# Presentation: 041593

## The Genome Center
Washington University
School of Medicine

- Washington University
- School of Medicine

**Richard K. Wilson, Ph.D.**

**GBM Mutation Analysis and**
**New Sequencing Technologies**

## Targeted re-sequencing in TCGA

- rwilson@wustl.edu

**~1500 genes of interest**

**~200 GBM tumors**
- (& matching normals)

**Sequencing Centers: BCM-HGSC, BI, WUGSC**
**Funded by NCI & NHGRI**

## Summary of Mutations in GBM

**A total of ****90 ****Mbp sequenced**
**A total of ****454 ****somatic non-synonymous mutations identified**
**94 GBM tumors sequenced: mutations found in ****85 ****tumors**
**601 candidate genes sequenced: mutations found in ****233 ****genes**
**A total of ****13 ****recurrent sites identified: ****4**** are novel**

| Types | Missense | Nonsense | Splice_site | Insertions | Deletions |
| --- | --- | --- | --- | --- | --- |
| # of mutations | 356 | 37 | 23 | 1 | 36 |

- rwilson@wustl.edu

## Significantly Mutated Genes in GBM

- rwilson@wustl.edu

**Notes:**
TP53 surprisingly high!
EGFR also “mutated” by deletion in many samples.

## Mutations in GBM

- rwilson@wustl.edu

## Mutations in GBM

- PIK3CA

- PIK3R1

- rwilson@wustl.edu

## PIK3CA/PIK3R1 Mutations

- rwilson@wustl.edu

## GBM and Lung Adeno Mutations

**Lung Adeno mutations: KRAS, STK11, APC, EPHA3, ATM, and KDR**
**GBM mutations: PTEN, ERBB2, PIK3R1, CHEK2, and PIK3CA**
**Overlap: TP53, RB1, EGFR, CDKN2A, and NF1**

**Li Ding, Washington University**

- rwilson@wustl.edu

## Limitations of our current approach?

**Hypothesis-driven (biased):**
- Gene sets with related functions: “kinome”, “phosphatome”
- Genes mutated in other cancers
- Closely related genes
- Investigator-driven ideas
**Data-driven (unbiased):**
- Use genomic platforms to identify loci with recurrent somatic alterations
- Array-based RNA profiling, exon arrays
- Array CGH (LOH and CNV analysis)

**But...****	****What are we missing outside of exons?**
**	****How does the cost and complexity of PCR limit us?**
**	****How can we effectively “shake the whole tree”?   **

- rwilson@wustl.edu

## Next Gen Sequencing Technology

- rwilson@wustl.edu

## Next Gen-based mutation detection

- rwilson@wustl.edu

- DNA from X tumor samples

- PCR amplification with Y primer pairs

- Pooled with equal concentration

- Pool PCR products

- 454 sequencing

- SNP/Indel Detection Using ssahaSNP and BreakPointRead

- Reads with G12 mutation in KRAS

- Reads with 15 bp deletion in EGFR

## Whole Cancer Genome Sequencing

- rwilson@wustl.edu

**“****AML1”**
**genome sequence**

**57 y/o Caucasian female**
***De novo***** M1 AML**
**Normal cytogenetics, no CNV/LOH by array**
**WGS: 32X tumor, 14X normal (Illumina)**
**3.7M variants detected, 1.3M novel**
**8 somatic mutations validated:**
**FLT3, NPMc**
**3 cancer-related genes**
**3 novel genes**

## Next Gen Sequencing in TCGA

- Three glioblastoma samples (tumor + matching normal) have been selected for whole genome sequencing.
- Broad and Wash U will utilize the Illumina technology, Baylor will utilize 454 + SOLiD.
- Twelve GBM samples have been selected for transcriptome analysis by cDNA sequencing.
- All three centers are currently working on applications to perform directed (targeted) sequencing using the various next gen platforms.  This will likely be done first for the second gene set for the ovarian samples.

- rwilson@wustl.edu

## Acknowledgments

**The Cancer Genome Atlas Consortium**
- _Genome sequencing centers_:
- Baylor Human Genome Sequencing Center
- Broad Institute of Harvard & MIT
- The Genome Center at Washington University
**The Genome Center**
- Li Ding, Elaine Mardis, Tim Ley, David Dooling

- rwilson@wustl.edu