Using exome-wide next generation sequencing to identify genetic changes in wild type GIST

Martin G. Belinsky Ph.D.

Kinase mutations in adult and pediatric GIST

Tarn and Godwin 2005

What gene(s) are mutated?Effect of mutation?

Targeted therapeutically?

Imatinib mesylate

Exome-sequencing: focus on wild type GIST

Mutant | Wild type

Mutant | Wild type

Genome-stable

High IGF1R

Approach: NexGen Exome Sequencing

• Exome = the ~ 1% of the genome formed by exons, the coding portion of genes

• Exome-capture with Agilent Human All Exon SureSelect library

• High-throughput sequencing on Illumina Genome Analyzer IIX

• Data transformation and analysis through Fox Chase Institute for Personalized Medicine, Biostatistics and Bioinformatics Facility

Pilot study: germline plus tumor DNA

Maternal germline

Paternal germline

Patient germline

GIST

Sequence variants vs. reference genome

69,239

71,050

69,444

72,003

Filter data->Substitution: G->A

Reference sequence(~30 million bases)

Number of variants

71,050Total exome variants

Data filtering: tumor example

15,002

4,191

448

Causes protein change

Not in FCCC controls

Not in variant databases

Biological query 1: somatic mutations

What variants are in tumor only?

Maternal germline

Paternal germline

Patient germline

GIST

71,050

15,002

4,191

448

Causes protein change

Total exome variants

Not in FCCC controls

Filtering GIST sequence variants:biological query

Not in variant databases

54

24

18

3

Not found in patient

Not found in parents

Visual inspection

All confirmed

Discard duplicates

Mutations are confirmed by conventional sequencing

Tumor Patient

Sanger sequencing (conventional)

Tumor

Patient

Tviewer

3 confirmed somatic mutations

1. MAP-kinase pathway, cell proliferation

2. Cell cycle regulation

3. Mitotic spindle assembly checkpoint

Query 2: inherited gene variations: cancer susceptibility?

Query 3: de novo mutations

What variants are found only in patient germline?

Maternal germline

Paternal germline

Patient germline

GIST

Results of de novo analysis

Summary and Directions

• Confirmation...somatic mutations in GIST tumors, and potential disease-susceptibility variants and mutations in patient germline DNA

• Validation… in larger set of wild type GIST

• Modeling..effect of mutation on protein structure and function

• Testing…effects on cell signaling and cell growth

• Translation… to the clinic

Acknowledgements

Institute for Personalized Medicine • Jeff Boyd• Biao Luo• Beth Wirchansky• Caitlyn Forsythe• Lucinda Hughes

Andy Godwin Lisa Vanderveer

Meg von MehrenLori RinkAlexis Cordero

Biosample Repository• Joellen Weaver

Bioinformatics •Suraj Peri•Alla Lapidus•Michael Slifker•Olga Tchuvatkina

Cytogenetics•Joe Testa•Jianming Pei

Funding•GCRF•FCCC•SFA •NIH•RTOG