma Subtypes Sturm et al, Cancer Cell, 2012 MAST • BOLOGNA, 25-26 OCTOBER, 2016

Co-amplification of Receptor Tyrosine Kinase genes in GBM MAST • BOLOGNA, 25-26 OCTOBER, 2016

Co-amplification of Receptor Tyrosine Kinase genes in GBM: Mosaic heterogeneity EGFR PDGFRA MET MET Snuderl et al, Cancer Cell, 2011 MAST • BOLOGNA, 25-26 OCTOBER, 2016

In GBM, each subclone is proliferating and gene amplification leads to protein overexpression EGFR EGFR MET MET pHH3 (M phase marker) MET (protein) Snuderl et al, Cancer Cell, 2011 MAST • BOLOGNA, 25-26 OCTOBER, 2016

Mosaic heterogeneity subclones in GBM share early genetic mutations EGFR MET PDGFRA CDKN2A CDKN2A CDKN2A EGFR MET P53 Snuderl et al, Cancer Cell, 2011 MAST • BOLOGNA, 25-26 OCTOBER, 2016

The size of glioma subclones varies from tumor to tumor MAST • BOLOGNA, 25-26 OCTOBER, 2016

Mosaic heterogeneity in GBM: Collaboration or Competition? Darwinian model of tumor evolution MAST • BOLOGNA, 25-26 OCTOBER, 2016

Functional implications of GBM heterogeneity EGFR PDGFRA Snuderl et al, Cancer Cell, 2011 MAST • BOLOGNA, 25-26 OCTOBER, 2016

Extreme mosaic heterogeneity EGFR Up to four DISTINCT subclones present in a single GBM: MET three mutually exclusive populations with RTK amplifications PDGFRA plus one GBM subclone without amplification MAST • BOLOGNA, 25-26 OCTOBER, 2016

Or is this really the most extreme…? MAST • BOLOGNA, 25-26 OCTOBER, 2016

Deeper (and deeper) insights into genetic heterogeneity of GBM PDGFRA Same, or different subclones….? MAST • BOLOGNA, 25-26 OCTOBER, 2016

Can DEPArray help us dissect mosaic heterogeneity in GBM (and other tumors)? Goals: - Identify & isolate glioma subclones using predicted targets from whole tumor profiling such as overexpressed RTKs and mutation specific markers (FFPE) - Identify cells with functional overexpressed RTK (FFPE/Fresh) - Unbiased single cell profiling of glioma cells to identify the true extent of heterogeneity missed by whole genome profiling (FFPE) - Identify and isolate stromal cells in malignant gliomas and correlate their profile with underlying GBM genetic features (FFPE/Fresh) MAST • BOLOGNA, 25-26 OCTOBER, 2016

Some notes for DEPArray users from the NYU experience: Brain (tumor) requires different approach than other tissues - Optimizing digestion enzymes and timing for optimal cell suspension - Clusters vs debris (too little vs too much digestion) - High in fat and low in collagen (collagenase might not be the best enzymes for dissociation of brain tissues) - Necrosis (?) -> debris that creates obstacles - Size of the cells, multinucleation, cells within the cells Single cell Single cell MAST • BOLOGNA, 25-26 OCTOBER, 2016

Current workflow 1 ) Whole genome methylation/CNV profiling and targeted mutation profiling to identify targets 2) Cell suspension 3) IF for presumed markers 4) Inspection of the suspension quantity/quality and quality of the staining 5) DEPArray run, subclone selection and isolation 6) Molecular studies MAST • BOLOGNA, 25-26 OCTOBER, 2016

Identifying expression of proteins predicted by the whole genome profiling (Illumina Methylation 450k BeadChip Array) CDK4 EGFR MAST • BOLOGNA, 25-26 OCTOBER, 2016

Identifying mosaic heterogeneity subclones in GBM by DEPArray CDK4 EGFR CDK4 EGFR MAST • BOLOGNA, 25-26 OCTOBER, 2016

Identifying mosaic heterogeneity subclones in GBM by DEPArray CDK4 EGFR CDK4 EGFR MAST • BOLOGNA, 25-26 OCTOBER, 2016

Identifying expression of functional proteins predicted by the CNV analysis pPDGFRA pEGFR MAST • BOLOGNA, 25-26 OCTOBER, 2016

Identifying cancer cells with phosphorylated Receptor Tyrosine Kinases by DEPArray pEGFR pPDGFRA pEGFR pPDGFRA MAST • BOLOGNA, 25-26 OCTOBER, 2016

Glioblastoma: Critical role of tumor microenvironment DEPArray: Next steps: isolating metabolically different glioma subclones by DEPArray MAST • BOLOGNA, 25-26 OCTOBER, 2016

Goals for DEPArray at NYU Neuropathology • Become the leader for DEPArray technology in brain disorders (tumor and non-tumor) • Establish DEPArray protocols for extracting cancer cell subclones and tumor associated stromal cells (macrophages, endothelial cells) from brain tumors • Establish protocols for studying tumor-stroma interaction of live cells • Establish CTC protocols to correlate with our current cfDNA workflow MAST • BOLOGNA, 25-26 OCTOBER, 2016

Acknowledgements Luis Chiriboga, PhD Briana Zeck Matthias Karajannis, MD John G. Golfinos, MD Others: Andrew Chi, MD Jonathan Serrano Peter Wu, MD, PhD Kasthuri Kannan, PhD Funding: Adriana Heguy, PhD • The Friedberg Charitable Foundation Dimitris Placantonakis, MD, PhD • The Making Headway Foundation • Alice and Thomas J. Tisch Brain Tumor Research Fund MAST • BOLOGNA, 25-26 OCTOBER, 2016