Next Generation Sequencing Approach to Desmoid Tumors

Next Generation Sequencing Approach to Desmoid Tumors

Matt van de Rijn

Which questions do we hope to address?

• Can we distinguish aggressive desmoids from those that are indolent?

• Can we distinguish recurrent desmoid tumors from scars?

What do we need to know about biology?

• Each human cell contains a nucleus where the information for >20,000 genes resides

• Previously we could study genes one at a time• Now can look at all of them simultaneously

DNA RNA mRNA protein

Matt’s (simple) view of the cell:

Matt’s (simple) view of the cell:

In each human cell:DNA: 3 billion basepairsmRNA: >20,000 genes

What is “next generation sequencing”?

• New technology in molecular biology that allows us to do work that used to take several years in one week– First human genome took 10 years to sequence at

a cost of >2 billion dollars– Now can be done within a week at <$5,000

Dramatic shift in approach:

Rather than sequencing long stretches of DNA (~700 bases) in relatively few experiments, we now sequence millions of very short fragments and let the computer figure out the overlap

3SEQ Uses Illumina Genome Analyzer for precise

quantification of genome-wide mRNA expression levels on both frozen tissues and paraffin blocks

PLoS One 2010, 5:e8768)

What’s a Paraffin Tissue Block?

Gene expression profiling by 3SEQRNAseqDNAseq

Applications of paraffin block-based “3SEQ”

• Gene expression profiling-Characterization of fibroblastic lesions-Comparing desmoid tumors to scars

• Study of lncRNA expression as potential biomarker

Fibromatosis Elastofibroma Fibr Tendon Sheath Sol Fibrous tumor

collagenous fibroma (6) elastofibroma (4)infantile digital fibromatosis (3)palmar fibromatosis (8) nasopharyngeal angiofibroma (6)fibroma of tendon sheath (4)nodular fasciitis (6)dermatofibrosarcoma protuberans (4)desmoid type fibromatosis (7)solitary fibrous tumor (5)

We no longer rely on rare frozen tissue specimens and can use readily available paraffin blocks

Goal:To generate a clinically relevant predictor of desmoid behavior

Plan:Compare desmoids with known aggressive behavior with those that never recurred and determine which markers can be used to distinguish them

Applications of paraffin block-based “3SEQ”

• Gene expression profiling-Characterization of fibroblastic lesions-Comparing desmoid tumors to scars

• Study of lncRNA expression as potential biomarker

Problem:When a desmoid comes back, it is difficult to tell recurring desmoid from the scar of the previous surgery

Preliminary studies already identify possible markers

Plan:Extend these studies and validate findings

Applications of paraffin block-based “3SEQ”

• Gene expression profiling-Characterization of fibroblastic lesions-Comparing desmoid tumors to scars

• Study of lncRNA expression as potential biomarker

DNA RNA mRNA protein

Matt’s (simple) view of the cell:

lncRNA

lncRNA’s detected by 3SEQ as specifically expressed in different malignancies

Plan:Include desmoid tumors in this series to look for biologically relative lncRNAs

Laboratory:Xiangqian GuoSushama VarmaShirley ZhuMatt van de RijnRob WestSantosh GuptaTrip SweeneyErna Forgo

Stanford Oncology:Kristen Ganjoo

Stanford Surgery:Raffi Avedian

DANA-Farber Oncology:Suzanne George

Thank you!