AGBT 2010 - Timothy Triche - Children's Hospital Los Angeles

Unraveling the Complexity of Primary and Metastatic Ewing's Sarcoma Using Helicos Singele Molecule Sequencing

Came out of ongoing studies of high risk childhood cancers.
Ewing Sarcoma
* had no survivors, now has 50% survival rate.
* If it metastisize, there is no survival (poor outcome)
Started with 16 year old female
* metastasize 6 months later
* had a lot of DNA from stock piled bone marrow

Goal:
* use RNA/DNA/Epigenomics to understand cancer
Interested in just about all types of sequencing, and integrating it all. List pretty much every type of Next gen sequencing technique. [Not much they aren't interested in.]

Using Helicos to do sequencing
* Identify two p53 mutations - both previously known.
* Chimeric genes in sarcomas usually mean the rest of the genome is less rearranged. However, there were a fairly significant rearrangements in metastasis. (eg. Entire chr 7 & 8 duplicated, )

Metastasis is not just an explant.
CNV vs RNA:
* Statistically, there is a strong association between CNV duplications and RNA up expression of genes.

[something about cell adhesion molecules?]

Mechanisms of double strand breaks... uniform at nearly single base resolution.
* 11q24.3
* in middle of FLI1 gene.
* approximately 1Mb deletion in tumour, in metastasis, this completely disappears.

Breakpoint @ 22q12.2
* less defined... again CNV changes disappear.
18qter DEL & LOH in CHLA9 disappears in CHLA10: Is the metastasis derived from the primary?
* Deletions "disappear", so the the dominant clone in the primary is not likely the one that metastasized.

Comparing primary to metastasis

[Whoa... colours... orange fused to blue, turned to light blue.. something diluted... much too fast to take notes on this without pictures.]
* Dosage effects are seen.
* 22 chromosomes show LOH and profound Homozygosity in the Metastasis that is not seen in the primary. 16/20 chromosomes.
* This shows a major simplification in the genome.

Used RNA-Seq... some filtering on RNA.
* random primers, poly a Tail addition, Hybidize and ...
* look for fusion - get EWS-FLI1 fusion

overall RNA expression.
* far more complex, especially intronic, 5` and 3` of exons.
* This is regulated under controls that have yet to be discovered.
* more than 40% of transcription in the pirmary tumour and metastasis is non-exonic.
* Genes are up regulated in metastases
* Some times you see lots of intron expression, some times you see LOTS.

[ I'm going to go see another talk - Have to stop notes here.]