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

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

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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

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

DEP DEPArray rray™ User Meeting

Disse ssect cting G g Gen eneti tic and and Epigenetic Heter erog

  • gen

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

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

Precision Medicine: Tailoring the treatment for the patient based on underlying molecular biology

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

Tumour Biopsy Histological pathology Genomic pathology Glioma Therapy Mutation A Mutation B Mutation C

IDH1 EGFRvIII STAT3

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Molecular changes underlying recurrence and treatment failure: molecular progression and intratumoral heterogeneity

Initial diagnosis Recurrence Surgery, XTR+TMZ

WHO II – astrocytoma WHO III – anaplastic astrocytoma WHO IV– glioblastoma

p53 mut PDGFR 5-10 year 11p/19q loss CDK4/6 amp RB mut 2-3 year 10q loss PTEN mut 1 year

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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)
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Glioblastoma, WHO Grade IV, histological criteria

Hypercellularity, atypia, mitosis Microvascular proliferation Pseudopallisading necrosis Tumor microenvironment

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Molecular Glioblastoma Subtypes

Sturm et al, Cancer Cell, 2012

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Co-amplification of Receptor Tyrosine Kinase genes in GBM

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PDGFRA MET EGFR MET

Co-amplification of Receptor Tyrosine Kinase genes in GBM: Mosaic heterogeneity

Snuderl et al, Cancer Cell, 2011

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In GBM, each subclone is proliferating and gene amplification leads to protein overexpression

EGFR MET MET (protein) EGFR MET pHH3 (M phase marker) Snuderl et al, Cancer Cell, 2011

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Mosaic heterogeneity subclones in GBM share early genetic mutations

EGFR MET P53 EGFR CDKN2A MET CDKN2A PDGFRA CDKN2A Snuderl et al, Cancer Cell, 2011

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The size of glioma subclones varies from tumor to tumor

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Mosaic heterogeneity in GBM: Collaboration or Competition?

Darwinian model of tumor evolution

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Functional implications of GBM heterogeneity

PDGFRA EGFR

Snuderl et al, Cancer Cell, 2011

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Extreme mosaic heterogeneity

EGFR MET PDGFRA Up to four DISTINCT subclones present in a single GBM: three mutually exclusive populations with RTK amplifications plus one GBM subclone without amplification

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Or is this really the most extreme…?

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Deeper (and deeper) insights into genetic heterogeneity of GBM

PDGFRA

Same, or different subclones….?

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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)

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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

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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

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Identifying expression of proteins predicted by the whole genome profiling

(Illumina Methylation 450k BeadChip Array)

CDK4 EGFR

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Identifying mosaic heterogeneity subclones in GBM by DEPArray

EGFR CDK4 CDK4 EGFR

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EGFR CDK4 CDK4 EGFR

Identifying mosaic heterogeneity subclones in GBM by DEPArray

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pPDGFRA pEGFR

Identifying expression of functional proteins predicted by the CNV analysis

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pPDGFRA pEGFR

Identifying cancer cells with phosphorylated Receptor Tyrosine Kinases by DEPArray

pEGFR pPDGFRA

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Glioblastoma: Critical role of tumor microenvironment

DEPArray:

Next steps: isolating metabolically different glioma subclones by DEPArray

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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

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Acknowledgements

Jonathan Serrano Briana Zeck Luis Chiriboga, PhD John G. Golfinos, MD

Others: Andrew Chi, MD Peter Wu, MD, PhD Kasthuri Kannan, PhD Adriana Heguy, PhD Dimitris Placantonakis, MD, PhD

Matthias Karajannis, MD

Funding:

  • The Friedberg Charitable Foundation
  • The Making Headway Foundation
  • Alice and Thomas J. Tisch Brain Tumor Research Fund