# Presentation: 556749

## CGCC updateTCGA meetingDec 3-4, 2008

- Gad Getz
- Representing the Broad Institute/Dana-Farber Cancer InstituteCancer Genome Characterization Center

## Outline

- Determining purity, ploidy and absolute copy number in DNA from tumor samples
- First look at ovarian data:
- copy-number and expression

## Tumor purity and ploidy

- T = Tumor cellsN = Normal cells

- T

**Purity** = fraction of              tumor cells

- N

**N**

**T**

**T**

- 250ng of mixed
- tumor and normal
- DNA

**Ploidy** = mass of DNA
- in units of normal haploid genome mass. Here ~2.7.

- 70%

**Notes:**
A tumor sample is a mix of tumor and normal cells.
1) We define the purity as the faction of tumor cells
2) If you look at the genome of tumor and normal cells, from these SKY images, we can see that normal samples have 2 copies of each chromosome and tumor samples have different numbers of chromosomes.
We define the ploidy of a sample as the mass of DNA in units of normal haploid genome.

Once we take a fixed amount of DNA we loose the information on the number of cells.
And even if we had no normal cells, putting a fixed amount of DNA (and not a fixed number of cells), makes all measurements of copy-numbers relative to the ploidy of the sample.
This is independent of the technology, aCGH, SNP arrays, or shot gun sequencing.

Luckily tumor samples have alterations which can shed light at these two variables.
So, our goal is to estimate the ploidy and purity for each sample based on copy-number data.

This can help determine absolute copy-number, help characterize the sample, help choose samples for sequencing, help interpret other data (expression, mutations).

## Segmentation and histogram

- Copy ratio estimate at each position (level 2)
- Segment data (level 3)
- Generate a histrogram

- Histogram

## Effect of purity and ploidy on relative copy-number

- Purity

- (ploidy=2)

**Notes:**
Decrease purity  closer peaks
Increase ploidy  closer peaks
Ambiguous cases

## Effect of purity and ploidy on relative copy-number

- Purity

- Ploidy

- (ploidy=2)

- (purity=1)

- 1

- 2

- 3

- 3

- 4

- 2

- 4

- 5

- 3

- 2

**Notes:**
Decrease purity  closer peaks
Increase ploidy  closer peaks
Ambiguous cases

## Some samples are ambiguous

- 1

- 2

- 3

- 2

- 3

- 4

## Bayesian analysis producing probability of various purity/ploidy solutions

- Segmented data

- Graphical model

- Samples from
*P*(ploidy, purity | data)

- weight=0.5

- weight=0.5

- Ploidy

- Purity

## Allele-specific copy number data can disambiguate the solutions

- Only one of the two previous solutions is compatible with the ASCN for this sample.
- Color represents chromosome number

- Observed segments with negative allele-specific levels  impossible

- 0

- 1

- 2

- 3

- 4

## Results for GBM samples

- We ran the method on 110 GBMs and for 95 (~86%) we could get a unique solution using allele-specific data.
- The remaining samples are either very high ploidy or multi-clonal
- Low-purity samples enriched in mesenchymal cluster (X)

## First look at ovarian data

- Copy Number
- 38 Ovarian tumors w/ matched normals
- All 38 are primary tumors
- 35 pass quality control
- Expression
- 38 tumor samples and 10 normal samples
- All pass quality control

## Focal GISTIC analysis (n=35)

**PTEN**

**CCNE1**

**RB1**

## Similar profile

- TCGA ovarian n=35

- Internal ovarian n=118

- to other dataset

- 250K SNP array
- Etamoghadam et al.
- Bowtell group in Australia *CCR*, In press

- Amplification data
- Also similar for deletions

## Subtype discovery based on expression data

- 1) Filtered the genes:
- Top 2000 variating genes (MAD)
- 2) Consensus clustering:
- Clearly separates the normal samples from the tumors
- Identifies a subtype of tumors

- Normal

- Tumor

- subtype

## Analysis of subtype

- Correlation with copy-number events: No significant correlations
- 2) Differentially expressed genes and pathways:
- SOX11 is highly expressed in cluster (27-fold on average)
- SOX11 – a transcription factor involved in embryonic development
- A group of HLA genes and neighboring genes on chromosome 6 with no clear correlation to copy-number events
- Very similar to cluster C5 in Tothill et al., *CCR*, 2008

**Notes:**
 1      TCGA-13-0730-01
 2      TCGA-09-0365-01
 3      TCGA-13-0802-01
 4      TCGA-09-0364-01
 5      TCGA-13-0717-01
 6      TCGA-09-0369-01
 7      TCGA-13-0800-01
 8      TCGA-13-0794-01
 9      TCGA-13-0727-01
10      TCGA-09-0367-01

## Summary

- We can estimate purity and ploidy based on allele-specific copy-number data
- Data of high quality for ovarian cancer
- GISTIC and gene expression results are similar to previous reports

## Acknowledgements

- _Analysis Team_
- Scott L. Carter Michael O’KellyWendy WincklerRoel VerhaakMike LawrenceDerek Chiang
- Rameen Beroukhim
- Craig Mermel
- Jim Robinson

- _Broad Institute Genetic Analysis Platform_
- Brendan Blumenstiel
- Matt DeFelice
- Casey Gates
- Carrie Sougnez
- Huy Nguyen
- Mellisa Parkin
**Wendy Winckler**
**Stacey Gabriel**
- _Broad Institute Biological Samples Platform_
- Kristin Ardlie

**Jill Mesirov**
**Todd Golub**
**Eric Lander**
**Matthew Meyerson**

- Broad Institute of Harvard and MIT
- Dana Farber Cancer Institute